( A ) Tmem106b - / - Grn - / - mice do not show any changes in . of 5 - month - old mice of indicated genotypes were measured analyzed . n = 6 - 10 .

O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Tmem106b-/-Grn-/- mice do not show any changes in body weight. Body weight of 5-month-old mice of indicated genotypes were measured and analyzed. n=6-10.


( B Tmem106b - / - Grn - / - mice show reduced activities in open field . 4 . 5 - month - old mice were used in study .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(B Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study.


C ) Tmem106b - / - Grn - / - mice show reduced activities in open field . 4 . 5 - month - old mice were used in study . Total movement was quantified . n = 8 - 10 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * * , p < 0 . 0001 .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Tmem106b-/-Grn-/- mice show reduced activities in the open field test. 4.5-month-old mice were used in the study. Total movement was quantified. n=8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.


( D Tmem106b - / - Grn - / - mice show abnormal hindlimb clasping behavior . 5 - month - old mice were used in study .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(D Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study.


E ) Tmem106b - / - Grn - / - mice show abnormal hindlimb clasping behavior . 5 - month - old mice were used in study . Severity score was quantified . n = 8 - 10 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Tmem106b-/-Grn-/- mice show abnormal hindlimb clasping behavior. 5-month-old mice were used in the study. Severity score was quantified. n= 8-10. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.


( A , B ) Spinal cord sections ( C1 - C4 ) from 5 - month - old mice of indicated genotypes were stained with indicated antibodies ( A ) numbers of NeuN , or CHAT positive , as well as IBA1 GFAP intensities were quantified ( B ) . 3 - 5 sections / mouse were used for . n = 3 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar : 20 µm for CHAT , 200 µm for other .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(A,B) Spinal cord sections (C1-C4) from 5-month-old mice of indicated genotypes were stained with indicated antibodies (A) and the numbers of NeuN, or CHAT positive cells, as well as IBA1 and GFAP intensities were quantified (B). 3-5 sections/mouse were used for quantification. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Scale bar: 20 µm for CHAT staining, 200 µm for other images.


( A - D ) Western blot of NeuN , PSD95 , SYN , GFAP , TMEM106B , PGRN GAPDH in spinal cord ( C5 - C8 ) ( A , B ) brain ( C , D ) lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O

(A-D) Western blot analysis of NeuN, PSD95, SYN, GFAP, TMEM106B, PGRN and GAPDH in spinal cord (C5-C8) (A, B) and brain (C, D) lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.


( A , B ) Retinal sections from 5 - month - old mice were stained with indicated antibodies . Thickness of each layer , number of Brn3a positive ( retinal ganglion ) per section of outer segment as indicated by opsin are quantified . Data information : ONL : outer layer ; INL : Inner layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion layer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Retinal sections from 5-month-old mice were stained with indicated antibodies. Thickness of each layer, number of Brn3a positive cells (retinal ganglion cells) per section and length of outer segment as indicated by opsin staining are quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


( C Increase in apoptosis in Grn - / - Tmem106b - / - Grn - / - retina as indicated by TUNEL . TUNEL positive signals per section were quantified . Data information : ONL : outer layer ; INL : Inner layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion layer .

O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


D ) Increase in apoptosis in Grn - / - Tmem106b - / - Grn - / - retina as indicated by TUNEL . TUNEL positive signals per section were quantified .

O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

D) Increase in apoptosis in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by TUNEL assay. TUNEL positive signals per section were quantified.


( E Increase in Müller glial activation in Tmem106b - / - Grn - / - retina as indicated by GFAP . GFAP per section was quantified normalized to WT control . Data information : ONL : outer layer ; INL : Inner layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion layer .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


F ) Increase in Müller glial activation in Tmem106b - / - Grn - / - retina as indicated by GFAP . GFAP per section was quantified normalized to WT control .

O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

F) Increase in Müller glial activation in Tmem106b-/-Grn-/- retina as indicated by GFAP staining. GFAP intensity per section was quantified and normalized to WT control.


( G , H ) Increase in microglia activation in Grn - / - Tmem106b - / - Grn - / - retina as indicated by IBA1 . number of IBA1 positive per section was quantified . Data information : ONL : outer layer ; INL : Inner layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion layer .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) Increase in microglia activation in Grn-/- and Tmem106b-/-Grn-/- retina as indicated by IBA1 staining. The number of IBA1 positive cells per section was quantified. Data information: ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer.


( A illustrating gene expression changes in WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - cortex samples isolated from 2 . 7 - month - old mice . n = 3 . Genes with FDR p ≤ 0 . 05 , LogFC ≥ 0 . 5 or ≤ 0 . 5 between WT Tmem106b - / - Grn - / - groups are plotted on hierarchical clustering . Data information : Inflammation genes are highlighted in magenta lysosomal genes are highlighted in blue .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(A Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- cortex samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.


B ) illustrating gene expression changes in WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - spinal cord samples isolated from 2 . 7 - month - old mice . n = 3 . Genes with FDR p ≤ 0 . 05 , LogFC ≥ 0 . 5 or ≤ 0 . 5 between WT Tmem106b - / - Grn - / - groups are plotted on hierarchical clustering . Data information : Inflammation genes are highlighted in magenta lysosomal genes are highlighted in blue .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Heatmap illustrating gene expression changes in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples isolated from 2.7-month-old mice. n=3. Genes with FDR p≤0.05, LogFC≥0.5 or ≤0.5 between WT and Tmem106b-/-Grn-/- groups are plotted based on hierarchical clustering analysis. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.


( C ) illustrating gene expression changes in inflammation lysosome pathways in WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - spinal cord samples . Data information : Inflammation genes are highlighted in magenta lysosomal genes are highlighted in blue .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(C) Heatmap illustrating gene expression changes in the inflammation and lysosome pathways in WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- spinal cord samples. Data information: Inflammation genes are highlighted in magenta and lysosomal genes are highlighted in blue.


Western blot of Galectin - 3 GPNMB in spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of Galectin-3 and GPNMB in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.


Western blot of Galectin - 3 GPNMB in brain lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of Galectin-3 and GPNMB in the brain lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001.


( E ) Immunostaining of Galectin - 3 , CathD CD68 in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . Scale bar = 10 µm .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O

(E) Immunostaining of Galectin-3, CathD and CD68 in spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 10 µm.


( A - B ) Lipofuscin accumulation in retina : number of auto per section was quantified . ONL : outer layer ; INL : Inner layer ; OPL : outer plexiform layer ; IPL : inner plexiform layer ; GCL : ganglion layer . n = 3 - 6 . Scale bar = 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Lipofuscin accumulation in the retina: the number of auto fluorescent puncta per section was quantified. ONL: outer nuclear layer; INL: Inner nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer. n=3-6. Scale bar = 50 µm.


TMEM106B PGRN deletion results in increased autofluorescence in spinal cord of 5 - month - old mice . n = 3 . Scale bar = 10 µm .

B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

TMEM106B and PGRN deletion results in increased autofluorescence in the spinal cord of 5-month-old mice. n=3. Scale bar = 10 µm.


TMEM106B PGRN deletion results in increased autofluorescence in thalamus of 5 - month - old mice . n = 3 . Scale bar = 10 µm .

B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

TMEM106B and PGRN deletion results in increased autofluorescence in the thalamus of 5-month-old mice. n=3. Scale bar = 10 µm.


( G , H ) Western blot of lysosomal in spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . Asterisk indicates non - specific bands . n = 3 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(G, H) Western blot analysis of lysosomal proteins in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Asterisk indicates non-specific bands. n=3.


