A , deformed nucleus of migrating through narrow constrictions was bleached at front side to determine mobility . Inset are shown close - up views of bleached areas at front of nucleus . B , of recovery . Error bars correspond to standard error of mean , n = 24 over 3 biological replicates .

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A, The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility. Inset are shown close-up views of the bleached areas at the front of the nucleus. B, Quantification of the recovery. Error bars correspond to the standard error of the mean, n=24 over 3 biological replicates.


A , Expression of artificial mini - nesprin - 2G construct results in significant nesprin accumulation ( orange arrow ) in migrating through narrow constrictions .

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A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions.


C , of mini - nesprin - 2G accumulation of migrating through narrow constrictions . Note that mutant construct does not accumulate at front of nucleus . Error bars correspond to standard deviation . Mini - nesprin - 2G : n = 10 over 4 biological replicates ; Mutant : n = 11 over 4 biological replicates . A Wilcoxon signed - rank is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to measurement at side , pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to " before " time point .

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C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , Fixed labelled with phalloidin shows filamentous actin accumulation on edges of pillars along top bottom surfaces . Cross - sections along direction of migration ( yellow ) perpendicular to direction of migration ( blue , 1 - 4 ) are shown below .

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A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.


B , A confocal slice on surface of nucleus indicates that GFP - nesprin - 2 co - localizes with actin cables at surface of nucleus . Dashed lines represent outlines of constriction .

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B, A confocal slice on the surface of the nucleus indicates that GFP-nesprin-2 co-localizes with actin cables at the surface of the nucleus. Dashed white lines represent the outlines of the constriction.


C , Spinning disk confocal microscopy on live reveals elongation state of nesprins at envelope . Dashed lines represent outlines of constriction . between mCh - LAC GFP - nesprin - 2 was measured at back , at constriction , at front near constriction , at center of front . ( N = 17 , over 3 biological replicates . alignment of two channels was corrected using at least 28 beads . )

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C, Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope. Dashed white lines represent the outlines of the constriction. The distance between mCh-LAC and GFP-nesprin-2 was measured at the back, at the constriction, at the front near the constriction, and at the center of the front. (N = 17 cells, over 3 biological replicates. The alignment of the two channels was corrected using at least 28 fluorescent beads.)


D , Two - photon ablation reveals that ablation at front of nucleus results in a rearward movement of nucleus , but not ablation at back . nuclei of DN - KASH , which show a decrease of endogenous nesprin at envelope show a dampened rearward movement upon ablation at front of nuclei . Bars represent average value . Error bars represent standard error on mean . Back : N = 9 , Front : N = 30 , Front : N = 16 , DNKASH : N = 31 , 3 biological replicates .

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D, Two-photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus, but not ablation at the back. The nuclei of DN-KASH, which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei. Bars represent the average value. Error bars represent the standard error on the mean. Back: N= 9, Front: N= 30, Cell Front: N = 16, and DNKASH: N=31, 3 biological replicates.


( A ) BMDMs from WT ( Pml + / + ) Pml - / - mice were pre - treated with zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 30 min followed by co - treatment with AT - 406 ( 1 μM ) . viability was evaluated by ATP release after 18 h . Data show mean ± SD of four biological replicates . ( B ) WT Pml - / - BMDM were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 16 h . viability was determined by ATP release . Data show mean ± SD of three biological replicates .

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(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates.


( D ) WT Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) , GSK872 ( 10 μM ) , as indicated , for 8 h . viability was determined by ATP . Data show mean ± SD of three biological replicates . ( E ) WT Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , IKK inhibitor BMS - 345541 ( 10 mM ) Nec - 1 , as indicated , for 6 h then was assayed . Data show mean ± SD of five biological replicates , except Nec - 1 - containing experiment ( n = 2 ) .

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(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).


( A , B ) Pml + / + Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) ( A ) or TNF + zVAD ( Tz ) ( B ) , as in Fig . 1 , for indicated times . Contents of RIPK1 , p - RIPK1 [ S166 ] , RIPK3 , p - RIPK3 , MLKL , p - MLKL in lysates was analysed by immunoblots . Bottom panels , quantitation of p - RIPK1 [ S166 ] from three biological replicates using normalized of p - RIPK1 [ S166 ] in WT at 4 h as 1 . Data show mean ± SD .

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(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.


( D ) BMDMs were treated with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) for 18 h , TNF generated was then analyzed by ELISA . Data show mean ± SD of four biological replicates . ( E ) BMDMs were treated with zVAD + AT - 406 ( 1 μM ) , with or without anti - TNF for 18 h , viability quantitated . Data show mean ± SD of three biological replicates .

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(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.


( A ) Pml + / + Pml - / - BMDMs were treated with zVAD for 30 min , followed by AT - 406 ( zA ) treatment at indicated time - points , before preparing total lysates ( TCL ) . TCL were immunoprecipitated with anti - FADD amounts of p - RIPK1 [ S166 ] , RIPK1 caspase - 8 in precipitates TCL were analysed . ( B ) Pml + / + Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) as in ( A ) , TCL were then harvested . TCL were immunoprecipitated with anti - caspase - 8 , contents of p - RIPK1 [ S166 ] RIPK1 in precipitates TCL was analysed .

