Mice lungs were collected from PBS - ( mock ) vaccinated BALB / c mice at day 5 post - RSV infection , fixed , paraffin embedded , slides were processed for imaging . Immunohistochemistry of lung sections shows RSV localized to distal alveoli by Control rabbit sera ( left panels ) or rabbit anti - sera against RSV F ( center panels ) , or against RSV F - p27 ( 110 - 136 ) peptide ( right panels ) . Scale bar = 100 µm . of positive for RSV - F ( middle panel ) RSV - p27 ( right panel ) covering 500 x 500 µm of lung tissue for each mice for each antibody are shown . experiments were performed twice variation between two independent expeiremnts was < 9 % .

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

Mice lungs were collected from PBS-(mock) vaccinated BALB/c mice at day 5 post-RSV infection, fixed, paraffin embedded, and slides were processed for imaging. Immunohistochemistry staining of lung sections shows RSV protein localized to distal alveoli by Control rabbit sera (left panels) or rabbit anti-sera against RSV F protein (center panels), or against RSV F-p27 (110-136) peptide (right panels). Scale bar = 100 µm. The percentage of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) covering 500 x 500 µm of lung tissue for each mice for each antibody staining are shown. The experiments were performed twice and variation between the two independent expeiremnts was <9%.


( A ) Lung tissues from mice ( n = 5 ) at day 5 post - RSV challenge ( or uninfected control ) were used to evaluate CD4 / CD8 T separately for airway distal lungs . Lung slides were fixed stained with DAPI to visualize nuclei ( blue ) either anti - CD4 ( green ) or anti - CD8 ( red ) T . number of CD4 / CD8 T were determined in each lung airway distal region . CD4 + / CD8 + T data are presented as number of positive per 174 x 174 µm of lung tissue for each mice . Results are presented as box whisker plots , where boxes extends from 25th to 75th percentile , whiskers show minimum to maximum value central band represents median value for group .

O O O O 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 O O O O O B-MEASURED_VAR 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 O O O O O O O O O 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) Lung tissues from individual mice (n=5) at day 5 post-RSV challenge (or uninfected control) were used to evaluate CD4/CD8 T cells separately for airway and distal lungs. Lung slides were fixed and stained with DAPI to visualize nuclei (blue) and either anti-CD4 (green) or anti-CD8 (red) T cells. The number of CD4/CD8 T cells were determined in each lung airway and distal region. Individual CD4+/CD8+ T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.


B Phospholipid transport / synthesis , as measured by 3H - serine incorporation , in human fibroblasts ( mean ± SE ; n = 9 , with 5 replicates / experiment ) in mouse hippocampal neurons ( mean ± SE ; n = 3 , with 4 or 5 replicates / experiment ) . * , p < 0 . 05 vs E3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.


C Comparison of 3H - serine incorporation in ApoE ACM - treated WT - Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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

C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).


D Duramycin sensitivity in ApoE ACM - treated fibroblasts . Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4 - treated , which was blocked upon addition of 15 μM exogenous PtdEtn ( mean ± SD ; n = 3 , with 3 replicates / experiment ) .

O O O O B-CONTROLLED_VAR 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

D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 μM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).


B Fractions from discontinuous gradient were blotted with anti - ApoE ( WUE - 4 ) antibody ; doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [ 16 ] .

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

B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16].


C An ApoE - containing fraction ( fraction 5 [ + ] ) , an ApoE - negative fraction ( fraction 3 [ - ] ) , recombinant lipid - free ApoE were applied to human fibroblasts . Note increase in phospholipid synthesis using an ApoE4 - containing lipoprotein fraction , but not with either an ApoE4 - negative fraction or with lipoprotein - free recombinant ApoE3 or ApoE4 ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

O O B-CONTROLLED_VAR 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 B-CONTROLLED_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 B-CONTROLLED_VAR 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

C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).


A Cholesteryl ester synthesis , as measured by 3H - oleate incorporation . Note increased CE in ApoE4 - treated , whereas there was essentially no difference in triglyceride ( TAG ) production ( mean ± SE ; n = 3 . with 3 replicates / experiment ) . † , p = 0 . 06 vs E3 .

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

A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.


B Comparison of 3H - oleate incorporation in ApoE ACM - treated WT - Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 5 replicates / experiment ) .

B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).


C ApoE ACM - treated fibroblasts were stained with LipidTox green neutral lipid stain , lipid droplets were visualized ( example at left ) counted ( right ) . Note significantly more lipid droplets in ApoE4 - treated ( mean ± SD , n = 3 ) .

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-CONTROLLED_VAR O O O O O O O O O O O O

C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).


HeLa were transfected with plasmids to visualize ER ( in green ) mitochondria ( in red ) , then were treated with ApoE3 - ApoE4 - ACM for 24 hours . Shown is a box - - whisker plot comparing co - localization in ApoE3 ACM ( n = 15 analyzed [ gray circles , average of co - localization in ~ 5 in each image field ] ) compared to that in ApoE4 ACM ( n = 18 ) .

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 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

HeLa cells were transfected with plasmids to visualize ER (in green) and mitochondria (in red), and then were treated with ApoE3- and ApoE4-ACM for 24 hours. Shown is a box-and-whisker plot comparing co-localization in ApoE3 ACM (n = 15 images analyzed [gray circles, average of co-localization in ~5 cells in each image field]) compared to that in ApoE4 ACM (n = 18 images).


A . 293T were co - transfected with CMV - renilla , 8X - TBS ( eight repeats of TEAD - binding - ) luciferase reporter , Flag - YAP , HA - TEAD2 wild - type ( WT ) or catalytically inactive ( CS ) USP9X - V5 . One day after transfection , luciferase activity was measured normalized with respect to renilla activity . value for Flag - YAP / HA - TEAD - transfected ( 2nd column ) was adjusted to 1 . Shown below is a representative Western blot showing that expression were comparable between samples ( n = 4 ) . Error bars indicate S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired 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 B-MEASURED_VAR 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 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

A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).


B . 293T were transfected with control or USP9X - targeting siRNAs . One day after siRNA transfection , were co - transfected with CMV - renilla , 8X - TBS luciferase , Flag - YAP HA - TEAD2 . Reporter activity was quantified as described in ( A ) ( n = 4 ) . Error bars indicate S . E . M . ( * * * p < 0 . 001 ; paired Student ' s t - ) .

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 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

B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test).


C . RPE or MCF10A were transfected with indicated siRNAs for 24 hours then re - seeded to either sparse or dense culture conditions . At 48 hours after siRNA transfection , were harvested extracts were analyzed by Western blotting for indicated . L . E . , long exposure , S . E . , short exposure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re-seeded to either sparse or dense culture conditions. At 48 hours after siRNA transfection, the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins. L.E., long exposure, S.E., short exposure.


D . were treated as described in ( C ) , indicated mRNAs were analyzed with RT - qPCR , results were normalized with respect to β - actin mRNA ( n = 4 ) . Error bars indicate S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired 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

D. Cells were treated as described in (C), the indicated mRNAs were analyzed with RT-qPCR, and the results were normalized with respect to the β-actin mRNA (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).


E . RPE were transfected with indicated siRNAs for 24 hours , then co - transfected with CMV - renilla 8X - TBS luciferase . One day after latter transfection , were re - seeded to either sparse or dense conditions , reporter activity was measured at 24 hours after re - seeding ( n = 3 ) . Error bars indicate S . E . M . ( * * p < 0 . 01 ; paired 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

E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student's t-test).


A . Representative anti - YAP immunofluorescence of sparsely cultured RPE transfected with indicated siRNAs . Green , YAP . Blue , DAPI . Scale bar , 10 μm .

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

A. Representative anti-YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs. Green, YAP. Blue, DAPI. Scale bar, 10 μm.