( A ) Spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - Tmem106b - / - Grn - / - mice were stained with anti - neurofascin , anti - LAMP1 anti - CathD antibodies . Representative from three independent mice were shown . Scale bar = 10 µm . ( B ) of LAMP1 positive vacuoles ( > 2 µm ) for experiments in ( A ) . 4 - 6 sections / spinal cord from 3 mice were analyzed for each genotype . Data presented as mean ± . Unpaired Student ' s t : * * * * , p < 0 . 0001 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/- and Tmem106b-/-Grn-/- mice were stained with anti-neurofascin, anti-LAMP1 and anti-CathD antibodies. Representative images from three independent mice were shown. Scale bar = 10 µm. (B) Quantification of LAMP1 positive vacuoles (> 2 µm) for experiments in (A). 4-6 sections/spinal cord from 3 mice were analyzed for each genotype. Data presented as mean ± SEM. Unpaired Student's t test: ****, p<0.0001.


( A Immunostaining of GFAP with LAMP1 Cath D in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . Scale bar = 5 µm .

O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O

(A Immunostaining of GFAP with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.


B ) Immunostaining of IBA1 with LAMP1 Cath D in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . Scale bar = 5 µm .

B) Immunostaining of IBA1 with LAMP1 and Cath D in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Scale bar = 5 µm.


( A ) Accumulation of autophagosomes lysosomes in spinal cord sections from a 5 - month - old Tmem106b - / - Grn - / - mouse as shown by transmission electron microscope . Scale bar = 1µm . Arrows : autophagosome ; arrow heads : lysosomes ; ( a ) : axon .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Accumulation of autophagosomes and lysosomes in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: autophagosome; arrow heads: lysosomes; (a): axon.


( B ) Accumulation of myelin debris amorphous inclusions in microglia in spinal cord sections from a 5 - month - old Tmem106b - / - Grn - / - mouse as shown by transmission electron microscope . Scale bar = 1µm . Arrows : myelin debris ; arrow heads : amorphous inclusions ; ( m ) : microglia .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Accumulation of myelin debris and amorphous inclusions in the microglia in the spinal cord sections from a 5-month-old Tmem106b-/-Grn-/- mouse as shown by transmission electron microscope analysis. Scale bar = 1µm. Arrows: myelin debris; arrow heads: dark amorphous inclusions; (m): microglia.


( C ) Western blot of LC3 - I LC3 - II in spinal cord lysates from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . n = 3 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * * , p < 0 . 0001 .

O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blot analysis of LC3-I and LC3-II in the spinal cord lysates from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ****, p<0.0001.


( D Immunostaining of TFE3 IBA1 in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O

(D Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice.


E ) Immunostaining of TFE3 IBA1 in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . signals of TFE3 in IBA1 positive microglia were quantified . n = 3 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar = 10 µm .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E) Immunostaining of TFE3 and IBA1 in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Nuclear signals of TFE3 in IBA1 positive microglia were quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.


( A , B ) Western blot of p62 , ubiquitin ( Ub ) , TDP - 43 p - TDP - 43 in RIPA - urea - soluble fractions from spinal cord ( C5 - C8 ) brain of 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . Spinal cord : n = 3 ; brain : n = 4 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * , p < 0 . 05 ; * * , p < 0 . 01 * * * , p < 0 . 001 , * * * * , p < 0 . 0001 .

O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Western blot analysis of p62, ubiquitin (Ub), TDP-43 and p-TDP-43 in RIPA- and urea-soluble fractions from spinal cord (C5-C8) and brain of 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. Spinal cord: n=3; brain: n=4. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: *, p<0.05; **, p<0.01***, p<0.001, ****, p<0.0001.


( C , D ) Immunostaining of p62 ubiquitin in spinal cord sections from 5 - month - old WT , Tmem106b - / - , Grn - / - , Tmem106b - / - Grn - / - mice . number of p62 or Ub positive was quantified . n = 3 . Data presented as mean ± . One - way ANOVA with Bonferroni ' s multiple comparisons : * * * , p < 0 . 001 , * * * * , p < 0 . 0001 . Scale bar = 10 µm .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Immunostaining of p62 and ubiquitin in the spinal cord sections from 5-month-old WT, Tmem106b-/-, Grn-/-, and Tmem106b-/-Grn-/- mice. The number of p62 or Ub positive puncta was quantified. n=3. Data presented as mean ± SEM. One-way ANOVA tests with Bonferroni's multiple comparisons: ***, p<0.001, ****, p<0.0001. Scale bar = 10 µm.


A . H3122 expressing endogenous EML4 - ALK V1 HEK293 transfected with YFP - EML4 - ALK V1 WT KD were stained for either anti - ALK or anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown . B . Violin plots showing number of EML4 - ALK V1 foci per . Data represents 30 - 50 from three biological replicates . * * * P < 0 . 001 , * * * * P < 0 . 0001 in comparison to HEK293 YFP - EML4 - ALK - V1 KD by one - way ANOVA .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis.


C . H2228 expressing endogenous EML4 - ALK V3 HEK293 transfected with YFP - EML4 - ALK V3 WT KD were stained for either anti - ALK or anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown . D . Violin plots showing number of EML4 - ALK V3 foci per . Data represents 30 - 50 from four biological replicates . * * * P < 0 . 001 , * * * * P < 0 . 0001 in comparison to HEK293 YFP - EML4 - ALK - V3 KD by one - way ANOVA .

C. H2228 cells expressing endogenous EML4-ALK V3 and HEK293 transfected with YFP-EML4-ALK V3 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. D. Violin plots showing the number of EML4-ALK V3 cytoplasmic foci per cell. Data represents 30-50 counts from four biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V3 KD by one-way ANOVA analysis.


H . Whisker plot showing calculated velocity of single events in kymograph . Data represent 20 from 10 kymographs . n = 2 . Error bar represents SD of two biological replicates . * P < 0 . 05 in comparison to YFP EML4 - ALK V3 KD by unpaired t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O

H. Whisker plot showing the calculated velocity of single events in the kymograph. Data represent 20 counts from 10 kymographs. n=2. Error bar represents SD of two biological replicates. *P< 0.05 in comparison to YFP EML4-ALK V3 KD by unpaired t test.


A , B . H3122 H2228 were stained for anti - ALK ( green ) , anti - GRB2 ( red ) , anti - SOS1 ( red ) , anti - pC - KITY721 ( red ) , anti - PI3K p85β or anti - pPLCγ2Y759 DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A, B. H3122 and H2228 cells were stained for anti-ALK (green), anti-GRB2 (red), anti-SOS1 (red), anti-pC-KITY721 (red), anti-PI3K p85β or anti-pPLCγ2Y759 and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


C . HEK293 transfected with YFP - EM4L - ALK V1 WT or V3 WT for 48 hrs treated with 5 % or 10 % 1 , 6 - hexanediol . Representative still after 5 min ( taken from time - lapse movies ) are shown of treated with 10 % 1 , 6 - Hexanediol or DMSO . Scale bars , 10 μm ; arrowheads indicate foci . D . Box plot showing number of foci in DMSO 5 % or 10 % Hexanediol ( 5 min ) . Data represent 5 - 10 from 3 independent experiments ( n = 3 ) . Error bar represents SD of three biological replicates . * * * * P < 0 . 0001 in comparison to YFP - EML4 - ALK V3 WT DMSO by one - way ANOVA .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

C. HEK293 cells transfected with YFP-EM4L-ALK V1 WT or V3 WT for 48 hrs and treated with 5% or 10% 1,6-hexanediol. Representative still images after 5 min (taken from time-lapse movies) are shown of cells treated with 10% 1,6-Hexanediol or DMSO. Scale bars, 10 μm; the arrowheads indicate cytoplasmic foci. D. Box plot showing the number of cytoplasmic foci in DMSO and 5% or 10% Hexanediol (5 min). Data represent 5-10 counts from 3 independent experiments (n=3). Error bar represents SD of three biological replicates. ****P< 0.0001 in comparison to YFP-EML4-ALK V3 WT DMSO by one-way ANOVA analysis.