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(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.


( A , B ) WT ( Pml + / + ) Pml - / - mice ( n = 12 in each group ) were injected with mouse TNF ( 1 . 5 μg / g ) , rectal ( A ) survival ( B ) were determined at indicated time - points . # , of only six mice was measured at indicated time - points . Mean ± S . E . M is shown . * P < 0 . 05 as determined by two - way ANOVA ( A ) , or by Log - rank ( Mantel - Cox ) ( B ) .

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(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B).


( A ) Pml + / + Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for indicated timeframes . contents of p - IKKα / β , IKKα / β , IκBα , p - MK2 MK2 was analysed by immunoblotting . ( B ) Pml + / + Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for indicated times , of p - RIPK1 [ S321 ] , RIPK1 , p - JNK , JNK , p - ERK , ERK , p - p38 , p38 at indicated time points were then determined .

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(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting. (B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined.


( C , D , E , F ) WT ( Pml + / + ) Pml - / - mice were treated with DMSO or Nec - 1s ( 6 μg / g ) or MK2 inhibitor PF - 3644022 ( 75 μg / mouse ) by intraperitoneal injection 15 min before 60 min after intravenous TNF ( 1 . 5 μg / g ) injection . ( C , E ) survival ( D , F ) were monitored . Experiments C - F were conducted concurrently . Mean ± S . E . M is shown ( C , E ) .

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(C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E).


( A , B ) WT Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) ( A ) or with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) ( B ) , then contents of p - RIPK1 [ S321 ] , RIPK1 , p - p38 , p38 was determined at indicated time - points .

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(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.


( C ) WT , Pml - / - , Mk2 - / - Pml - / - Mk2 - / - BMDMs were treated with zVAD ( 20 μM ) , AT - 406 ( 1 μM ) , Nec - 1 ( 40 μM ) GSK872 ( 10 μM ) , as indicated , for 18 h . viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - according to Holm - Sidak method . ( D ) WT , Pml - / - , Mk2 - / - Pml - / - Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) GSK872 ( 10 μM ) , as indicated , for 16 h . viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - according to Holm - Sidak method .

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(C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method.


( F ) WT , Pml - / - , Mk2 - / - Pml - / - Mk2 - / - mice were injected i . v . with TNF ( 1 . 5 mg / kg ) , survival was monitored for 24 h . Data represent a combination of two independent experiments ( n = 10 ) . * P < 0 . 05 for Log - rank ( Mantel - Cox ) .

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(F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test.


( D ) WT Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for indicated times . p38 MAPK was immunoprecipitated , its association with MK2 was then analysed .

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(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.


( E ) Recombinant Flag - PML - I , GST - MK2 ( 60 ng ) His - p38 ( 60 ng ) were incubated , as indicated , at 4 ° C for 2 h . p38 was pulled down by anti - p38 then PML , MK2 p38 contents in precipitate reaction mixture ( input ) were detected by immunoblotting .

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(E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.


( F ) Recombinant Flag - PML - I , GST - MK2 ( 100 ng ) His - p38 ( 200 ng ) were incubated , as indicated , at 30 ° C in in vitro kinase buffer ( 20 mM HEPES , 10 mM MgCl2 , 100 nM ATP , 1 mM DTT ) for 20 min . of p - MK2 , MK2 , p38 , PML ( FLAG ) in reaction mixture were then determined .

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(F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.


( A ) mH2A1 . 2 different neurogenesis markers are present in cortical lysates collected at various time points ( E13 , E15 , E17 , P0 ) .

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(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).


( C ) A scatter diagram shows change trend of mH2A1 . 2 different neurogenesis markers . n = 5 mice , independent replicates .

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(C) A scatter diagram shows the change trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates.


( E ) Electroporation of mH2A1 . 2 - shRNA results in abnormal distribution in developing neocortex . Electroporation was performed at E13 . 5 , mouse was harvested at E16 . 5 . Scale bar represents 50 μm . ( F ) Graph shows of GFP - positive distributed in VZ / SVZ , IZ CP . n = 7 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(E) Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm. (F) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 7 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.01(**) or P < 0.001(***). n.s., not significant.


( D ) Abnormal distribution was observed in mH2A1 . 2 silenced neocortex . GFP plasmid was electroporated into WT KO mice brains at E13 . 5 , mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( E ) Graphs of of GFP - positive in VZ / SVZ , IZ CP . n = 5 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(D)Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (E) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( F ) Brain sections of WT KO mice at E16 . 5 were immunostained with mitotic marker pH3 DAPI . Scale bar represents 20 μm . ( G ) Statistics of pH3 + of cortex . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(F) Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm. (G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( H ) Representative of E16 . 5 coronal brain sections were immunostained for PAX6 . GFP plasmid was electroporated into WT KO mice brains at E13 . 5 , mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( I ) of GFP Pax6 double positive in VZ / SVZ . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