B . YAP intensities of shown in ( A ) were quantified using Image J software . At least 70 from five or six random fields were analyzed . Quantified values were normalized by adjusting average of " siControl Dense " to 1 . We performed two independent experiments obtained similar results . Data from one representative experiment are presented . red bar indicates average value . S , Sparse . D , Dense ( * * * p < 0 . 001 ; paired Student ' s t - ) .

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

B. The nuclear YAP intensities of the cells shown in (A) were quantified using the Image J software. At least 70 cells from five or six random fields were analyzed. Quantified values were normalized by adjusting the average of "siControl Dense cells" to 1. We performed two independent experiments and obtained similar results. Data from one representative experiment are presented. The red bar indicates the average value. S, Sparse. D, Dense (***p < 0.001; paired Student's t-test).


C . Sparsely cultured RPE or MCF10A were transfected with indicated siRNAs , fractionated into extracts , analyzed by Western blotting for indicated .

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. Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs, fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


D . Representative anti - YAP immunofluorescence of densely cultured RPE transfected with indicated plasmids . Green , GFP or V5 . Red , YAP . Blue , DAPI . Scale bar , 10 μm .

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

D. Representative anti-YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids. Green, GFP or V5. Red, YAP. Blue, DAPI. Scale bar, 10 μm.


E . YAP intensities of transfected in ( D ) were quantified using Image J software . At least 70 from five to six random fields were analyzed . Quantified values were normalized by adjusting average of " EGFP transfected " to 1 . Similar results were obtained from two independent experiments . Data from one representative experiment are presented . red bar indicates average value . ( * * * p < 0 . 001 ; paired Student ' s t - ) .

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

E. The nuclear YAP intensities of transfected cells in (D) were quantified using the Image J software. At least 70 cells from five to six random fields were analyzed. Quantified values were normalized by adjusting the average of "EGFP transfected cells" to 1. Similar results were obtained from two independent experiments. Data from one representative experiment are presented. The red bar indicates the average value. (***p < 0.001; paired Student's t-test).


F . Densely cultured stable RPE clones integrated with empty vector ( Vec ) , USP9X - V5 WT or CS mutant were fractionated into extracts , analyzed by Western blotting for indicated .

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

F. Densely cultured stable RPE clones integrated with empty vector (Vec), USP9X-V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


G . Densely cultured RPE transduced with CRISPR - SAM targeting USP9X were fractionated into extracts , analyzed by Western blotting for indicated .

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

G. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.


A . RPE were analyzed by Western blotting for indicated .

A. RPE cells were analyzed by Western blotting for the indicated proteins.


B . Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for indicated .

B. Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins.


C . Densely cultured RPE transduced with CRISPR - SAM targeting USP9X were analyzed by Western blotting for indicated .

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

C. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were analyzed by Western blotting for the indicated proteins.


D . RPE or MCF10A expressing control or USP9X shRNA were transfected with LATS1 / 2 siRNA as indicated . were re - seeded to low density harvested after one day .

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

D. RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1/2 siRNA as indicated. Cells were re-seeded to low density and harvested after one day.


E . RPE transduced with Flag - YAP WT or Flag - YAP 5SA were transfected with indicated siRNAs . were re - seeded to low density harvested after one day .

O O O O O O O B-MEASURED_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

E. RPE cells transduced with Flag-YAP WT or Flag-YAP 5SA were transfected with the indicated siRNAs. Cells were re-seeded to low density and harvested after one day.


F . RT - qPCR of CTGF mRNAs in described in ( E ) ( n = 3 ) . Error bars indicate S . E . M . ( * p < 0 . 05 , paired Student ' s t - ) .

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

F. RT-qPCR of CTGF mRNAs in the cells described in (E) (n=3). Error bars indicate the S.E.M. (*p< 0.05, paired Student's t-test).


A . RPE or MCF10A were transfected with indicated combinations of siRNAs re - seeded to low density , extracts were analyzed by Western blotting for indicated .

O O O O 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. RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re-seeded to low density, and cell extracts were analyzed by Western blotting for the indicated proteins.


B . described in ( A ) were analyzed by RT - qPCR for indicated target genes ( n = 4 ) . Error bars indicate S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 ; paired 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

B. The cells described in (A) were analyzed by RT-qPCR for the indicated target genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).


C . Sparsely cultured RPE transfected with indicated siRNAs were immunoprecipitated with an anti - AMOTL2 antibody analyzed by Western blotting for indicated . WCL , whole lysates .

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

C. Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti-AMOTL2 antibody and analyzed by Western blotting for the indicated proteins. WCL, whole cell lysates.


A . 293T were transfected with indicated siRNAs , cultured for 24 hours , then transfected with indicated DNAs . 48 hours after siRNA transfection , were harvested ubiquitination of Flag - AMOTL2 was examined . C , control siRNA .

O O O O O O O O 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

A. 293T cells were transfected with the indicated siRNAs, cultured for 24 hours, and then transfected with the indicated DNAs. 48 hours after siRNA transfection, the cells were harvested and ubiquitination of Flag-AMOTL2 was examined. C, control siRNA.


B . 293T were transfected with indicated combinations of DNAs , cultured for 24 hours , subjected to in vivo ubiquitination assays .

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

B. 293T cells were transfected with the indicated combinations of DNAs, cultured for 24 hours, and subjected to in vivo ubiquitination assays.


C . RPE transduced with vector ( control ) or Flag - AMOTL2 were seeded under sparse ( S ) or dense ( D ) conditions , an in vivo ubiquitination was performed .

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

C. RPE cells transduced with vector (control) or Flag-AMOTL2 were seeded under sparse (S) or dense (D) conditions, and an in vivo ubiquitination assay was performed.


D . RPE transduced with Flag - AMOTL2 were transfected with a 1 : 1 mixture of two USP9X siRNAs . One day after siRNA transfection , were re - seeded to sparse condition an in vivo ubiquitination was performed after one day .

O O O O O O O B-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 O O O O O

D. RPE cells transduced with Flag-AMOTL2 were transfected with a 1:1 mixture of the two USP9X siRNAs. One day after siRNA transfection, the cells were re-seeded to the sparse condition and an in vivo ubiquitination assay was performed after one day.


E . 293T were transfected with indicated combinations of DNAs , a co - immunoprecipitation was performed .

E. 293T cells were transfected with the indicated combinations of DNAs, and a co-immunoprecipitation assay was performed.


F . Sparsely cultured RPE or MCF10A were immunoprecipitated with an anti - AMOTL2 antibody .

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

F. Sparsely cultured RPE or MCF10A cells were immunoprecipitated with an anti-AMOTL2 antibody.


A . Flag - AMOTL2 constructs were designed to express regions spanning from N - terminus to coiled - coil domain ( AA 1 - 306 ) , coiled - coil domain ( AA 307 - 581 ) , C - terminal portion ( AA 582 - 781 ) . These constructs were co - transfected into 293T with Myc - Ub , an in vivo ubiquitination was conducted . * , non - specific band .

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

A. Flag-AMOTL2 constructs were designed to express regions spanning from the N-terminus to the coiled-coil domain (AA 1-306), the coiled-coil domain (AA 307-581), and the C-terminal portion (AA 582-781). These constructs were co-transfected into 293T cells with Myc-Ub, and an in vivo ubiquitination assay was conducted. *, non-specific band.