E . Representative western blots of HEK293 transfected EML4 - ALK V1 WT V3 WT for 48 hrs . were treated with either 5 % or 10 % 1 , 6 - Hexanediol for 5 minutes . DMSO was used a control . Lysates were analysed for phosphorylation expression of indicated antibodies . GAPDH was used as a loading control . Data representative of n = 2 experiments .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Representative western blots of HEK293 transfected EML4-ALK V1 WT and V3 WT for 48 hrs. Cells were treated with either 5% or 10% 1,6-Hexanediol for 5 minutes. DMSO was used a control. Lysates were analysed for the phosphorylation and expression of the indicated antibodies. GAPDH was used as a loading control. Data representative of n=2 experiments.


A , B , C , D . HEK293 were transfected with EML4 - ALK V1 WT or V3 WT or Kinase Dead ( KD ) for 48 hours . were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation with anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown .

O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B, C, D. HEK293 cells were transfected with EML4-ALK V1 WT or V3 WT or Kinase Dead (KD) for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


B . HEK293 were transfected with EML4 - ALK V1 deletion 12N blade for 48 hours . were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation with anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HEK293 cells were transfected with EML4-ALK V1 deletion 12N blade for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown.


D . HEK293 were transfected with EML4 - ALK V3 ∆ JM 1058 - 1100 for 48 hours . were either untreated ( DMSO ) or treated with ALK inhibitors for 4 hours before fixation with anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown . E . Violin plot representing number of foci counted per from D . Data represent from at least 30 , n = 3 . * * * * P < 0 . 0001 in comparison to DMSO by one - way ANOVA .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. HEK293 cells were transfected with EML4-ALK V3 ∆JM 1058-1100 for 48 hours. Cells were either untreated (DMSO) or treated with ALK inhibitors for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. E. Violin plot representing the number of cytoplasmic foci counted per cell from D. Data represent counts from at least 30 cells, n=3. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.


A - F HEK293 were transfected with A . YFP - EML4 - ALK V3 K1150M , C . R1275Q or E . F1174L constructs treated with ALK inhibitors or DMSO for 4 hours before fixation with anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown . B , D , F . Violin plots representing number of foci counted per from A , C E . Data represent from at least 30 , n = 3 or 4 . * * * * P < 0 . 0001 in comparison to DMSO by one - way ANOVA .

O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F HEK293 cells were transfected with A. YFP-EML4-ALK V3 K1150M, C. R1275Q or E. F1174L constructs and treated with ALK inhibitors or DMSO for 4 hours before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B, D, F. Violin plots representing the number of cytoplasmic foci counted per cell from A, C and E. Data represent counts from at least 30 cells, n=3 or 4. ****P< 0.0001 in comparison to DMSO by one-way ANOVA.


B . HEK293 transfected with YFP - EML4 - GyrB either untreated ( DMSO ) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation with anti - GFP ( green ) , anti - α - tubulin ( red ) , DAPI ( blue ) . Scale bars , 10 μm ; magnified views of a selected are shown . C . Violin plot showing number of foci per HEK293 transfected with YFP - EML4 - GyrB . Data represent from > 30 , n = 4 . * * * * P < 0 . 0001 in comparison to DMSO by unpaired t .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. HEK293 cells transfected with YFP-EML4-GyrB and either untreated (DMSO) or treated with Coumermycin A1 to induce dimerization of GyrB before fixation and staining with anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. C. Violin plot showing the number of cytoplasmic foci per HEK293 cell transfected with YFP-EML4-GyrB. Data represent counts from >30 cells, n= 4. ****P< 0.0001 in comparison to DMSO by unpaired t test.


( A ) HEK293T ‐ Fpn were incubated with FAC ( 10 μM Fe ) for 24 h followed by incubation for 6 h in absence or presence of 10 μM Ponasterone A . were then incubated with without 100 μM chloroquine or 10 μM leupeptin for 10 h harvested . ferritin content was determined by ELISA . Induction of Fpn resulted in decreased ferritin this was not prevented by treatment with chloroquine or leupeptin . data are presented as standard deviation from three different experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then incubated with and without 100 μM chloroquine or 10 μM leupeptin for 10 h and harvested. The ferritin content was determined by ELISA. Induction of Fpn resulted in decreased ferritin levels and this was not prevented by treatment with chloroquine or leupeptin. The data are presented as the standard deviation from three different experiments.


( B ) treated as in ( A ) were incubated in presence of 1 μg / ml EGF for 2 h . were fixed processed for immunofluorescence using mouse anti ‐ EGF receptor Alexa 594 conjugated goat anti ‐ mouse IgG . Chloroquine leupeptin effectively inhibit degradation of EGF receptor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O

(B) Cells treated as in (A) were incubated in the presence of 1 μg/ml EGF for 2 h. Cells were fixed and processed for immunofluorescence using mouse anti‐EGF receptor and Alexa 594 conjugated goat anti‐mouse IgG. Chloroquine and leupeptin effectively inhibit degradation of EGF receptor.


( A ) HEK293T ‐ Fpn were incubated with FAC ( 10 μM Fe ) for 24 h followed by incubation for 6 h in absence or presence of 10 μM Ponasterone A . were then treated with or without 10 μM MG132 or 10 μM lactacystin in presence of Ponasterone A for 10 h harvested . ferritin content was determined by ELISA . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(A) HEK293T‐Fpn cells were incubated with FAC (10 μM Fe) for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 or 10 μM lactacystin in the presence of Ponasterone A for 10 h and harvested. The ferritin content was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


( B ) Samples treated as in ( A ) were immunoprecipitated using anti ‐ ferritin antibodies immunoprecipitate examined for presence of ferritin or ubiquitin by Western blot . arrows indicate migration of H L chains .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

(B) Samples treated as in (A) were immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ferritin or ubiquitin by Western blot analysis. The arrows indicate the migration of H and L chains.


( C ) were incubated with 1 . 0 × 10 − 7 M Tf ( 59Fe ) 2 for 24 h followed by incubation for 6 h in absence or presence of 10 μM Ponasterone A . were then treated with or without 10 μM MG132 in presence of Ponasterone A for 10 h harvested amount of 59Fe in immunoprecipitatedferritin was determined .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Cells were incubated with 1.0 × 10−7 M Tf(59Fe)2 for 24 h followed by incubation for 6 h in the absence or presence of 10 μM Ponasterone A. Cells were then treated with or without 10 μM MG132 in the presence of Ponasterone A for 10 h and harvested and the amount of 59Fe in immunoprecipitatedferritin was determined.


( D ) were treated as described in ( A ) extracts were applied to exclusion chromatography ferritin content , in selected fractions , was determined by ELISA . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(D) Cells were treated as described in (A) and cell extracts were applied to size exclusion chromatography and the ferritin content, in selected fractions, was determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


( E ) were treated as described in ( C ) , ferritin was then immunoprecipitated , eluted using 100 mM glycine , pH 2 . 5 measured by ELISA . amount of ferritin ‐ associated 59Fe was measured specific activity of ferritin determined . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(E) Cells were treated as described in (C), ferritin was then immunoprecipitated, eluted using 100 mM glycine, pH 2.5 and measured by ELISA. The amount of ferritin‐associated 59Fe was measured and the specific activity of ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.