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(H) Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (I) Quantification of GFP and Pax6 double positive cells in the VZ/SVZ. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( A ) Representative of E16 . 5 coronal brain sections immune stained for Tbr2 BrdU . BrdU was injected intraperitoneally into pregnant mice at E16 . 5 for 2h of labeling . Scale bar represents 20 μm . ( B ) Statistics of TBR2 + per 100μm2 surface of VZ / SVZ . number of TBR2 + increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . ( C ) Statistics of BrdU + per 100μm2 surface of VZ / SVZ . number of BrdU + increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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(A) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of pulse labeling. Scale bar represents 20 μm. (B) Statistics of TBR2+ cells per 100μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. (C) Statistics of BrdU+ cells per 100μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( E ) cycle exit was decreased in KO mice . E16 . 5 brain sections were stained with anti - BrdU anti - Ki67 in WT mice KO mice that were administered BrdU ( 100 mg / kg ) for 24 h harvested at E16 . 5 . Scale bar represents 20 μm . ( F ) of cycle exit ( BrdU + Ki67 − / BrdU + ) in WT mice KO mice . n = 7 mice , independent replicates . Date information : Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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(E) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice and KO mice that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm. (F) Percentage of cell cycle exit (BrdU+Ki67−/ BrdU+) in WT mice and KO mice. n=7 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( G ) Western blot showed that expression of neural marker , including TBR2 , PCNA , PAX6 are increased in mH2A1 . 2 KO mice compared with WT . ( H ) Statistics of normalized density of TBR2 , PCNA , PAX6 . n = 5 mice , independent replicates . Date information : Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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(G)Western blot analysis showed that the protein expression levels of neural stem cell marker, including TBR2, PCNA, and PAX6 are increased in mH2A1.2 KO mice compared with WT. (H) Statistics of the normalized density of TBR2, PCNA, and PAX6. n=5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( K ) Coronal brain slices of E16 . 5 WT mice KO mice were immunostained using anti - TUJ1 . Scale bar represents 20 μm . ( L ) Relative TUJ1 + in VZ of WT mice KO mice . TUJ1 was reduced in cortical . n = 6 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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(K) Coronal brain slices of E16.5 WT mice and KO mice were immunostained using anti-TUJ1. Scale bar represents 20 μm. (L) Relative TUJ1+ cells in the VZ of WT mice and KO mice. TUJ1 was reduced in cortical. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( M ) apoptosis between WT KO brains showed no significant difference . E16 . 5 brain sections of WT KO mice were subjected to TUNEL . Scale bar represents 50 μm . ( N ) Graph shows numbers of apoptotic per field . n = 4mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) . n . s . , not significant .

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(M) The apoptosis levels between WT and KO brains showed no significant difference. E16.5 brain sections of WT and KO mice were subjected to TUNEL assay. Scale bar represents 50 μm. (N) Graph shows the numbers of apoptotic cells per field. n=4mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( A ) Representative of neurons after 4 days of culture in vitro . GFP expressing plasmid was electroporated into E13 . 5 cerebral cortices of WT KO mice to label neural progenitor . After 24 h , GFP + were isolated cultured in differentiation medium for 4 days . Scale bar represents 15 μm . ( B ) Graph shows that total dendritic of neurons is decreased when mH2A1 . 2 is deleted . n = 25 from four samples . ( C ) Statistics show that number of branch points is reduced upon mH2A1 . 2 deletion . n = 25 from four samples . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) .

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

(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).


( F ) brain sections were immunostained with CUX1 CTIP2 ( marker of layer I ; marker of layer V ) at P0 . Scale bar represents 20 μm . n = 4 mice , independent replicates . ( G ) Statistics of CUX1 + in upper layer . CUX1 + were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + in deep layer . CTIP2 + were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 (

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(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(


( A ) Representative tracing path of WT mice KO mice in open field . ( B ) total traveling was not different between WT KO mice . ( C ) KO mice spent less time in center than WT mice . Date information : mice were , 8 - 12 old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . was performed by researcher blinded to experimental conditions .

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(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.


( E ) Graph shows of GFP + in VZ / SVZ , IZ CP . number of GFP + in VZ / SVZ region increased significantly , number of GFP + in CP layer decreased significantly . n = 5 mice , independent replicates . Date information : Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


( I ) Representative of E16 . 5 cortices that had been electroporated with GFP or GFP + NKX2 . 2 into WT KO brains at E13 . 5 . Scale bar represents 50 μm . ( J ) Graphs of of GFP + in VZ / SVZ , IZ CP . Overexpression of NKX2 - 2 restored distribution of GFP + to normal state . n = 5 mice , independent replicates . Date information : Representative from at least three independent experiments . Error bars represent means ± S . E . M . ; Two - unpaired t - , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

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

(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm. (J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state. n=5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


A ) ALS from UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , hippocampus . indicates associated T - statistic for each voxel , with blue representing highest value .

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A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value.