B . SBP - AMOTL2 coiled - coil domain was expressed in 293T , subjected to tandem affinity purification , fractionated by SDS - PAGE stained with Coomassie blue . Bands corresponding to AMOTL2 coiled - coil domain were excised analyzed by mass spectrometry for identification of ubiquitinated sites ( K - G - G linkages ) . Shown are MS / MS spectra recoded using an LTQ - Orbitrap mass spectrometer for doubly charged peptide , IEK * LESEIQR ( MH + = 1358 . 72 , z = 2 + ; upper ) , triply charged peptide , LASK * TQEAQAGSQDMVAK ( MH + = 1992 . 96 , z = 3 + ; lower ) . These peptides contain K347 K408 ubiquitination sites , respectively ( marked by asterisks ) . Fragmented ions are annotated according to nomenclature for peptide fragmentation in mass spectrometry .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The SBP-AMOTL2 coiled-coil domain was expressed in 293T cells, subjected to tandem affinity purification, fractionated by SDS-PAGE and stained with Coomassie blue. Bands corresponding to the AMOTL2 coiled-coil domain were excised and analyzed by mass spectrometry for identification of ubiquitinated sites (K-G-G linkages). Shown are MS/MS spectra recoded using an LTQ-Orbitrap mass spectrometer for the doubly charged peptide, IEK*LESEIQR (MH+ = 1358.72, z = 2+; upper), and the triply charged peptide, LASK*TQEAQAGSQDMVAK (MH+ = 1992.96, z = 3+; lower). These peptides contain the K347 and K408 ubiquitination sites, respectively (marked by asterisks). Fragmented ions are annotated according to the nomenclature for peptide fragmentation in mass spectrometry.


C . indicated Flag - AMOTL2 WT or mutant were subjected to an in vivo ubiquitination in 293T .

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

C. The indicated Flag-AMOTL2 WT or mutant proteins were subjected to an in vivo ubiquitination assay in 293T cells.


D . RPE or MCF10A were transduced with Flag - AMOTL2 WT or Flag - AMOTL2 K347 / 408R , immunoprecipitated with an anti - Flag antibody . Immunoprecipitated WCL samples were analyzed by Western blotting for indicated .

O O O O O O O O O O B-MEASURED_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

D. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 K347/408R, and immunoprecipitated with an anti-Flag antibody. Immunoprecipitated and WCL samples were analyzed by Western blotting for the indicated proteins.


E . RPE or MCF10A were transduced with Flag - AMOTL2 WT or Flag - AMOTL2 Y213A , processed as ( D ) .

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

E. RPE or MCF10A cells were transduced with Flag-AMOTL2 WT or Flag-AMOTL2 Y213A, and processed as (D).


A . MCF10A expressing shRNA against AMOTL2 were complemented with shRNA - resistant Flag - AMOTL2 WT or K347 / 408R mutant . indicated were harvested analyzed by Western blotting for indicated .

O O O O O O B-CONTROLLED_VAR 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

A. MCF10A cells expressing shRNA against AMOTL2 were complemented with shRNA-resistant Flag-AMOTL2 WT or the K347/408R mutant. The indicated cells were harvested and analyzed by Western blotting for the indicated proteins.


B . mRNAs isolated from in ( A ) were analyzed by RT - qPCR against indicated EMT marker genes ( n = 4 ) . Error bars indicate S . E . M . ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; paired 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

B. mRNAs isolated from cells in (A) were analyzed by RT-qPCR against indicated EMT marker genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01, ***p < 0.001; paired Student's t-test).


C . Transwell migration was performed using in ( A ) ( n = 8 ) . We analyzed 5 random fields for each sample . Error bars indicate S . E . M . ( * * p < 0 . 01 , paired 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

C. Transwell migration assay was performed using cells in (A) (n=8). We analyzed 5 random fields for each sample. Error bars indicate the S.E.M. (**p< 0.01, paired Student's t-test).


D . Representative of soft agar results using in ( A ) . Scale bar , 1 mm .

D. Representative images of soft agar assay results using cells in (A). Scale bar, 1 mm.


E . Soft agar was performed using in ( A ) ( n = 8 ) . Error bars indicate S . E . M . ( * * p < 0 . 01 , * * * p < 0 . 001 , paired Student ' s t - ) .

E. Soft agar assay was performed using cells in (A) (n=8). Error bars indicate the S.E.M. (**p < 0.01, ***p < 0.001, paired Student's t-test).


B , C . 293T were transfected with indicated DNAs immunoprecipitated with an anti - Flag antibody or pulled down with streptavidin - binding beads .

O O 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, C. 293T cells were transfected with the indicated DNAs and immunoprecipitated with an anti-Flag antibody or pulled down with streptavidin-binding beads.


D , E . 293T were transfected as indicated immunoprecipitated with an anti - HA antibody . LATS2 immune complexes were subjected to a kinase with His - YAP cold ATP .

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-MEASURED_VAR O O O

D, E. 293T cells were transfected as indicated and immunoprecipitated with an anti-HA antibody. LATS2 immune complexes were subjected to a kinase assay with His-YAP and cold ATP.


F . Lats1 - / - ; Lats2 fl / fl ; SV40 mouse embryonic fibroblasts complemented with vector , LATS2 WT or ΔUBA mutant were infected with empty ( control ) or Zeocin - Cre retroviruses selected with 400 μg / ml of Zeocin for 4 days . Harvested were analyzed by Western blotting for indicated .

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

F. Lats1 -/-; Lats2 fl/fl; SV40 mouse embryonic fibroblasts complemented with vector, LATS2 WT or ΔUBA mutant were infected with empty (control) or Zeocin-Cre retroviruses and selected with 400 μg/ml of Zeocin for 4 days. Harvested cells were analyzed by Western blotting for the indicated proteins.


Paneth on Lgr5 - DTReGFP duodenum ( Duo ) Ileum ( Ile ) . Left panel : Representative of Lendrum ' s that evidence Paneth granules . Arrows show differentiated Paneth . Right panel : of Paneth per 10 intervilli regions . Each symbol indicates value for a given .

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

Paneth cell quantification on Lgr5-DTReGFP duodenum (Duo) and Ileum (Ile). Left panel: Representative images of Lendrum's staining that evidence Paneth cell granules. Arrows show differentiated Paneth cells. Right panel: quantification of the Paneth cells per 10 intervilli regions. Each symbol indicates the value for a given embryo.


Expression by qRT - PCR of indicated Paneth markers in Lgr5 - DTReGFP ileums ( n = 8 WT , 12 HE , 10 KO ) .

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

Expression analysis by qRT-PCR of the indicated Paneth cell markers in Lgr5-DTReGFP ileums (n= 8 WT, 12 HE, 10 KO).


X - gal in Axin2Lac / + - Lgr5 - GFP - CreERT2 WT or KO ileums .

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

X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums.


Gene expression by qRT - PCR of markers in Lgr5 - DTReGFP ileums ( n = 3 WT , 6 HE , 6 KO ) .

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

Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTReGFP ileums (n= 3 WT, 6 HE, 6 KO).


FACS of number of eGFP + ve ( ISC ) per small intestine at indicated developmental stages in Lgr5 HEs Lgr5 KOs . Each symbol indicates value for a given . Mean value for each group is depicted on graph .

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

FACS quantification of the number of eGFP+ve (ISC) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs. Each symbol indicates the value for a given embryo. Mean value for each group is depicted on the graph.


Plating efficiency of ex vivo cultured E18 . 5 small intestines quantified 6 days after initial seeding . Each symbol indicates value for a given .

Plating efficiency of ex vivo cultured E18.5 small intestines quantified 6 days after initial seeding. Each symbol indicates the value for a given embryo.


Influence of mouse Rspondin 1 on growth of replated Lgr5 - DTReGFP WT Lgr5 KO organoids after 5 days of culture .

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

Influence of mouse Rspondin 1 concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 5 days of culture.


Paneth on Lgr5 - DTReGFP ileums . Each symbol indicates value for a given .

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

Paneth cell quantification on Lgr5-DTReGFP ileums. Each symbol indicates the value for a given embryo.