( F ) Samples were treated as in ( D ) , ferritin was immunoprecipitated analyzed by Western blot using an antibody to ubiquitin .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O

(F) Samples were treated as in (D), and ferritin was immunoprecipitated and analyzed by Western blot analysis using an antibody to ubiquitin.


( A ) FM3A ( black ) ts85 ( grey ) were transiently transfected with a plasmid containing CMV ‐ regulated Fpn ‐ GFP incubated in presence of FAC ( 10 μM Fe ) 0 . 5 μM hepcidin for 24 h . Hepcidin was either maintained ( + ) or removed ( − ) to allow Fpn ‐ GFP localization at plasma . were maintained at permissive ( 33 ° C ) or moved to restrictive ( 39 ° C ) incubated for 6 h . were harvested ferritin content determined by ELISA . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(A) FM3A (black) and ts85 (grey) cells were transiently transfected with a plasmid containing CMV‐regulated Fpn‐GFP and incubated in the presence of FAC (10 μM Fe) and 0.5 μM hepcidin for 24 h. Hepcidin was either maintained (+) or removed (−) to allow Fpn‐GFP localization at the plasma membrane. Cells were maintained at the permissive temperature (33°C) or moved to the restrictive temperature (39°C) and incubated for 6 h. Cells were harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


( B ) FM3A ts85 were treated as in ( A ) but FAC was replaced with 1 . 0 × 10 − 7 M Tf ( 59Fe ) 2 . Ferritin was immunoprecipitated specific activity of 59Fe ‐ ferritin determined . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(B) FM3A and ts85 cells were treated as in (A) but FAC was replaced with 1.0 × 10−7 M Tf(59Fe)2. Ferritin was immunoprecipitated and the specific activity of 59Fe‐ferritin determined. Error bars represent the standard deviation from three different experiments in duplicate.


( C ) ts85 were treated as in ( B ) extracts applied to exclusion chromatography . Ferritin content in selected fractions was measured by ELISA . Black bars represent assembled ferritin ( > 400 kDa ) , gray bars represent monomeric ( 50 kDa ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(C) ts85 cells were treated as in (B) and cell extracts applied to size exclusion chromatography. Ferritin content in selected fractions was measured by ELISA. Black bars represent assembled ferritin (>400 kDa), gray bars represent monomeric (50 kDa).


( D ) FM3A ts85 were treated as in ( A ) but that had been incubated at restrictive ( 39 ° C ) were then returned to permissive ( 33 ° C ) incubated in presence of cycloheximide ( 75 μg / ml ) 10 μM FAC for 1 h . were then harvested ferritin determined by ELISA . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(D) FM3A and ts85 cells were treated as in (A) but cells that had been incubated at the restrictive temperature (39°C) were then returned to the permissive temperature (33°C) and incubated in the presence of cycloheximide (75 μg/ml) and 10 μM FAC for 1 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


( A ) were then incubated at restrictive ( 39 ° C ) for 6 h in presence or absence of 100 μM DFO , with or without 100 μM chloroquine or 10 μM MG132 . were then harvested ferritin content determined by ELISA . Error bars represent standard deviation from three different experiments in duplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(A) Cells were then incubated at the restrictive temperature (39°C) for 6 h in the presence or absence of 100 μM DFO, with or without 100 μM chloroquine or 10 μM MG132. Cells were then harvested and ferritin content determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate.


( B ) treated as in ( A ) were incubated in presence or absence of 5 mM 3 ‐ methyladenine .

O O O O O O O O O O O O O O O O O O O O O O

(B) Cells treated as in (A) were incubated in the presence or absence of 5 mM 3‐methyladenine.


( A ) Δccc1 / pGAL ‐ H + L were transformed with a plasmid containing a methionine regulated CCC1 ( pMET3CCC1 ) . were grown in medium with galactose for 20 h , washed incubated in galactose ( • ) , glucose with 10 × methionine ( ▵ ) , glucose ( ○ ) or glucose without methionine ( ▾ ) for 10 h . were then harvested ferritin determined by ELISA . Error bars represent standard deviation from three different experiments in duplicate . absence of methionine leads to expression of vacuolar iron transporter Ccc1p .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Δccc1/pGAL‐H+L cells were transformed with a plasmid containing a methionine regulated CCC1 (pMET3CCC1). Cells were grown in medium with galactose for 20 h, washed and incubated in galactose (•), glucose with 10 × methionine (▵), glucose (○) or glucose without methionine (▾) for 10 h. Cells were then harvested and ferritin levels determined by ELISA. Error bars represent the standard deviation from three different experiments in duplicate. The absence of methionine leads to expression of the vacuolar iron transporter Ccc1p.


( B ) erg6 ‐ 2 erg6 ‐ 2 pGAL ‐ H + L strains were grown in medium with galactose 250 μM FeSO4 for 24 h . were then washed incubated in galactose or glucose in absence or presence of 50 μM MG132 for 7 h .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) erg6‐2 and erg6‐2 pGAL‐H+L strains were grown in medium with galactose and 250 μM FeSO4 for 24 h. Cells were then washed and incubated in galactose or glucose in the absence or presence of 50 μM MG132 for 7 h.


( C ) were then harvested , ferritin determined by ELISA immunoprecipitated using anti ‐ ferritin antibodies immunoprecipitate examined for presence of ubiquitin by Western . with erg6 ‐ 2 allele permit entry of MG132 ( Lee Goldberg , 1996 ) .

O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

(C) Cells were then harvested, ferritin levels determined by ELISA and immunoprecipitated using anti‐ferritin antibodies and the immunoprecipitate examined for the presence of ubiquitin by Western analysis. Cells with the erg6‐2 allele permit the entry of MG132 ( Lee and Goldberg, 1996).


( A ) Mitochondrial extracts from 143B , COX1 ∆ COX2 ∆ mutant cybrids their respective isogenic controls ( CON - 1 CON - 2 ) were analyzed by SDS - PAGE immunoblotting with indicated antibodies . graphs ( right ) show densitometric quantifications of signals normalized to CII subunit SDHA . mean of three independent controls ( CON ) was set to 100 measurements were referenced to that value . values represent mean ± SD ( n = 3 - 8 ) . Mann - Whitney U was applied for statistical analyses . Variations between controls mutants : α , p < 0 . 05 ; β , p < 0 . 01 ; γ , p < 0 . 001 . Specific variations between COX1 ∆ COX2 ∆ mutants : ∗ p < 0 . 05 ; ∗ ∗ p < 0 . 01 .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(A) Mitochondrial extracts from 143B cells, COX1∆ and COX2∆ mutant cybrids and their respective isogenic controls (CON-1 and CON-2) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The graphs (right) show densitometric quantifications of the signals normalized to CII subunit SDHA. The mean of three independent controls (CON) was set to 100 and all measurements were referenced to that value. The values represent mean ± SD (n=3-8). Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; β, p<0.01; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05; ∗∗p<0.01.


( B ) Digitonized mitochondria ( digitonin - to - of 2 : 1 ) were analyzed by BN - PAGE , followed by CI in gel activity ( IGA ) assays Coomassie . SC I + III2 + IV1 band ( b1 ) was excised from control ( 143B ) lane SC I + III2 bands ( b2 to b4 ) were excised from COX1 ∆ COX2 ∆ lanes . Their compositions were subsequently analyzed by nano - LC / ESI - MS ( see also Figure EV2 ) . relative position of molecular marker is indicated on left .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Digitonized mitochondria (digitonin-to-protein ratio of 2:1) were analyzed by BN-PAGE, followed by CI in gel activity (IGA) assays and Coomassie staining. The SC I+III2+IV1 band (b1) was excised from the control (143B) lane and the SC I+III2 bands (b2 to b4) were excised from the COX1∆ and COX2∆ lanes. Their protein compositions were subsequently analyzed by nano-LC/ESI-MS (see also Figure EV2). The relative position of the molecular weight marker is indicated on the left.