Sites that bound either HIF - 1α ( A & B ) or HIF - 2α ( C & D ) in both replicates were identified by MACS peak caller ordered on y - axis according to HIF - 1β signal . Heatmaps show HIF ChIP - seq signal ( read per million mapped reads , CPM ; expressed as colour , averaged across two independent ChIP - seq experiments ) at HIF binding sites across flanking ± 5kb regions ( x - axis ) . ( A ) HIF - 1β signal ( B ) HIF - 1α signal at HIF - 1α binding sites ( C ) HIF - 1β signal ( D ) HIF - 2α signal at HIF - 2α binding sites . HIF - 1β signal above local background is observed at HIF - 1α HIF - 2α binding sites . ( E & F ) plots showing average ( n = 2 , independent ChIP - seq experiments ) HIF - 1β signal ( solid blue lines ) within MACS defined ( E ) HIF - 1α ( F ) HIF - 2α binding sites compared to average background HIF - 1β binding signal ( dashed black ) at non - HIF - α - binding accessible sites ( defined by FAIRE - seq ) . Sites are ranked on x - axis according to HIF - 1β signal . HIF - 1β signal at both HIF - 1α HIF - 2α binding sites was consistently above genome - wide background . ( G ) HIF - 1β signal was plotted against total HIF - α ( HIF - 1α + HIF - 2α ) signal for sites that bound one or more HIF subunits . Strong correlation was observed between HIF - α HIF - 1β signal intensities . ( H ) of total HIF - α signal to HIF - 1β signal ( x - axis ) was determined for each site plotted as a frequency distribution ( y - axis ) showing a tight unimodal distribution . ( I ) non - reproducibility of total HIF - α / HIF - 1β was assessed by plotting in replicate 1 versus that in replicate

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

Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2


For each , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in - seq analyses ( n = 3 ) . Enrichment of genes ( TSS ) closest to each canonical HIF - α / β binding site ( i . e . identified in both HIF - 1α ( or HIF - 2α ) both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment ( ES = enrichment score , NES = normalized enrichment score )

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For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)


Immunoblots show progressive induction of HIF - 1α HIF - 2α with increasing severity of

Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxia


HIF - 1α ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for canonical HIF - binding sites that bound at one oxygen or other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen no new sites were

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HIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


( C ) HIF - 2α ( D ) HIF - 1β ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for canonical HIF - binding sites that bound at one oxygen or other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen no new sites were

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(C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


Frequency distribution of ChIP - seq signals at 0 . 5 % oxygen compared to 3 % oxygen . of total HIF - α signal was unimodally distributed ( purple ) . Upper tertile HIF - α sites ( solid red ) had a significantly ( p = 10 - 7 , Wilcox rank sum ) higher of HIF - 1β signal ( dotted red ) vice versa ( blue lines )

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Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)


of HIF - 1α to HIF - 2α ChIP - seq signal for canonical HIF - binding sites at 0 . 5 % oxygen was plotted against that at 3 % oxygen . Strong correlation in HIF - 1α to HIF - 2α was observed between two oxygen

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The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations


Immunoblots show induction of HIF - 1α HIF - 2α level , which is highest at 6 hours falls by 48

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Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours


HIF - 1α , ( C ) HIF - 2α ( D ) HIF - 1β ChIP - seq signal ( averaged across two independent ChIP - seq experiments ) at 16 hours is plotted against that at 6 hours for canonical HIF - binding sites that bound at any of three timepoints . ChIP - seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia , but correlated well between two timepoints no novel sites were observed at either timepoint . ( E - F ) same plots comparing ( E ) HIF - 1α , ( F ) HIF - 2α ( G ) HIF - 1β ChIP - seq signals at 48 hours 6 hours hypoxia . ChIP - seq signal at 48 hours correlated well with that at 6 hours again no novel sites were observed at either

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HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepoint


Frequency distribution of ChIP - seq signals at 48 hours compared to 6 hours hypoxia . of total HIF - α signal had a unimodal distribution ( purple ) . However , upper tertile HIF - α sites ( solid red ) had a comparable of HIF - 1β signal ( dotted red ) to of HIF - 1β signal in lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological

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Frequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological difference


of HIF - 1α to HIF - 2α ChIP - seq signal for canonical HIF - binding sites at ( I ) 16 hours hypoxia at ( J ) 48 hours hypoxia was plotted against that at 6 hours hypoxia . Strong correlation in HIF - 1α to HIF - 2α was observed between various

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The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints


were incubated in 0 . 5 % ambient oxygen for 16 hours . Immunoblots show HIF - 1α , HIF - 2α HIF - 1β in wild type single clones of CRISPR - Cas9 engineered

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Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines


HIF - 1α ChIP - seq signal in HIF - 2α KO was plotted against that in wild - type for canonical HIF - 1 binding sites in either

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HIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell type


HIF - 2α ChIP - seq signal in HIF - 1α KO was plotted against that in wild - type for canonical HIF - 2 binding sites in either type . No significant increase ( Wilcoxon rank sum ) in binding of either isoform was observed following deletion of

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HIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the other


Box - - whisker plots showing : ( D ) effect of HIF - 2α inactivation on HIF - 1α ChIP - seq signal at sites that specifically bound HIF - 2α , but not HIF - 1α in wild - type showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank ) decrease rather than increase ( compare red boxes ) consistent with reduction in HIF - 1α observed in panel

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Box-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel A


effect of HIF - 1α inactivation on HIF - 2α ChIP - seq signal ( compare blue boxes ) at sites that specifically bound HIF - 1α , but not HIF - 2α in wild - type showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank )

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The effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test)


effect , at sites that bind both isoforms in wild - type , of ( F ) HIF - 2α inactivation on HIF - 1α binding showing a similar small but significant reduction rather than increase ( p = 2 x 10 - 16 , Wilcoxon Signed rank ) as

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The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as abov


effect , at sites that bind both isoforms in wild - type , o HIF - 1α inactivation on HIF - 2α binding showing no significant effect ( p = 0 . 4 , Wilcoxon signed rank )

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The effect, at sites that bind both isoforms in wild-type cells, o HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test)


Canonical HIF - 1 HIF - 2 binding sites were determined by overlap of HIF - α HIF - 1β binding sites identified by MACS peak caller as for Figure 4 . from each binding site to nearest annotated gene promoter was determined plotted as a frequency distribution for ( A ) HIF - 1 ( B ) HIF - 2 sites in HKC - 8 , ( C ) HIF - 1 ( D ) HIF - 2 sites in RCC4 , ( E ) HIF - 1 ( F ) HIF - 2 sites in HepG2 . distribution of HIF - 1 binding sites was consistently more promoter proximal than HIF - 2 sites despite sites themselves differing between

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Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types


distribution of accessible HRE motifs was also plotted in HepG2 differed significantly from that of both HIF - 1 ( p ~ 10 - 50 ) HIF - 2 ( p ~ 10 - 80 ) binding sites ( Chi - squared )

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The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)


Immunoblots showing HIF - 1α , HIF - 2α HIF - 1β in HKC - 8 , HepG2 , RCC4 MCF - 7 lines in normoxia following 16 hours incubation at 0 . 5 %

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Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen


Sites that bound ( B ) canonical HIF - 1 ( i . e . identified in both HIF - 1α both HIF - 1β ChIP - seq experiments ) or ( C ) canonical HIF - 2 ( i . e . identified in both HIF - 2α both HIF - 1β ChIP - seq experiments ) were identified from ChIP - seq datasets in HepG2 HKC - 8 following 16 hours incubation in 0 . 5 % oxygen from normoxic RCC4 compared with previously published data from MCF - 7 . ( D ) proportion of total HIF - 1 or HIF - 2 sites that were shared between 2 , 3 or 4 lines is plotted . For sites that are shared between ( E ) HepG2 HKC - 8 , ( F ) HKC - 8 RCC4 , or ( G ) RCC4 HepG2 , of HIF - 1α / HIF - 2α signal for one was plotted against other . of HIF - 1α / HIF - 2α in one correlates well with that in other . ChIP - seq signal is averaged across two independent

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Sites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments


Heatmaps showing average ( n = 2 , independent ChIP - seq experiments ) ChIP - seq signal ( read per million mapped reads , CPM ; expressed as colour ) at canonical HIF - 1 HIF - 2 binding sites across flanking ± 5kb regions ( x - axis ) in HepG2 incubated in 0 . 5 % oxygen for 16 hours . Sites are ordered on y - axis according to HIF signal derived from HepG2 incubated in hypoxia . ChIP - seq signal for histone modifications was obtained from ENCODE was derived from HepG2 incubated in

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Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia


Bar chart showing enrichment of transcription factor binding sites ( TFBS ) at canonical HIF - 1 HIF - 2 sites in HepG2 ( identified as in Figure 4 ) . TFBSs for 61 additional - binding were determined from ENCODE ChIP - seq data in normoxic HepG2 . significance of overlap between each set of binding sites canonical HIF - 1 HIF - 2 sites , was determined using a hypergeometric . Accessible sites determined from ENCODE DNAse - seq data in HepG2 was used as a negative

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Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control


Flow cytometric evaluation of CXCR4 surface expression using an anti - CXCR4 - PE antibody . Left : positive ; right : negative ; representative data are shown . gating strategy is depicted in Supplementary Fig S1 .

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Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.


Representative histograms revealing magnitude of CXCR4 expression in myeloma as compared to indicated bone marrow subtype .

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Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.


Median of surface CXCR4 expression relative to isotype control ( n = 7 - 10 ) . Horizontal bars indicate mean of values ; asterisks indicate statistical significance ( plasma vs B : P = 0 . 0082 ; plasma vs T : P = 0 . 0091 ; plasma vs CD34 + : P = 0 . 011 ; plasma vs monocytes : P = 0 . 0423 ; Student ' s t - to compare mean plasmacell relative expression with mean relative expression of each further indicated subtype ) . MM judged positive for CXCR4 expression were selected for this .

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Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7-10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.


Representative MM bone marrow of a positive for MM CXCR4 expression : hematoxylin eosin ( H & amp ; amp ; E ) . Immunohistochemistry for CD138 , CXCR4 , chain kappa , chain lambda .

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Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&amp;E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.


Real - time PCR of cxcr4 transcript expression in HeLa ( negative control ) in MM lines MM . 1S OPM - 2 . Shown is mean relative expression ± ( n = 3 independent experiments ) . Values are normalized to expression of ubiquitin ( Ub ) . asterisk indicates statistically significant differences ( HeLa vs MM . 1S : P < 0 . 001 ; HeLa vs OPM - 2 : P < 0 . 0001 ; Student ' s t - ) .