Immunofluorescence showing Olfm4 + ve in Lgr5 - DTReGFP duodenums . membranes are shown with b - catenin nuclei were counterstained with DAPI . of Olfm4 + ve per intervilli regions in duodenum ileum . Each symbol indicates value for a given .

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

Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervilli regions in duodenum and ileum. Each symbol indicates the value for a given embryo.


Axin2 expression detected by RNAscope on Lgr5 - DTReGFP proximal intestine .

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

Axin2 expression levels detected by RNAscope on Lgr5-DTReGFP proximal intestine.


Gene expression by qRT - PCR of differentiation markers in Lgr5 - DTReGFP ileums ( vehicle - treated : 7 WT 7 KO ; LGK974 - treated : 3 WT 7 KO ) .

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

Gene expression analysis by qRT-PCR of stem cell and differentiation markers in Lgr5-DTReGFP ileums (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 7 KO).


Representative pictures of lineage tracing in Lgr5 - expressing or Lgr5 cKO intestine after 10 days of chase of number of traced clones per recombined surface at PN18 ( ileum ) . Each symbol indicates value for a given mouse .

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 O O O O

Representative pictures of lineage tracing in Lgr5-expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 (ileum). Each symbol indicates the value for a given mouse.


Representative immunofluorescence pictures showing Paneth ( Plyz + ve ) in traced clones ( RFP + ve ) from control ( Lgr5Cre / + ) cKO ( Lgr5Cre / flox ) PN18 adult ileums . Arrow heads point to Plyz + ve in traced clones . of number of Paneth number per recombined RFP + ve crypt on control cKO ileums in PN18 . Each symbol indicates value for a given mouse .

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

Representative immunofluorescence pictures showing Paneth cells (Plyz+ve) in traced clones (RFP+ve) from control (Lgr5Cre/+) and cKO (Lgr5Cre/flox) PN18 and adult ileums. Arrow heads point to Plyz+ve cells in traced clones. Quantification of the number of Paneth cell number per recombined RFP+ve crypt on control and cKO ileums in PN18. Each symbol indicates the value for a given mouse.


ISC ( eGFP + ve ) were FACS - sorted from Lgr5 - DTReGFP at E16 . 5 subjected to RNAseq . of differentially regulated genes in HE KO ISCs at E16 . 5 . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO )

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 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

ISC (eGFP+ve) cells were FACS-sorted from Lgr5-DTReGFP embryos at E16.5 and subjected to RNAseq analysis. Heatmap of differentially regulated genes in HE and KO ISCs at E16.5. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO)


Compared Hallmarks for upregulated downregulated genes in transcriptome of E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE adult Lgr5 - Cre cKO vs HEs . * Asterisks refer to given pathways for which differentially modulated genes were found upregulated downregulated . Right panels : GSEA showing enrichment of epithelial to mesenchymal transition ( EMT ) data set in E16 . 5 Lgr5 KOs vs E16 . 5 HEs , adult HEs vs E18 . 5 HE , adult Lgr5 cKO vs adult HE modulated genes . NES : Normalized Enrichment Score . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 ( n = 2 HE ) Lgr5 - DTReGFP adult ( n = 2 HE ) ; Lgr5 - Cre / flox adult ( n = 2 HE , 3 cKO ) .

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 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 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 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 B-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

Compared Hallmarks for upregulated and downregulated genes in the transcriptome of E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos and adult Lgr5-Cre cKO vs HEs. * Asterisks refer to given pathways for which differentially modulated genes were found upregulated and downregulated. Right panels: GSEA showing enrichment of the epithelial to mesenchymal transition (EMT) data set in E16.5 Lgr5 KOs vs E16.5 HEs, adult HEs vs E18.5 HE embryos, and adult Lgr5 cKO vs adult HE modulated genes. NES: Normalized Enrichment Score. Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO).


Expression of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16 . 5 Lgr5 adult vs E18 . 5 HEs . RPKM : reads per kilobase of transcript per million mapped reads . Below : GeneOntology of downregulated genes identified in Epithelial Mesenchymal Transition Gene Set ( Hallmark MSigDB collection ) . Biological replicates for RNAseq experiments on ISC Lgr5 - DTReGFP E16 . 5 independent embryonic pools , each obtained from independent litters ( n = 2 HE , 2 KO ) ; Lgr5 - DTReGFP E18 . 5 ( n = 2 HE ) Lgr5 - DTReGFP adult ( n = 2 HE ) ; Lgr5 - Cre / flox adult ( n = 2 HE , 3 cKO ) . Data are represented as means ± ( C ) .

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

Expression levels of genes commonly downregulated in ISCs of Lgr5 KOs vs Lgr5 HEs at E16.5 and Lgr5 adult vs E18.5 HEs. RPKM: reads per kilobase of transcript per million mapped reads. Below: GeneOntology analysis of downregulated genes identified in the Epithelial Mesenchymal Transition Gene Set (Hallmark MSigDB collection). Biological replicates for RNAseq experiments on ISC Lgr5-DTReGFP E16.5 independent embryonic pools, each obtained from independent litters (n= 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n=2 HE) and Lgr5-DTReGFP adult individual animals (n=2 HE); Lgr5-Cre/flox adult individual animals (n= 2 HE, 3 cKO). Data are represented as means ± sem (C).


Expression profile of Col1a1 , Zeb2 Cxcl10 genes detected by RNAscope in embryonic adult intestines . Insets with higher magnification are depicted on right of each corresponding picture , boundaries between epithelium mesenchyme are shown with dotted lines arrows point to epithelial expression .

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

Expression profile of Col1a1, Zeb2 and Cxcl10 genes detected by RNAscope in embryonic and adult intestines. Insets with higher magnification are depicted on the right of each corresponding picture, boundaries between epithelium and mesenchyme are shown with dotted lines and arrows point to epithelial expression.


Expression of Axin2 , Lgr5 , Lgr4 , Rspo1 , Rspo2 , Rspo3 Rspo4 genes was analyzed on E15 , E16 . 5 adult stages by RNAscope . Arrows evidence expressing .

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

Expression of Axin2, Lgr5, Lgr4, Rspo1, Rspo2, Rspo3 and Rspo4 genes was analyzed on E15, E16.5 and adult stages by RNAscope. Arrows evidence expressing cells.


Representative pictures showing influence of mouse Rspondin type on growth of replated Lgr5 - DTReGFP WT Lgr5 KO organoids after 4 days of culture . Arrows show alive elements arrow heads dead elements .

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

Representative pictures showing the influence of mouse Rspondin type and concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 4 days of culture. Arrows show alive elements and arrow heads dead elements.


Organoid depends on Rspondin ligand type . Left panel : growth quantified as of elements present at day1 surviving at day4 . A mean of 100 elements were analyzed per sample per condition ( n : 4 WT , 2 KO organoid culture samples , each originating from a given ) . Right panel : organoid complexity measured by branching coefficient . Each symbol indicates value of a given organoid ( was done on 4 WT 2 KO organoid culture samples , each originating from a given ) .

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

Organoid morphology depends on Rspondin ligand type and concentration. Left panel: growth quantified as the percentage of elements present at day1 surviving at day4. A mean of 100 elements were analyzed per sample and per condition (n: 4 WT, 2 KO organoid culture samples, each originating from a given embryo). Right panel: organoid complexity measured by branching coefficient. Each symbol indicates the value of a given organoid (analysis was done on 4 WT and 2 KO organoid culture samples, each originating from a given embryo).


showing impact of Rspondin type ( ng / ml ) on relative Wnt target gene expression ( list from Sansom APC data set ) in Lgr5 - DTReGFP WT KO organoids analyzed by RNAseq ( n = 3 WT 2 organoid culture samples , each originating from a given ) .