( C ) BN - PAGE was followed by CI - IGA immunoblotting with indicated antibodies ( in brackets ) . identity of MRC complexes SCs is : I + III2 + IV1 , SC containing CI , CIII2 , CIV ; I + III2 , SC containing CI CIII2 ; I + III2plus , SC containing CI , CIII2 a COX1 - submodule ; III2 + IV , SC containing CIII2 CIV ; I , I ; III2 , CIII dimer ; IV2 , CIV dimer ; subCOX1 , COX1 - containing subcomplexes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O

(C) BN-PAGE was followed by CI-IGA assay and immunoblotting with the indicated antibodies (in brackets). The identity of the MRC complexes and SCs is: I+III2+IV1, SC containing CI, CIII2, and CIV; I+III2, SC containing CI and CIII2; I+III2plus, SC containing CI, CIII2 and a COX1-submodule; III2+IV, SC containing CIII2 and CIV; I, complex I; III2, CIII dimer; IV2, CIV dimer; subCOX1, COX1-containing subcomplexes.


( A ) Control 143B ( left panels ) , COX1 ∆ cybrids ( middle panels ) , COX2 ∆ cybrids ( right panels ) . right lane highlights structural subunits of CI ( yellow ) , CIII ( green ) , CIV ( orange brown ) , assembly factors ( orange ) . - absolute ( iBAQ ) values were normalized to sum of control values . Top lanes indicate gel slice numbers their relative native masses .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Control 143B cells (left panels), COX1∆ cybrids (middle panels), and COX2∆ cybrids (right panels). The right lane highlights structural subunits of CI (yellow), CIII (light green), and CIV (orange brown), and assembly factors (orange). Intensity-based absolute quantification (iBAQ) values were normalized to the sum of all control values. Top lanes indicate gel slice numbers and their relative native masses.


( B ) profiles showing abundance relative distribution of structural subunits from CI ( upper graphs ) , CIII ( middle graphs ) , CIV ( bottom graphs ) . identities of MRC complexes SCs are as in Figure 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Intensity profiles showing abundance and relative distribution of structural subunits from CI (upper graphs), CIII (middle graphs), and CIV (bottom graphs). The identities of MRC complexes and SCs are as in Figure 1.


( A ) Excerpts of complexome profiling data showing distribution of CIV subunits assembly factors HIGD1A , HIGD2A , CMC1 COA3 in control ( 143B ) , COX1 ∆ COX2 ∆ cybrids . relative abundance distribution of detected is shown in lower graphs . localization of SC I + III2plus in COX2Δ cybrids is highlighted with a red . approximate molecular weights of CIV - containing structures are indicated on top ( in kDa ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(A) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A, HIGD2A, CMC1 and COA3 in control (143B), COX1∆ and COX2∆ cybrids. The relative abundance and distribution of detected proteins is shown in the lower graphs. The localization of SC I+III2plus in COX2Δ cybrids is highlighted with a red line. The approximate molecular weights of CIV-containing structures are indicated on the top (in kDa).


( B ) 2D - BN / SDS - PAGE from control ( 143B ) COX2Δ cybrids followed by immunoblotting with indicated antibodies . I + III2 + IV1 , SC containing CI , CIII , CIV ; I + III2plus , SC containing CI , CIII a COX1 - submodule ; III2 + IV , SC containing CIII CIV ; IV2 , CIV dimer ; IV , CIV monomer . presence of a COX1 - CMC1 - COA3 - containing subcomplex is indicated .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(B) 2D-BN/SDS-PAGE analysis from control (143B) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies. I+III2+IV1, SC containing CI, CIII, and CIV; I+III2plus, SC containing CI, CIII and a COX1-submodule; III2+IV, SC containing CIII and CIV; IV2, CIV dimer; IV, CIV monomer. The presence of a COX1-CMC1-COA3-containing subcomplex is indicated.


Mitochondrial extracts from control 143B ( left ) , COX1 ∆ cybrids ( right ) , COX2 ∆ cybrids ( middle ) were immunoprecipitated using antibodies against : ( A ) CIV subunits COX1 COX5B . ( B ) CI subunit NDUFB7 . Samples were subsequently analyzed by SDS - PAGE immunoblotting with indicated antibodies . In , input . IP , immuno - precipitate . FT , flow - through . Ig , unrelated immunoglobulin used as a negative control .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (A) CIV subunits COX1 and COX5B. (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.


Mitochondrial extracts from control 143B ( left ) , COX1 ∆ cybrids ( right ) , COX2 ∆ cybrids ( middle ) were immunoprecipitated using antibodies against : ( B ) CI subunit NDUFB7 . Samples were subsequently analyzed by SDS - PAGE immunoblotting with indicated antibodies . In , input . IP , immuno - precipitate . FT , flow - through . Ig , unrelated immunoglobulin used as a negative control .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.


Mitochondrial extracts from control 143B ( left ) , COX1 ∆ cybrids ( right ) , COX2 ∆ cybrids ( middle ) were immunoprecipitated using antibodies against : ( C ) CIII subunit CORE2 . Samples were subsequently analyzed by SDS - PAGE immunoblotting with indicated antibodies . In , input . IP , immuno - precipitate . FT , flow - through . Ig , unrelated immunoglobulin used as a negative control .

Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (C) CIII subunit CORE2. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.


( A ) Digitonin - solubilized mitochondria from doxycycline - treated 143B , COX1 ∆ COX2 ∆ cybrids were analyzed by BN - PAGE followed by CI - IGA assays ( upper panels ) or immunoblotting ( medium lower panels ) with antibodies against CI subunits NDUFA9 NDUFS1 . identities of MRC complexes SCs are as in Figure 1 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O

(A) Digitonin-solubilized mitochondria from doxycycline-treated 143B cells, COX1∆ and COX2∆ cybrids were analyzed by BN-PAGE followed by CI-IGA assays (upper panels) or immunoblotting (medium and lower panels) with antibodies against CI subunits NDUFA9 and NDUFS1. The identities of MRC complexes and SCs are as in Figure 1.


( B ) Mean incorporation rates of NDUFA9 NDUFS1 into SC I + III2 + IV1 from 143B into SCs I + III2 from CIV - KO cybrids . Signals from at least three independent experiments were quantified normalized by CII subunit SDHA . Time - point values are expressed as percentages of untreated ( SS ) indicated as mean ± SD .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Mean incorporation rates of NDUFA9 and NDUFS1 into SC I+III2+IV1 from 143B cells and into SCs I+III2 from the CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.


( C ) Samples were subsequently analyzed by 2D - BN / SDS - PAGE immunoblotting with indicated antibodies . identities of MRC complexes SCs are as in Figure 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Samples were subsequently analyzed by 2D-BN/SDS-PAGE and immunoblotting with the indicated antibodies. The identities of MRC complexes and SCs are as in Figure 1.


( D ) Mean incorporation rates of CIII subunits CORE2 RISP into SC I + III2 + IV1 from control into SCs I + III2 from CIV - KO cybrids . Signals from at least three independent experiments were quantified normalized by CII subunit SDHA . Time - point values are expressed as percentages of untreated ( SS ) indicated as mean ± SD .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Mean incorporation rates of CIII subunits CORE2 and RISP into SC I+III2+IV1 from control cells and into SCs I+III2 from CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.


( A ) Digitonized mitochondria from doxycycline - treated 143B COX2 ∆ cybrids were analyzed by 2D - BN / SDS - PAGE western blot with indicated antibodies .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies.