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Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).


CXCR4 expression assessed by immunoblotting ( one representative blot out of 3 is shown ) .

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CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).


Binding of [ 68Ga ] Pentixafor to MM . 1S OPM - 2 after indicated incubation periods ( n = 3 per time point ) . Shown is mean ± . difference between OPM - 2 groups is statistically significant ; * P < 0 . 0017 ( one - way ANOVA ) .

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Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).


Mean - to - background ( TBR ) for [ 18F ] FDG ( left ) for [ 68Ga ] Pentixafor ( right ) in MM . 1S OPM - 2 xenograft - bearing NOD SCID mice . Shown is mean ± , n = 8 tumors ( 4 mice ) ; * P = 0 . 0111 for [ 18F ] FDG * P = 0 . 0113 for [ 68Ga ] Pentixafor ( Student ' s t - ) . One - way ANOVA revealed significant differences between groups ; P < 0 . 0001 ( not graphically shown ) .

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Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).


Representative [ 68Ga ] Pentixafor PET of three mice bearing MM . 1S ( right shoulder ) OPM - 2 ( left shoulder ) tumors .

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Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.


Flow cytometric of CXCR4 surface expression on resected MM . 1S OPM - 2 tumors . Data are mean ± , n = 4 . * P = 0 . 0286 ; Mann - Whitney U - .

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Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test.


Correlation of [ 68Ga ] Pentixafor PET mean TBR CXCR4 surface expression assessed by flow cytometry . n = 8 tumors were analyzed .

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Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.


Mice ( n = 4 ) bearing OPM - 2 MM . 1S xenografts were coinjected with AMD3100 ( right image , one representative mouse ) or not pretreated ( left image ) before undergoing [ 68Ga ] PentixaforPET . arrows point to bladder . is shown in Supplementary Fig S3A . Source data are available online for this figure .

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Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.


[ 68Ga ] PentixaforPET / CT [ 18F ] FDGPET / CTA - D Maximum projections ( MIP ) of [ 68Ga ] Pentixafor ( A ) [ 18F ] FDGPET / CT ( B ) of a 68 - year - old with histologically proven multiple myeloma indicating better lesion - to - background contrast for [ 68Ga ] Pentixafor in corresponding myeloma manifestations . Trans - axial views of upper thorax ( C ) pelvis ( D ) underline higher uptake values of bone manifestations ( yellow arrows ) of [ 68Ga ] Pentixafor compared to [ 18F ] FDG .

[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.


Visual comparison of [ 18F ] FDG - [ 68Ga ] Pentixafor PET scansNumber of with visual positivity for indicated PET tracer ( total : n = 14 ) . Number of ( total n = 14 ) for whom imaging with [ 18F ] FDG PET ( FDG , n = 2 ) or [ 68Ga ] Pentixafor PET ( Pentixafor , n = 7 ) was superior , with comparable positivity ( comparable , n = 3 ) , with dual imaging providing complementary visual information ( complementary , n = 2 ) .

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Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).


[ 68Ga ] PentixaforPET / MRimagesA - C Coronal views of [ 68Ga ] Pentixafor ( A ) , T2 STIR weighted MRI ( B ) CTbone window ( C ) of a maleuptake with histologically proven multiple myeloma . increased [ 68Ga ] Pentixaforbone correlates with hyperintense T2 STIR signal ; however , myeloma manifestations are underestimated in corresponding bone window CT .

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[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.


( A ′ ) HeLa transfected with pEGFP - RAB11 were fixed in basal conditions stained for mATG9 . Pearson ' s coefficient : 0 . 452 ± 0 . 034 ( ) . ( A ″ ) HeLa were transfected with ATG9L1 - pEGFP , fixed in basal condition , labeled for RAB11 . Pearson ' s coefficient : 0 . 413 ± 0 . 020 ( ) .

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(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson's coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson's coefficient: 0.413 ± 0.020 (SEM).


( B ) HeLa were incubated for 1 hr at 37 ° C or 18 ° C to reach correct , loaded with transferrin Alexa - 555 for 1 hr , fixed , labeled with anti - ATG9 anti - TGN46 ( marker for Trans Golgi Network ) . Histogram shows Pearson ' s coefficient between ATG9 transferrin ( Tf ) or TGN46 . Error bar , . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification .

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(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson's coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.


( C ) HeLa were incubated in serum - free medium containing Dynasore 100 μM or DMSO for 2 hr loaded for 30 min with transferrin Alexa - 555 . were then fixed labeled for mATG9 TGN46 . Pearson ' s coefficient between mATG9 transferrin or TGN46 was quantified . Error bar , . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification .

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(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.


( D ) treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed then analyzed by TIRF microscopy . Pictures are at same magnification brightness . transfected with Dynamin mutant were identified by abnormal transferrin accumulation at plasma .

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(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.


( E ) HeLa were incubated for 1 hr at 4 ° C with ice - cold NHS - LC - Biotin ( Pierce ) solution , rinsed , lysed in RIPA buffer . Biotinylated were precipitated using streptavidin - agarose beads ( Pierce ) blotted for mATG9 to assess localization at plasma .