O 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

Heatmap showing the impact of Rspondin type and concentration (ng/ml) on relative Wnt target gene expression (list from the Sansom APC data set) in Lgr5-DTReGFP WT and KO organoids analyzed by RNAseq (n= 3 WT and 2 organoid culture samples, each originating from a given embryo).


Gene expression by qRT - PCR of markers in Lgr5 - DTREeGFP WT KO organoids after 5 days of culture ( expression relative to reference genes ) . Each symbol indicates value for an organoid culture originated from a given .

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

Gene expression analysis by qRT-PCR of stem cell markers in Lgr5-DTREeGFP WT and KO organoids after 5 days of culture (expression relative to reference genes). Each symbol indicates the value for an organoid culture originated from a given embryo.


Immunofluorescence showing Olfm4 + ve in Lgr5 - DTReGFP WT KO organoids cultured in Rspo2 conditions . Epithelium is delineated with b - catenin nuclei counterstained with DAPI ( Merge ) . Insets : arrowheads evidence Olfm4 + ve concentrated in organoid protrusions . of Olfm4 + ve with respect to total epithelial . Each symbol indicates value for an organoid culture originating from a given .

O O B-MEASURED_VAR O O O B-CONTROLLED_VAR 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 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

Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP WT and KO organoids cultured in Rspo2 conditions. Epithelium is delineated with b-catenin and nuclei counterstained with DAPI (Merge). Insets: white arrowheads evidence Olfm4+ve cells concentrated in organoid protrusions. Quantification of Olfm4+ve area with respect to the total epithelial area. Each symbol indicates the value for an organoid culture originating from a given embryo.


A Representative of alkaline phosphatase ( ALP ) , Alizarin Red S ( ARS ) Van Gieson ' s of human

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

A Representative images of alkaline phosphatase (ALP), Alizarin Red S (ARS) and Van Gieson's staining of human MSCs


B , C Quantitative analyses of ALP activity calcium mineralization . Results are shown as mean ± ; n = 6 ; * * : p < 0 . 01 * * * : p < 0 . 001 by t

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

B, C Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=6; **: p<0.01 and ***: p<0.001 by t test


D , E Quantitative RT - PCR western blot analyses of expression of osteogenic markers . were cultured in osteogenic medium for 5 days . Results are shown as mean ± ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 * * * : p < 0 . 001 by 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

D, E Quantitative RT-PCR and western blot analyses of the expression of osteogenic markers. Cells were cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test


F H & E of MSCs - mediated ectopic bone formation . Scale bar , 50 μm

F H&E staining of the MSCs-mediated ectopic bone formation. Scale bar, 50 μm


G Quantitative of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± ; n = 6 . * : p < 0 . 05 by 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

G Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=6. *: p<0.05 by t test


A Scatter plot of - seq expression . 508 genes were upregulated 391 genes were downregulated . Human MSCs were transfected with USP34 or control siRNAs cultured in osteogenic medium for 2 days . Two biological replicates per group

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

A Scatter plot of RNA-seq expression analysis. 508 genes were upregulated and 391 genes were downregulated. Human MSCs were transfected with USP34 or control siRNAs and cultured in osteogenic medium for 2 days. Two biological replicates per group


C of representative genes associated with BMP2 signaling pathway

C Heatmap of representative genes associated with BMP2 signaling pathway


D Relative activity of BRE luciferase . 293T were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± ; n = 3 . * * * : p < 0 . 001 by t

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

D Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. ***: p<0.001 by t test


E Immunoblot of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f Usp34f / f control MSCs were starved overnight then stimulated with 100 ng / ml BMP2 for 24

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

E Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours


F Quantitative RT - PCR of BMP2 - induce Sp7 transcription . were depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± ; n = 3 ; * : p < 0 . 05 * * : p < 0 . 01 by t

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

F Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Cells were depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by t test


G Representative of ALP ARS . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 10 21 days ,

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

G Representative images of ALP and ARS staining. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 10 and 21 days, respectively


H , I Quantitative analyses of ALP activity ( n = 4 ) calcium mineralization ( n = 5 ) . Results are shown as mean ± ; * : p < 0 . 05 , * * : p < 0 . 01 * * * : p < 0 . 001 by t

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

H, I Quantitative analyses of the ALP activity (n=4) and calcium mineralization (n=5). Results are shown as mean ± SEM; *: p<0.05, **: p<0.01 and ***: p<0.001 by t test


J , K Representative microCT of newly formed bone in response to BMP2 . Results are shown as mean ± ; n = 8 . * : p < 0 . 05 by t . Scale bar , 2500 μm

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

J, K Representative microCT images and quantification of newly formed bone in response to BMP2. Results are shown as mean ± SEM; n=8. *: p<0.05 by t test. Scale bar, 2500 μm


L H & E of BMP2 - induced newly formed bone . Scale bar , 50

L H&E staining of BMP2-induced newly formed bone. Scale bar, 50 μm


M Quantitative of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± ; n = 8 ; * * : p < 0 . 01 by 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

M Quantitative analysis of bone volume versus total tissue volume (BV/TV). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


N H & E of calvarial organ cultures stimulated with or without 100 ng / ml BMP2 . Red dotted lines indicate interface between original newly formed bone . Scale bar , 50

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

N H&E staining of calvarial organ cultures stimulated with or without 100 ng/ml BMP2. Red dotted lines indicate the interface between original and newly formed bone. Scale bar, 50 μm


O Quantitative of newly formed bone volume versus original bone volume ( % ) . Results are shown as mean ± ; n = 8 ; * * : p < 0 . 01 by t

O Quantitative analysis of newly formed bone volume versus original bone volume (%). Results are shown as mean ± SEM; n=8; **: p<0.01 by t test


A Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged Smad1 in 293T

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

A Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged Smad1 in 293T cells


B Immunoblot showing decreased FLAG - Smad1 after USP34 knockdown

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

B Immunoblot analysis showing decreased FLAG-Smad1 after USP34 knockdown


C Immunoblot of Smad1 - linked polyUb . 293T were co - transfected with FLAG - Smad1 overexpression plasmid USP34 siRNA , treated with 10 µM MG132 for 4 hours before collection

O O O B-MEASURED_VAR 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

C Immunoblot of Smad1-linked polyUb. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 10 µM MG132 for 4 hours before collection


D Immunoblot of degradation of FLAG - Smad1 . 293T were co - transfected with FLAG - Smad1 overexpression plasmid USP34 siRNA , treated with 50 µg / ml cycloheximide ( CHX ) before collection at designated time points . Right : relative of FLAG - Smad1 at different time

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

D Immunoblot analysis of the degradation of FLAG-Smad1 protein. 293T cells were co-transfected with FLAG-Smad1 overexpression plasmid and USP34 siRNA, and treated with 50 µg/ml cycloheximide (CHX) before collection at designated time points. Right: relative quantification of FLAG-Smad1 protein levels at different time points


E Co - immunoprecipitation of USP34 with ectopically expressed FLAG - tagged RUNX2 in 293T

E Co-immunoprecipitation of USP34 with ectopically expressed FLAG-tagged RUNX2 in 293T cells


F Co - immunoprecipitation of USP34 with endogenous RUNX2 in mouse

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

F Co-immunoprecipitation of USP34 with endogenous RUNX2 in mouse MSCs


G Immunoblot of RUNX2 - linked polyUb . Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection . Prx1 - Cre ( + ) indicates MSCs isolated from Prx1 - Cre ; Usp34f / f mice , Prx1 - Cre ( - ) indicates those from Usp34f / f control

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

G Immunoblot of RUNX2-linked polyUb. Differentiated mouse MSCs were treated with 10 µM MG132 for 4 hours before collection. Prx1-Cre(+) indicates MSCs isolated from Prx1-Cre;Usp34f/f mice, and Prx1-Cre(-) indicates those from Usp34f/f control mice