( B ) Mean incorporation rates of COX subunits into SC I + III2plus from COX2 ∆ cybrids . Signals from three independent experiments were quantified normalized by CII . Time - point values are expressed as percentages of untreated ( SS ) indicated as mean ± SD . Control ( 143B ) values for subunits COX4 , COX5A COX6C were obtained from ( Moreno - Lastres et al . , 2012 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

(B) Mean incorporation rates of COX subunits into SC I+III2plus from COX2∆ cybrids. Signals from three independent experiments were quantified and normalized by CII. Time-point values are expressed as percentages of the untreated cells (SS) and indicated as mean ± SD. Control (143B) values for subunits COX4, COX5A and COX6C were obtained from (Moreno-Lastres et al., 2012).


C - D SDS - PAGE ( C ) BN - PAGE ( D ) analyses from 143B COX2 ∆ cybrids transiently transfected either with a COX5B - Myc - DDK construct or with empty vector , followed by immunoblotting with antibodies against COX5B CII subunit SDHA ( loading control ) . identities of MRC complexes SCs are as in Figure 1 . red arrow points to accumulation of over expressed COX5B bound to SC I + III2 in control .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

C-D SDS-PAGE (C) and BN-PAGE (D) analyses from 143B cells and COX2∆ cybrids transiently transfected either with a COX5B-Myc-DDK construct or with the empty vector, followed by immunoblotting with antibodies against COX5B and CII subunit SDHA (loading control). The identities of MRC complexes and SCs are as in Figure 1. The red arrow points to the accumulation of over expressed COX5B bound to SC I+III2 in control cells.


Mitochondrial translation products were - labelled in control 143B ( left panel ) , COX1 ∆ ( middle panel ) COX2 ∆ ( right panel ) cybrids with [ 35S ] - methionine for 30 minutes in presence of anisomycin to inhibit translation . For chase , were washed incubated with fresh culture medium for indicated time points ( T0 - T24 hours ) . Radiolabeled mitochondrial ( indicated on right ) were separated by 2D - BN / SDS - PAGE visualized by autoradiography . A enlarged section of SCs region at T24 ( delimited by a square ) shows co - migration of COX1 with SC I + III2plus ( black arrow ) in COX2 ∆ cybrids relative to position of canonical SC I + III2 ( gray arrow ) . identities of MRC complexes SCs ( bottom ) are as in Figure 1 . Molecular markers are indicated on left .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mitochondrial translation products were pulse-labelled in control 143B cells (left panel), COX1∆ (middle panel) and COX2∆ (right panel) cybrids with [35S]-methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation. For chase, cells were washed and incubated with fresh culture medium for the indicated time points (T0-T24 hours). Radiolabeled mitochondrial proteins (indicated on the right) were separated by 2D-BN/SDS-PAGE and visualized by autoradiography. A enlarged section of the SCs region at T24 (delimited by a square) shows the co-migration of COX1 with SC I+III2plus (black arrow) in the COX2∆ cybrids relative to the position of canonical SC I+III2 (gray arrow). The identities of MRC complexes and SCs (bottom) are as in Figure 1. Molecular weight markers are indicated on the left.


( A ) Respiratory chain enzyme activities of control ( blue ) , COX1 ∆ ( red ) COX2 ∆ ( green ) cybrids . Enzyme activities were measured in three experimental replicates . Data are expressed as percentages of control , indicated as means ± SD . Mann - Whitney U was applied for statistical analyses . Variations between controls mutants : α , p < 0 . 05 ; γ , p < 0 . 001 . Specific variations between COX1 ∆ COX2 ∆ mutants : ∗ p < 0 . 05 .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O

(A) Respiratory chain enzyme activities of control (blue), COX1∆ (red) and COX2∆ (green) cybrids. Enzyme activities were measured in three experimental replicates. Data are expressed as the percentages of control cells, and indicated as means ± SD. Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05.


Colorectal outcomes in WT ( n = 7 ) , IL - 33KO ( n = 4 ) ST2KO ( n = 3 ) mice at completion of AOM / DSS carcinogenesis protocol . ( A ) volumes at endpoint ( each dot represents a , mice with no are marked as zero ) , Skin outcomes in WT ( n = 7 ) , IL - 33KO ( n = 6 ) ST2KO ( n = 7 ) mice at completion of DMBA / DNFB carcinogenesis protocol . ( D ) volumes at endpoint ( each dot represents a , mice with no are marked as zero ) ,

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero),


H ) of IL - 33 + epidermal dermal in DNFB - versus acetone - treated WT skin . Dots represent from three randomly selected high power field ( HPF ) per sample ( n = 5 per group ) . J ) of IL - 33 + epithelial stromal of colon treated with AOM / DSS versus no treatment . Dots represent from three randomly selected HPF per sample ( n = 4 per group ) . Data information : Graphs show mean + SD , unpaired t - .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group). J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group). Data information: Graphs show mean + SD, unpaired t-test.


( H ) ChIP - qPCR for Smad6 in presence of IL - 33 full or cytokine domain using an anti - RUNX2 antibody . After Pam212 were transfected with IL - 33 full or cytokine domain for 24 hours , lysates were subjected to - immunoprecipitation with anti - RUNX2 antibody eluted RUNX2 - bound was used for qPCR with primers against Smad6 promoter region . Anti - IgG ChIP - qPCR results are shown as negative controls ( n = 4 per group ) .

O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).


( D ) Immunoblot of p - SMAD2 / 3 SMAD2 / 3 in response to TGF - β poly ( I : C ) treatment . Pam212 were treated with poly ( I : C ) or PBS followed by incubation with without TGF - β ( 5nM ) . Data represent three independent experiments with similar results . ( E ) Immunoblot of p - SMAD2 / 3 SMAD2 / 3 in response to TGF - β + poly ( I : C ) after knocking down of IL - 33 . Pam212 were transfected with siIl33 knockdown construct or siRNA control ( siCon ) for 30 hours followed by incubation with poly ( I : C ) TGF - β . Data represent three independent experiments with similar results . ( F ) Immunoblot of p - SMAD2 / 3 SMAD2 / 3 upon expression of IL - 33 full or cytokine domain . Pam212 were transfected with IL - 33 full or cytokine domain for 24 hours followed by incubation with without TGF - β . Data represent three independent experiments with similar results . Data information : GAPDH is used as control housekeeping in D - F ) . Graphs show mean + SD , NS : not significant , unpaired t - .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.


( G ) Impact of IL - 33 knockdown on proliferation in response to TGF - β . Pam212 were treated with siIl33 or siCon followed by TGF - β treatment ( n = 7 in each group ) . ( H ) Impact of IL - 33 knockdown SB431542 treatment on proliferation . Pam212 were treated with siIl33 or siCon in combination with SB431542 or DMSO ( carrier control , n = 7 in each group ) .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group).


( I ) SMAD signaling in WT K14 - IL33tg , ST2KO skin treated with acetone ( control ) at completion of DMBA / TPA carcinogenesis protocol . Tissue lysates prepared from whole back skin of were subjected to immunoblot with p - SMAD2 / 3 , p - SMAD1 / 5 , SMAD6 , SMAD2 / 3 SMAD1 antibodies . GAPDH is used as control housekeeping ( n = 5 in each group ) .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).


( J ) Representative of IL - 33 p - SMAD2 / 3 immunostaining on adjacent sections of human squamous carcinoma ( SCC ) normal skin ( scale bar : 100 μm ) . Data information : Graphs show mean + SD , NS : not significant , unpaired t - .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm). Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.


( B ) Il33 Smad6 mRNA in caerulein - treated WT pancreas compared with PBS - treated controls at completion of chronic pancreatitis protocol ( n = 8 in caerulein n = 9 in PBS group for Il33 , n = 9 in caerulein n = 10 in PBS group for Smad6 , unpaired t - ) .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).