(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.


( F ) HeLa treated as in ( C ) were processed for immunogold labeling on cryosections stained with anti - mATG9 antibody . Arrows show clathrin - coated structures . Scale bar in a c , 150 nm ; scale bar in b , 100 nm .

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(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.


( G ) HeLa transfected with GFP - ATG16L1 were fixed , labeled for mATG9 , then analyzed by TIRF microscopy . Control were cotransfected with GFP - ATG16L1 Straw - ATG16L1 . Pictures are at same magnification brightness . See Figure S1J for .

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(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.


( H ) HeLa treated with Dynasore were processed for immunogold labeling on cryosections double labeled with ATG16L1 ( 10 nm ) mATG9 ( 15 nm ) . Scale bar , 100 nm .

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(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.


( I ) HeLa treated as in ( H ) were processed for triple labeling on cryosections ( clathrin , 5 nm ; ATG16L1 , 10 nm ; mATG9 , 15 nm ) . Scale bar , 150 nm . ( J ) Table shows of numbers of clathrin - coated structures carrying mATG9 ATG16L1 alone or together or neither . A minimum of 50 clathrin - coated structures were examined from three different experiments . We constructed a 2 × 2 table with 0 or 1 - 3 gold particles for mATG9 or ATG16L1 for a X2 to avoid statistical with 0 . X2 = 10 . 4 , df = 1 , p = 0 . 001 .

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(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1-3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.


( A ) HeLa transfected with GFP - ATG16L1 for 24 hr were incubated for 15 min with 2 . 5 μg / ml with HRP - cholera toxin subunit B at 4 ° C then for 10 min at 37 ° C . were then fixed treated for immunogold labeling on cryosections . ATG16L1 was detected with anti - ATG16L1 antibody ( 15 nm ) , cholera toxin was detected using anti - HRP antibody . Scale bar , 150 nm .

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(A) HeLa cells transfected with GFP-ATG16L1 for 24 hr were incubated for 15 min with 2.5 μg/ml with HRP-cholera toxin subunit B at 4°C and then for 10 min at 37°C. Cells were then fixed and treated for immunogold labeling on cryosections. ATG16L1 was detected with anti-ATG16L1 antibody (15 nm), and cholera toxin was detected using anti-HRP antibody. Scale bar, 150 nm.


( B ) HeLa were transiently transfected with GFP - ATG16L1 for 20 hr . in basal conditions were fixed labeled with anti - RAB11 antibody .

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(B) HeLa cells were transiently transfected with GFP-ATG16L1 for 20 hr. Cells in basal conditions were fixed and labeled with anti-RAB11 antibody.


( C ) HeLa transfected with mStrawberry - ATG16L1 ( 20 hr ) starved ( 4 hr ) were processed for immunogold labeling on cryosections . Double labeling was performed with rabbit anti - ATG16L1 ( 10 nm gold ) rabbit anti - RAB11 ( 15 nm gold ) . Scale bar , 150 nm . Schematic diagram shows outlines from EM . Green lines indicate structures positive for both ATG16L1 RAB11 , whereas red lines are only positive for RAB11 . Histogram shows by EM of ATG16L1 tubulovesicular structures that carry RAB11 . 281 tubulovesiclular structures from different experiments were analyzed . Error bars , SD .

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(C) HeLa cells transfected with mStrawberry-ATG16L1 (20 hr) and starved (4 hr) were processed for immunogold labeling on cryosections. Double labeling was performed with rabbit anti-ATG16L1 (10 nm gold) and rabbit anti-RAB11 (15 nm gold). Scale bar, 150 nm. Schematic diagram shows membrane outlines from EM. Green lines indicate structures positive for both ATG16L1 and RAB11, whereas red lines are only positive for RAB11. Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11. 281 tubulovesiclular structures from different experiments were analyzed. Error bars, SD.


( D ) HeLa were transfected with EGFP - ATG16L1 mCherry - RAB11 , 5 min movies were recorded . Representative from movies are shown .

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(D) HeLa cells were transfected with EGFP-ATG16L1 and mCherry-RAB11, and 5 min movies were recorded. Representative images from movies are shown.


( E ) HeLa were loaded with mouse anti - transferrin receptor antibody ( 5E9C11 ) for 30 min fixed for immunogold labeling for cryosections . sample was labeled with rabbit anti - mouse antibody to recognize transferrin receptor ( 5 nm ) , anti - ATG16L1 ( 10 nm ) , anti - mATG9 ( 15 nm ) . Scale bar , 150 nm .

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(E) HeLa cells were loaded with mouse anti-transferrin receptor antibody (5E9C11) for 30 min and fixed for immunogold labeling for cryosections. The sample was labeled with rabbit anti-mouse antibody to recognize transferrin receptor (5 nm), anti-ATG16L1 (10 nm), and anti-mATG9 (15 nm). Scale bar, 150 nm.


( F ) HeLa were transfected with pmStrawberry - ATG16L1 ATG9L1 - pEGFP , 5 min movies were recorded . Representative from movies are shown .

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(F) HeLa cells were transfected with pmStrawberry-ATG16L1 and ATG9L1-pEGFP, and 5 min movies were recorded. Representative images from the movies are shown.