H Immunoblot of degradation of endogenous RUNX2 in MSCs . Prx1 - Cre ; Usp34f / f Usp34f / f control MSCs were stimulated with BMP2 , treated with 50 µg / ml CHX , collected at designated time points . Right : relative of RUNX2 at different time

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

H Immunoblot analysis of the degradation of endogenous RUNX2 protein in MSCs. Prx1-Cre;Usp34f/f and Usp34f/f control MSCs were stimulated with BMP2, treated with 50 µg/ml CHX, and collected at designated time points. Right: relative quantification of RUNX2 protein levels at different time points


I Representative of ALP ARS . Usp34f / f Prx1 - Cre ; Usp34f / f MSCs were transduced with GFP , Smad1 , Runx2 or Smad1 + Runx2 adenoviruses , then cultured in osteogenic medium for 5 10 days ,

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

I Representative images of ALP and ARS staining. Usp34f/f and Prx1-Cre;Usp34f/f MSCs were transduced with GFP, Smad1, Runx2 or Smad1+Runx2 adenoviruses, and then cultured in osteogenic medium for 5 and 10 days, respectively


J , K Quantitative analyses of ALP activity calcium mineralization . Results are shown as mean ± ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

J, K Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


L , M H & E quantitative of MSCs - mediated ectopic bone formation . Scale bar , 50 μm . Results are shown as mean ± ; n = 8 ; * : p < 0 . 05 * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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

L, M H&E staining and quantitative analysis of MSCs-mediated ectopic bone formation. Scale bar, 50 μm. Results are shown as mean ± SEM; n=8; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


A Relative activity of BRE luciferase . 293T were depleted with serum overnight followed by a treatment with BMP2 for 6 hours . Results are shown as mean ± ; n = 3 . * : p < 0 . 05 by ANOVA with Tukey ' s post hoc

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

A Relative activity of BRE luciferase assay. 293T cells were depleted with serum overnight followed by a treatment with BMP2 for 6 hours. Results are shown as mean ± SEM; n=3. *: p<0.05 by ANOVA with Tukey's post hoc test


B Immunoblot of BMP2 - induce Sp7 expression . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , starved overnight then stimulated with 100 ng / ml BMP2 for 24

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

B Immunoblot analysis of BMP2-induce Sp7 expression. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, starved overnight and then stimulated with 100 ng/ml BMP2 for 24 hours


C Quantitative RT - PCR of BMP2 - induce Sp7 transcription . Prx1 - Cre ; Usp34f / f MSCs were treated with Smurf1 or control siRNAs , depleted from serum overnight before BMP2 stimulation . Results are shown as mean ± ; n = 3 ; * : p < 0 . 05 * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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

C Quantitative RT-PCR analysis of BMP2-induce Sp7 transcription. Prx1-Cre;Usp34f/f MSCs were treated with Smurf1 or control siRNAs, and depleted from serum overnight before BMP2 stimulation. Results are shown as mean ± SEM; n=3; *: p<0.05 and **: p<0.01 by ANOVA with Tukey's post hoc test


D ALP quantitative measurement of ALP activity . MSCs were cultured with normal medium supplemented with 100 ng / ml BMP2 for 5 days . Results are shown as mean ± ; n = 8 ; * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

O B-MEASURED_VAR O O 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 O O O 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 ALP staining and quantitative measurement of ALP activity. MSCs were cultured with normal medium supplemented with 100 ng/ml BMP2 for 5 days. Results are shown as mean ± SEM; n=8; **: p<0.01 by ANOVA with Tukey's post hoc test


E Representative of ARS . were cultured with normal medium supplemented with 100 ng / ml BMP2 for 14 days . Results are shown as mean ± ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 by ANOVA with Tukey ' s post hoc

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

E Representative images of ARS staining and quantification. Cells were cultured with normal medium supplemented with 100 ng/ml BMP2 for 14 days. Results are shown as mean ± SEM; n=8; *: p<0.05, and **: p<0.01 by ANOVA with Tukey's post hoc test


F Representative of ALP ARS . Mouse MSCs were treated with Smurf1 or control siRNAs , cultured in osteogenic medium for 5 10 days ,

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 O O

F Representative images of ALP and ARS staining. Mouse MSCs were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 and 10 days, respectively


G , H Quantitative analyses of ALP activity calcium mineralization . Results are shown as mean ± ; n = 8 ; * : p < 0 . 05 , * * : p < 0 . 01 * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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

G, H Quantitative analyses of the ALP activity and calcium mineralization. Results are shown as mean ± SEM; n=8; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


I , J Quantitative RT - PC analyses of expression of osteogenic markers . were treated with Smurf1 or control siRNAs , cultured in osteogenic medium for 5 days . Results are shown as mean ± ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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

I, J Quantitative RT-PC analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


western blot analyses of expression of osteogenic markers . were treated with Smurf1 or control siRNAs , cultured in osteogenic medium for 5 days . Results are shown as mean ± ; n = 3 ; * : p < 0 . 05 , * * : p < 0 . 01 * * * : p < 0 . 001 by ANOVA with Tukey ' s post hoc

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

western blot analyses of the expression of osteogenic markers. Cells were treated with Smurf1 or control siRNAs, and cultured in osteogenic medium for 5 days. Results are shown as mean ± SEM; n=3; *: p<0.05, **: p<0.01 and ***: p<0.001 by ANOVA with Tukey's post hoc test


A , B , Western blotting of co - precipitated with anti - FLAG antibody from testis extracts of E15 . 5 wild - type transgenic expressing FLAG - tagged NANOS2 with or without RNase ( A ) or from extracts of HeLa transfected with FLAG - tagged DND1 with or without HA - tagged NANOS2 ( B ) .

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

A, B, Western blotting analysis of proteins co-precipitated with anti-FLAG antibody from testis extracts of E15.5 wild-type embryos and transgenic embryos expressing FLAG-tagged NANOS2 with or without RNase (A) or from extracts of HeLa cells transfected with FLAG-tagged DND1 and with or without HA-tagged NANOS2 (B).


Precipitates were analyzed with indicated antibodies . C , GST pull - down using E . coli extracts expressing GST , GST - fused NANOS2 , or NANOS3 mixed with MBP - tagged LacZ or DND1 . Precipitates were analyzed by Coomassie Brilliant Blue ( CBB ) or western blotting with an anti - MBP antibody . An arrowhead indicates MBP - DND1 co - precipitated with GST - tagged NANOS2 or NANOS3 .

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

Precipitates were analyzed with the indicated antibodies. C, GST pull-down assay using E. coli extracts expressing GST, GST-fused NANOS2, or NANOS3 mixed with MBP-tagged LacZ or DND1. Precipitates were analyzed by Coomassie Brilliant Blue (CBB) staining or western blotting with an anti-MBP antibody. An arrowhead indicates MBP-DND1 co-precipitated with GST-tagged NANOS2 or NANOS3.


D , E , Western blotting analyses of co - precipitated with anti - FLAG antibody from extracts of HeLa transfected with FLAG - tagged NANOS2 , NANOS2 ( ΔN10 ) , NANOS2 ( ΔC10 ) , NANOS2 ( C61A , C96A ) or NANOS3 , HA - tagged DND1 ( D )

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

D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D)


D , E , Western blotting analyses of co - precipitated with anti - FLAG antibody from extracts of HeLa transfected with FLAG - tagged NANOS2 , NANOS2 ( ΔN10 ) , NANOS2 ( ΔC10 ) , NANOS2 ( C61A , C96A ) or NANOS3 , HA - tagged DND1 ( D ) or with FLAG - tagged NANOS2 , NANOS2 ( C61A ) , NANOS2 ( C96A ) or NANOS2 ( C61A , C96A ) , HA - tagged DND1 ( E ) .