( D ) Pancreatic - free survival of WT ( n = 4 ) , IL - 33KO ( n = 5 ) ST2KO ( n = 6 ) mice at completion of DMBA / caerulein carcinogenesis protocol ( log - rank ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).


( G , H ) of ( G ) IL - 33 + ( H ) p - SMAD2 / 3 + epithelial per HPF in matched human pancreatic tissues . Each dot represents average across three randomly selected HPF per sample ( n = 18 , paired t - ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test).


A . Representative FACS plot ( left ) showing a mixed - lineage clone with a T - lymphoid ( CD25 + / CD11b - Gr1 - ) , myeloid ( CD11b + Gr1 + / CD25 - ) , biphenotypic ( CD11b + Gr1 + / CD25 + ) fraction derived from a single Myc / Bcl2 - transformed DN2cell grown on 1 : 1 OP9 : OP9 - Dll1 co - culture with lineage - promiscuous cytokines IL - 2 , IL - 3 , IL - 6 , IL - 7 , SCF , GM - CSF , Flt3 . of indicated flow - sorted fractions was analyzed by Giemsa ( right ) .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative FACS plot (left) showing a mixed-lineage clone with a T-lymphoid (CD25+/CD11b-Gr1-), myeloid (CD11b+Gr1+/CD25-), and biphenotypic (CD11b+Gr1+/CD25+) fraction derived from a single Myc/Bcl2-transformed DN2cell grown on 1:1 OP9:OP9-Dll1 co-culture with lineage-promiscuous cytokines IL-2, IL-3, IL-6, IL-7, SCF,GM-CSF, Flt3. Morphology of the indicated flow-sorted cell fractions was analyzed by Giemsa staining (right).


B . Phenotype frequencies of clones from Myc / Bcl2 - transformed single ( grown phenotyped as mentioned in A ) . Analyzed were 31 clones from LSKs , 107 from GMPs 28 from DN2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Phenotype frequencies of clones from Myc/Bcl2-transformed single cells (grown and phenotyped as mentioned in A). Analyzed were 31 clones from LSKs, 107 from GMPs and 28 from DN2 cells.


C . Cumulative survival of 31 ± 2 27 ± 6 days of mice that received 5104 to 5105 T - lymphoid or myeloid , respectively , that were flow - sorted from mixed Myc / Bcl2 + DN2 - derived clones . summarized data are from 3 mice injected with T - lymphoid 4 mice injected with myeloid fraction of two different clones .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Cumulative survival of 31±2 and 27±6 days of mice that received 5104 to 5105 T-lymphoid cells or myeloid cells, respectively, that were flow-sorted from mixed Myc/Bcl2+ DN2-derived clones. The summarized data are from 3 mice injected with the T-lymphoid and 4 mice injected with the myeloid fraction of two different clones.


D . Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc / Bcl2 - transformed LSK or GMPcells on OP9 : OP9 - Dll1 co - cultures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc/Bcl2-transformed LSK or GMPcells on OP9:OP9-Dll1 co-cultures.


E . ( top panels ) Serial replating of Myc - Bcl2 + DN2 in methylcellulose generated myeloid clones with stable genomic reassembly of Dβ1 TCR locus in vitro ( gel : left two lanes ) . Upon transplantation , same clones induced myeloid leukemia that retained initial rearrangement in vivo ( gel : right two lanes ) as assessed by nested PCR . GL = germline . ( bottom panels ) Methylcellulose - replating of Myc - Bcl2 + GMPs generated myeloid clones that exclusively displayed TCRß locus in germline configuration .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

E. (top panels) Serial replating of Myc-Bcl2+DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro (gel: left two lanes). Upon transplantation, the same clones induced myeloid leukemia that retained the initial rearrangement in vivo (gel: right two lanes) as assessed by nested PCR. GL = germline. (bottom panels) Methylcellulose-based replating of Myc-Bcl2+GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration.


F . Cumulative survival of mice that received 106cells of TCRβ - rearranged myeloid DN2 - derived clones leading to within 33 ± 4 days after transplantation . Two DN2 clones were transplanted into 5 recipient mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Cumulative survival of mice that received 106cells of TCRβ-rearranged myeloid DN2-derived clones leading to death within 33±4 days after transplantation. Two DN2 clones were transplanted into 5 recipient mice.


A . Representative FACS plots of spleens from leukemic recipient mice that had received LSK , GMP or DN2 grafts immediately after retroviral Myc / Bcl2 transduction . Numbers indicate frequencies ( % ) of CD45 . 2 donor within indicated gates summarizing mean ± of at least 8 recipients per group from a total of 10 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments.


B . Cumulative survival of mice that received 2104 to 2105 freshly Myc / Bcl2 - transduced LSK , GMP or DN2 ; Graph shows n = 11 Myc - Bcl2 + LSK - , n = 14 DN2 - , n = 8 GMP - transplanted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+LSK-, n=14 DN2-, and n=8 GMP-transplanted animals.


C . Histological spleen sections of Myc - Bcl2 + LSK - , GMP - or DN2 - transplanted mice displaying myeloperoxidasestaining . Leukemic engraftment in spleens ranged from 75 . 1 % - 95 . 5 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Histological spleen sections of Myc-Bcl2+LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidasestaining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%.


D . Multiplex quantitative RT - PCR demonstrating strong expression of PU . 1 in myeloid ( M3L : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) biphenotypic T / myeloid ( BL : CD11b + Gr1 + / CD3 + CD4 + CD8 + ) fractions but not in T - lymphoid ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) fraction that were flow - sorted from diseased Myc - Bcl2 + DN2 - transplanted mice . Myeloid progeny of Myc / Bcl2 + GMP - transplanted or T - lymphoid progeny of Myc / Bcl2 + DN3 - transplanted as well as healthy thymocytes ( Thy ) BM from WT mice served as controls . Results are shown as fold over actin transcripts .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3-CD4-CD8-) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b-Gr1-/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts.


E . D - J rearrangement PCR of flow - sorted T - lymphoid ( TL ) , biphenotypic ( BL ) myeloid ( ML ) fractions of donor splenocytes of a diseased Myc / Bcl2 + DN2 - transplanted mouse . three fractions revealed a stable genomic reassembly of Dβ1 TCR locus ( Dβ1Jβ1 . 5 ) demonstrating clonality in DN2 leukemia . thy = thymus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. D-J rearrangement PCR of flow-sorted T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus.


F . FACS plot of diseased intrathymic grafts ( CD45 . 2 - gated ) , demonstrating that Myc / Bcl2 + DN2 could produce multi - lineage leukemia within a T - supporting environment ; Myc / Bcl2 + GMPs remained myeloid restricted under these conditions . Numbers indicate frequencies within gates ( mean ± ) representing six independent experiments .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments.


G . Intrathymic transplantation of mock - infected non - leukemic DN2 produced predominantly T - lymphoid but also myeloid progeny 23 days after injection . This differentiation outcome resembles that of freshly isolated DN2 ( Bell Bhandoola , 2008 ; Richie Ehrlich et al . , 2011 ) , indicates that short - term culture necessary for retroviral transduction did not interfere with physiological lineage potential of DN2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.


A . GSEA demonstrates that Myc / Bcl2 + DN2 - derived myeloid blasts were significantly ( FDR > 0 . 05 ) enriched for expression of a pan - dendritic gene set . Gene sets were published in ( Schonheit et al . , 2013 ) .

A. GSEA demonstrates that Myc/Bcl2+DN2-derived myeloid blasts were significantly (FDR >0.05) enriched for expression of a pan-dendritic cell gene set. Gene sets were published in (Schonheit et al., 2013).