( G ) HeLa were transfected with pmStrawberry - ATG16L1 pEGFP - LC3 , starved for 2 hr , fixed , labeled for mATG9 .

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(G) HeLa cells were transfected with pmStrawberry-ATG16L1 and pEGFP-LC3, starved for 2 hr, fixed, and labeled for mATG9.


( A B ) ( A ) HeLa were incubated for 4 hr at 37 ° C or 18 ° C processed for LC3II western blot . Some were incubated overnight with 20 mM NH4Cl in full medium ( to block lysosomal degradation ) at 37 ° C or 18 ° C ( B ) .

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(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).


( C ) HeLa were transfected with pEGFP - LC3 for 20 hr incubated for 4 hr at 37 ° C or 18 ° C .

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(C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C.


( D E ) ( D ) HeLa were transfected with pEGFP - ATG16L1 for 20 hr incubated for 4 hr at 37 ° C or 18 ° C . number ( E ) of vesicles were scored ( a minimum of 20 were examined for each condition ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 ∗ ∗ p < 0 . 01 .

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(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.


( F G ) ( F ) HeLa were transfected with pEGFP - ATG16L1 for 20 hr , incubated for 4 hr at 37 ° C or 18 ° C , labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes . Histogram in ( G ) shows amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment ( Manders ' coefficient ) . Error bar , . NS , not significant ; ∗ ∗ p < 0 . 01 .

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(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders' coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01.


( H ) HeLa were transfected with pmStrawberry - ATG16L1 for 20 hr , fixed , labeled for mATG9 . histograms show of colocalization of vesicles labeled with ATG16L1 mATG9 at 37 ° C or 18 ° C . Error bar , . ∗ ∗ ∗ p < 0 . 001 ∗ ∗ p < 0 . 01 .

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(H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.


( I ) HeLa were transfected with pEGFP - RAB11 or pEGFP - Myosin Vb tail ( MVb ) incubated during last 16 hr with Bafilomycin A1 ( Baf ) or DMSO processed for LC3 - II western blot . Control were transfected with pEGFP empty vector .

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(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.


( J ) HeLa were transfected with ATG16L1 - mStrawberry RAB11 - EGFP or MVb tail - EGFP . of ATG16L1 vesicles was scored in RAB11 MVb tail overexpression conditions . inserts show just ATG16L1 vesicles as quantified in histogram . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ∗∗∗p<0.001.


( K ) HeLa were transfected as in ( C ) loaded with transferrin Alexa - 647 for 30 min . Histogram inserts show correlation between ATG16L1 loaded transferrin ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( L ) HeLa were transfected as in ( C ) labeled for mATG9 . Histogram inserts show correlation between ATG16L1 mATG9 in RAB11 MVb tail overexpression conditions ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( M ) HeLa were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium , loaded with transferrin Alexa - 488 ( Tf ) . Pictures histogram show correlation between ATG16L1 transferrin in basal starving condition ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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(M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( N ) HeLa were transfected with mStrawberry - ATG16L1 for 20 hr , starved in HBSS for 4 hr , or maintained in full medium then fixed labeled with anti - mATG9 antibody . Pictures histogram show correlation between ATG16L1 mATG9 in basal starved ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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 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

(N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( A ) HeLa were transfected with mStrawberry - ATG16 for 20 hr treated for 10 min with N - ethylmaleimide ( NEM ; 100 μM ) in full medium , fixed , stained for mATG9 . Histogram shows correlation between mATG9 ATG16L1 ( Pearson ' s coefficient ) . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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

(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson's coefficient). Error bar, SEM. ∗∗∗p < 0.001.


( C D ) ( C ) Representative fields of in vitro fusion of ATG16L1 mATG9 vesicles . in ( D ) was performed on ten fields ( similar to that shown in figure ) per experiment . is expressed as Pearson ' s coefficient . Error bar , . ∗ ∗ p < 0 . 001 .

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

(C and D) (C) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles. The quantification in (D) was performed on ten fields (similar to that shown in the figure) per experiment. The quantification is expressed as Pearson's coefficient. Error bar, SEM. ∗∗p < 0.001.


( A ) HeLa were transfected with siRNAs for different R - SNAREs , we assessed of autophagy substrate p62 versus actin .

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

(A) HeLa cells were transfected with siRNAs for different R-SNAREs, and we assessed the levels of the autophagy substrate p62 versus actin.


( B ) HeLa transfected with control or VAMP3 siRNA ( oligo A ) for 4 days were treated during last 16 hr with Bafilomycin A1 ( Baf A1 ) or DMSO ( basal ) processed for LC3 - II western blots .

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 B-MEASURED_VAR O O O O O

(B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots.


( C ) Endogenous LC3 immunocytochemistry in HeLa transfected with control or VAMP3 siRNA for 4 days . histogram shows number of vesicles ( LC3 positive ) in a minimum of 30 . Error bar , . ∗ ∗ ∗ p < 0 . 001 .

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

(C) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days. The histogram shows the number of vesicles (LC3 positive) in a minimum of 30 cells. Error bar, SEM. ∗∗∗p < 0.001.