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

D, E, Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with FLAG-tagged NANOS2, NANOS2 (ΔN10), NANOS2 (ΔC10), NANOS2 (C61A, C96A) or NANOS3, and HA-tagged DND1 (D) or with FLAG-tagged NANOS2, NANOS2 (C61A), NANOS2 (C96A) or NANOS2 (C61A, C96A), and HA-tagged DND1 (E).


Precipitates were analyzed with indicated antibodies . F , GST pull - down using E . coli extracts expressing GST - fused DND1 mixed with MBP - tagged LacZ , NANOS2 , NANOS2 ( C61A , C96A ) or NANOS2 ( 60 - 114 ) . Arrowheads indicate MBP - tagged NANOS2 ( lane 2 ) or NANOS2 ( 60 - 114 ) ( lane 4 ) .

O O O O O O 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 B-CONTROLLED_VAR 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

Precipitates were analyzed with the indicated antibodies. F, GST pull-down assay using E. coli extracts expressing GST-fused DND1 mixed with MBP-tagged LacZ, NANOS2, NANOS2 (C61A, C96A) or NANOS2 (60-114). Arrowheads indicate MBP-tagged NANOS2 (lane 2) or NANOS2 (60-114) (lane 4).


Western blotting analyses of co - precipitated with anti - FLAG antibody from extracts of HeLa transfected with HA - tagged NANOS1 , NANOS2 or NANOS3 , FLAG - tagged DND1 ( A )

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

Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A)


Western blotting analyses of co - precipitated with anti - FLAG antibody from extracts of HeLa transfected with HA - tagged NANOS1 , NANOS2 or NANOS3 , FLAG - tagged DND1 ( A ) or with FLAG - tagged PUMILIO1 or PUMILIO2 , HA - tagged NANOS1 , NANOS2 or NANOS3 with or without MYC - tagged DND1 ( B ) .

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

Western blotting analyses of proteins co-precipitated with anti-FLAG antibody from extracts of HeLa cells transfected with HA-tagged NANOS1, NANOS2 or NANOS3, and FLAG-tagged DND1 (A) or with FLAG-tagged PUMILIO1 or PUMILIO2, and HA-tagged NANOS1, NANOS2 or NANOS3 with or without MYC-tagged DND1 (B).


C , Characterization of antibodies against PUMILIO1 PUMILIO2 . Western blotting analyses of Flag - tagged PUMILIO1 PUMILIO2 in HeLa transfected with FLAG - tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG , PUMILIO1 or PUMILIO2 . Note that antibodies against PUMILIO1 specifically recognized FLAG - tagged PUMILIO1 while anti - PUMILIO2 antibody could detect both Flag - tagged PUMILIO1 PUMILIO2 but less actively .

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

C, Characterization of antibodies against PUMILIO1 and PUMILIO2. Western blotting analyses of Flag-tagged PUMILIO1 and PUMILIO2 in HeLa cells transfected with FLAG-tagged PUMILIO1 or PUMILIO2 using antibodies against FLAG, PUMILIO1 or PUMILIO2. Note that the antibodies against PUMILIO1 specifically recognized FLAG-tagged PUMILIO1 while the anti-PUMILIO2 antibody could detect both Flag-tagged PUMILIO1 and PUMILIO2 but less actively.


D , FLAG - tagged NANOS2 was immunoprecipitated with an anti - FLAG antibody from E15 . 5 testicular extracts from transgenic mice expressing FLAG - tagged NANOS2 . Precipitates were analyzed by western blotting with indicated antibodies . Note that co - precipitation of both PUMILIO1 PUMILIO2 were not detected even though both CNOT1 DND1 were clearly co - precipitated .

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

D, FLAG-tagged NANOS2 was immunoprecipitated with an anti-FLAG antibody from E15.5 testicular extracts from transgenic mice expressing FLAG-tagged NANOS2. Precipitates were analyzed by western blotting with the indicated antibodies. Note that co-precipitation of both PUMILIO1 and PUMILIO2 were not detected even though both CNOT1 and DND1 were clearly co-precipitated.


Figure 3 | DND1 co - localizes with NANOS2 in P - bodies . A − C , Sections of gonads from E15 . 5embryos were immunostained with antibodies against DND1 ( green ) DCP1a ( red ) . D − F , Squash preparation of a gonocyte from E16 . 5embryoimmunostained with antibodies against DND1 ( red ) DCP1a ( green ) . Arrowheads indicate co - localization of DND1 DCP1a . G − I , Sections of gonads from E15 . 5embryos were immunostained with antibodies against DND1 ( red ) NANOS2 ( green ) . Arrowheads indicate co - localization of DND1 NANOS2 . J − M , NIH3T3 transfected with HA - tagged Dnd1 FLAG - tagged Nanos2 were then immunostained with antibodies against DND1 ( J ) ( magenta ) , NANOS2 ( K ) ( red ) , DCP1a ( L ) ( green ) . N − Y , Biomolecular complementation . NIH3T3 transfected with VENUS - C - fused Dnd1 VENUS - N - fused Nanos2 ( N − U ) or Nanos2 ( C61A , C96A ) ( V - γ ) were immunostained with antibodies against DND1 ( N , S , V ) , NANOS2 ( O , W , α ) or DDX6 ( R , Z ) . Then , signals of VENUS fusion were visualized ( P , T , X , β ) . Arrowheads in Z indicate P - bodies . was labeled with DAPI ( blue ) . Scale bars : 50 m in A for A − C ; 10 m in D for D - F , G for G − I , J for J − M , N for N − γ . Insets in A - C J - γ show an enlarged vision of each picture to better depict localization of each . See also Fig . EV2T - Y .

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

Figure 3 | DND1 co-localizes with NANOS2 in P-bodies. A−C, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (green) and DCP1a (red). D−F, Squash preparation of a male gonocyte from E16.5embryoimmunostained with antibodies against DND1 (red) and DCP1a (green). Arrowheads indicate co-localization of DND1 and DCP1a. G−I, Sections of male gonads from E15.5embryos were immunostained with antibodies against DND1 (red) and NANOS2 (green). Arrowheads indicate co-localization of DND1 and NANOS2. J−M, NIH3T3 cells transfected with HA-tagged Dnd1 and FLAG-tagged Nanos2 were then immunostained with antibodies against DND1 (J) (magenta), NANOS2 (K) (red), and DCP1a (L) (green). N−Y, Biomolecular fluorescence complementation assay. NIH3T3 cells transfected with VENUS-C-fused Dnd1 and VENUS-N-fused Nanos2 (N−U) or Nanos2 (C61A, C96A) (V-γ) were immunostained with antibodies against DND1 (N, S, V), NANOS2 (O, W, α) or DDX6 (R, Z). Then, the signals of VENUS fusion protein were visualized (P, T, X, β). Arrowheads in Z indicate P-bodies. DNA was labeled with DAPI (blue). Scale bars: 50 m in A for A−C; 10 m in D for D-F, G for G−I, J for J−M, N for N−γ. Insets in A-C and J-γ show an enlarged vision of each picture to better depict localization of each protein. See also Fig. EV2T-Y.


A , Western blotting analyses of in testes from E13 . 5 to E16 . 5 of Dnd1flox / flox or Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) each administered with tamoxifen at E13 . 5 E15 . 5 of Nanos2 + / - or Nanos2 - / - with indicated antibodies .

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

A, Western blotting analyses of proteins in testes from E13.5 to E16.5 embryos of Dnd1flox/flox or Dnd1flox/flox_Tg(Oct4PE-CreERT2) each administered with tamoxifen at E13.5 and E15.5 embryos of Nanos2+/- or Nanos2-/- with the indicated antibodies.