B . Heat map of top 100 differentially expressed genes ( adjusted p - value of ≤ 0 . 05 ) in each myeloid fraction ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from GMP - , LSK - DN2 - derived leukemias .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Heat map of the top 100 differentially expressed genes (adjusted p-value of ≤ 0.05) in each myeloid fraction (ML: CD11b+Gr1+/CD3-CD4-CD8-) from GMP-, LSK- and DN2-derived leukemias.


D . GSEA demonstrates significant enrichment of a normal DN2 - specific gene set ( see methods section ) in Myc / Bcl2 + DN2 - derived myeloid blasts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. GSEA demonstrates significant enrichment of a normal DN2-specific gene set (see methods section) in the Myc/Bcl2+DN2-derived myeloid blasts.


A . Western blot illustrating abundant Bcl11b Gata3protein expression in Myc / Bcl2 + DN2 - but not LSK - or GMP - derived blasts . Thymus ( Thy ) bone marrow ( BM ) extracts from healthy WT mice served as controls . Detection of α - tubulin or valosin - containing ( VCP ) ensured comparable loading .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Western blot illustrating abundant Bcl11b and Gata3protein expression in Myc/Bcl2+DN2- but not LSK- or GMP-derived blasts. Thymus (Thy) and bone marrow (BM) extracts from healthy WT mice served as controls. Detection of α-tubulin or valosin-containing protein (VCP) ensured comparable loading.


B , C . Proliferation assays illustrating growth of Myc / Bcl2 + DN2 - ( B ) or GMP - derived ( C ) blasts after lentiviral transduction with indicated shRNAs . shRNA - transduced were flow - sorted on co - expression of RFP - reporter seeded on OP9 / OP9Dll1 1 : 1 layers . Growing were RFP - sorted for 3 rounds within 21 days of culture growth was calculated over initially seeded number in relation to Luc control . Shown are results of 4 independent experiments with from 4 different DN2 - 3 different GMP - leukemic . Luc = control shRNA targeting luciferase ( Suzuki et al . , 2003 ) . At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated . * = p ≤ 0 . 05 , Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

B,C. Proliferation assays illustrating growth of Myc/Bcl2+ DN2- (B) or GMP-derived (C) blasts after lentiviral transduction with the indicated shRNAs. The shRNA-transduced cells were flow-sorted based on co-expression of the RFP-reporter and seeded on OP9/OP9Dll1 1:1 layers. Growing cells were RFP-sorted for 3 rounds within 21 days of culture and percent growth was calculated over the initially seeded cell number in relation to the Luc control. Shown are results of 4 independent experiments with cells from 4 different DN2- and 3 different GMP-leukemic animals. Luc= control shRNA targeting luciferase (Suzuki et al., 2003). At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated. * = p≤0.05, Student's t-test.


D . Representative FACS plots showing CD45 . 2 - donor splenocytes of a secondary ( CD45 . 1 ) recipient 23 after transplantation with 4103RFP + Myc - Bcl2 + DN2 - leukemia blasts carrying indicated shRNAs . Luc : luciferase control shRNA . data are representative of 2 Gata3 , 4 Bcl11b , 2 luciferase shRNA - transplanted .

O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O

D. Representative FACS plots showing CD45.2-donor splenocytes of a secondary (CD45.1) recipient 23 weeks after transplantation with 4103RFP+Myc-Bcl2+DN2-leukemia blasts carrying the indicated shRNAs. Luc: luciferase control shRNA. The data are representative of 2 Gata3, 4 Bcl11b, and 2 luciferase shRNA-transplanted animals.


A . Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow - sorted myeloid leukemia blasts ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from sick Myc / Bcl2 + LSK - , GMP - or DN2 - transplanted mice . percentages in indicated gates are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow-sorted myeloid leukemia blasts (ML: CD11b+Gr1+/CD3-CD4-CD8-) from sick primary Myc/Bcl2+LSK-, GMP- or DN2-transplanted mice. Cell percentages in indicated gates are shown.


B . Table summarizing phenotype frequencies within spleens of secondary recipients that were retransplanted with Myc / Bcl2 + LSK - , GMP - or DN2 - derived myeloid blasts as described in A . T - lymphoid leukemia : ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) , biphenotypic leukemia : BL ( CD11b + Gr1 + / CD3 + CD4 + CD8 + ) , myeloid leukemia : ML ( CD11b + Gr1 + / CD3 - CD4 - CD8 - ) . Numbers before dash represent mice holding indicated leukemia fraction ; numbers after dash indicate total mouse analyzed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

B. Table summarizing the phenotype frequencies within the spleens of secondary recipients that were retransplanted with Myc/Bcl2+LSK-, GMP- or DN2-derived myeloid blasts as described in A. T-lymphoid leukemia: (TL: CD11b-Gr1-/CD3+CD4+CD8+), biphenotypic leukemia: BL (CD11b+Gr1+/CD3+CD4+CD8+), myeloid leukemia: ML (CD11b+Gr1+/CD3-CD4-CD8-). Numbers before the dash represent mice holding the indicated leukemia fraction; numbers after the dash indicate the total mouse count analyzed.


C . Single myeloid blasts from 4 different Myc / bcl2 + DN2 - derived leukemias were flow - sorted from transplanted mice grown on irradiated OP9 : OP9DL1 stroma . clones were analyzed at two different time points . FACS plots of two representative clones , one with strong ( top ) one with weak ( bottom ) lineage switching potential are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Single myeloid blasts from 4 different Myc/bcl2+ DN2-derived leukemias were flow-sorted from transplanted mice and grown on irradiated OP9:OP9DL1 stroma. The clones were analyzed at two different time points. FACS plots of two representative clones, one with strong (top) and one with weak (bottom) lineage switching potential are shown.


D . Table summarizing lineage switching outcome of 20 independent clones that were grown from single DN2 - derived myeloid blasts of 4 different leukemias . Percentages at right indicate relative amounts of with T - lymphoid ( TL ) , biphenotypic ( BL ) or myeloid ( ML ) phenotype as analyzed by FACS at end of experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Table summarizing the lineage switching outcome of 20 independent clones that were grown from single DN2-derived myeloid blasts of 4 different leukemias. Percentages at the right indicate the relative amounts of cells with T-lymphoid (TL), biphenotypic (BL) or myeloid (ML) phenotype as analyzed by FACS at the end of the experiment.


E . FACS plot of DN2 - derived leukemic transduced with a retrovirus carrying GFP alone or GFP together with PU . 1 as a bicistronic . transduced were grown on OP9 / OP9Dll1 1 : 1 layer for 8 - 13 days analyzed using a GFP - gate . data are representative of 3 different experiments with similar outcome .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. FACS plot of DN2-derived primary leukemic cells transduced with a retrovirus carrying GFP alone or GFP together with PU.1 as a bicistronic RNA. The transduced cells were grown on OP9/OP9Dll1 1:1 layer for 8-13 days and analyzed using a GFP-gate. The data are representative of 3 different experiments with similar outcome.


A . Heat map depicting human AML samples from Haferlach study ( Haferlach et al . , 2010 ) whose transcriptomes resembled DN2 - leukemia by a Spearman ' s rank correlation coefficient ≥ 0 . 5 . top 100 up - downregulated gene signature of Myc / Bcl2 + DN2 - derived myeloid blasts was applied ( see also Figure 3B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Heat map depicting all human AML samples from the Haferlach study (Haferlach et al., 2010) whose transcriptomes resembled DN2-leukemia by a Spearman's rank correlation coefficient ≥ 0.5. The top 100 up- and downregulated gene signature of Myc/Bcl2+DN2-derived myeloid blasts was applied (see also Figure 3B).