B - P , Sections of testes from Dnd1flox / flox ( B , E , H , K , N ) , Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) ( C , F , I , L , O ) or Nanos2 - / - ( D , G , J , M , P ) were prepared at E16 . 5 then immunostained with antibodies against pH3 ( B - D ) , STRA8 ( E - G ) , SYCP3 ( H - J ) , activated - Caspase3 ( K - M ) or LAMININ ( N - P ) ( green ) . Germ were immunostained with TRA98 ( B - D ) or DAZL ( N - P ) ( red ) , was labeled with DAPI ( blue ) . Tamoxifen was administered at E13 . 5 . Scale bars : 50 m in B for B − D K - P , 50 m in E for E − J . See also Fig . EV3 .

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 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 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 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 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

B-P, Sections of testes from Dnd1flox/flox (B, E, H, K, N), Dnd1flox/flox_Tg(Oct4PE-CreERT2) (C, F, I, L, O) or Nanos2-/- (D, G, J, M, P) embryos were prepared at E16.5 and then immunostained with antibodies against pH3 (B-D), STRA8 (E-G), SYCP3 (H-J), activated-Caspase3 (K-M) or LAMININ (N-P) (green). Germ cells were immunostained with TRA98 (B-D) or DAZL (N-P) (red), and DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in B for B−D and K-P, 50 m in E for E−J. See also Fig. EV3.


A - L , Sections of testes from Dnd1flox / flox ( A , B , C , G , H , I , M , N , O ) Dnd1flox / flox _ Tg ( Oct4PE - CreERT2 ) ( D , E , F , J , K , L , P , Q , R ) were prepared at E16 . 5 then immunostained with antibodies against NANOS2 ( A , D ) ( green ) , DCP1a ( G , J ) ( green ) , DDX6 ( M , P ) ( green ) TRA98 ( B , E , H , K , N , Q ) . was labeled with DAPI ( blue ) . Tamoxifen was administered at E13 . 5 . Scale bars : 50 m in A for A − F ; 50 m in G for G - L ; 50 m in M for M - R . Insets show an enlarged vision of each picture to better depict localization of NANOS2 , DCP1a DDX6 .

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

A-L, Sections of testes from Dnd1flox/flox (A, B, C, G, H, I, M, N, O) and Dnd1flox/flox_Tg(Oct4PE-CreERT2) (D, E, F, J, K, L, P, Q, R) embryos were prepared at E16.5 and then immunostained with antibodies against NANOS2 (A, D) (green), DCP1a (G, J) (green), DDX6 (M, P) (green) and TRA98 (B, E, H, K, N, Q). DNA was labeled with DAPI (blue). Tamoxifen was administered at E13.5. Scale bars: 50 m in A for A−F; 50 m in G for G-L; 50 m in M for M-R. Insets show an enlarged vision of each picture to better depict localization of NANOS2, DCP1a and DDX6.


S - V , Immunoprecipitation with an anti - FLAG antibody from E15 . 5 gonadal extracts of wild - type transgenic mouse expressing FLAG - tagged DND1 ( S , T ) , or with an anti - NANOS2 antibody from E15 . 5 gonadal extracts of Dnd1flox / flox mice with or without Oct4PE - CreERT2 ( U , V ) . Precipitates were analyzed by western blotting ( S , U ) or by RT - qPCR ( T , V ) . RT - qPCR data are shown as average relative mRNA ± SEs ( n = 3 ) .

O O O 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 B-MEASURED_VAR 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

S-V, Immunoprecipitation with an anti-FLAG antibody from E15.5 male gonadal extracts of wild-type and the transgenic mouse line expressing FLAG-tagged DND1 (S, T), or with an anti-NANOS2 antibody from E15.5 male gonadal extracts of Dnd1flox/flox mice with or without Oct4PE-CreERT2 (U, V). Precipitates were analyzed by western blotting (S, U) or by RT-qPCR (T, V). The RT-qPCR data are shown as average relative mRNA levels ± SEs (n=3).


W , X , Western blotting analyses of in NANOS2 - depleted ( W ) or DND1 - depleted ( X ) testis extracts from E15 . 5 . were analyzed with indicated antibodies .

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 O O

W, X, Western blotting analyses of proteins in NANOS2-depleted (W) or DND1-depleted (X) testis extracts from E15.5 embryos. Proteins were analyzed with the indicated antibodies.


A . Whole chromosomes imaged by conventional . Data information : Scale bars :

A. Whole chromosomes imaged by conventional TEM. Data information: Scale bars: 1µm


B . Slice from a large tomographic volume ; part of carbon film surrounding a hole with vitrified ice containing plates is indicated with an asterisk ; inset illustrates that slice of a plate corresponds to a in x - y plane ( perpendicular to direction of electron beam ) . Data information : Scale bars : 200

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Slice from a large tomographic volume; part of the carbon film surrounding a hole with vitrified ice containing the plates is indicated with an asterisk; the inset illustrates that the slice of a plate corresponds to a line in the x-y plane (perpendicular to the direction of the electron beam). Data information: Scale bars: 200 nm


C - G Slices from different tomograms corresponding 3D segmentations showing plates with different sizes shapes . In addition to a typical slice through x - y plane ( yellow ) , two slices through x - z ( red ) y - z ( green ) planes ( orthogonal to x - y plane ) are shown in ( C ) . Data information : Scale bars : 200 nm D ) , 100 nm ( E - G ) , 50 nm ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Slices from different tomograms and the corresponding 3D segmentations showing plates with different sizes and shapes. In addition to a typical slice through the x-y plane (yellow), two slices through the x-z (red) and y-z (green) planes (orthogonal to the x-y plane) are shown in (C). Data information: Scale bars: 200 nm D), 100 nm (E-G), 50 nm (C).


A . Nucleosomes ( N ) decorating a relaxed plate ( P1 ) ; insets in upper right show additional examples . Nucleosomes are not visible as units in typical compact plates ( P2 ) . Short compact interdigitated solenoids are shown in main image ( S ) in bottom - left insets . Data information : Scale bars : 50

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Nucleosomes (N) decorating a relaxed plate (P1); insets in the upper right show additional examples. Nucleosomes are not visible as individual units in typical compact plates (P2). Short compact interdigitated solenoids are shown in the main image (S) and in the bottom-left insets. Data information: Scale bars: 50 nm


B - E Slice ( B ) segmentations in three different orientations ( C ) of a large relaxed plate decorated with many nucleosomes on its right side . Slice ( D ) segmentation ( E ) of a relaxed plate forming a tube decorated with nucleosomes . Data information : Scale bars : 50 nm insets , B - E )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Slice (B) and segmentations in three different orientations (C) of a large relaxed plate decorated with many nucleosomes on its right side. Slice (D) and segmentation (E) of a relaxed plate forming a tube decorated with nucleosomes. Data information: Scale bars: 50 nm insets, B-E)


A , B Plates with several layers that are not closely appressed . C - E Large multilayer plates having of human metaphase chromatids [ ~ 0 . 6 µm diameter ] ( C ) ; inset schematically shows perpendicular orientation of layers with respect to chromatid axes proposed in thin - plate model In other slices ( D , E ) , multilayer structures shown in ( C ) are more compact layers are not visible as separate elements . Data information : Scale bars : 50 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Plates with several layers that are not closely appressed. C-E Large multilayer plates having the size of human metaphase chromatids [~0.6 µm diameter ] (C); the inset schematically shows the perpendicular orientation of chromatin layers with respect to the chromatid axes proposed in the thin-plate model In other slices (D,E), the multilayer structures shown in (C) are more compact and the individual layers are not visible as separate elements. Data information: Scale bars: 50 nm.


A - C Slices showing lateral association between two plates corresponding 3D segmentations . D . Edge - to - edge interactions form cylindrical structures . Data information : Scale bars : 50 nm .

O O O O O O O O O O O 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-C Slices showing lateral association between two plates and the corresponding 3D segmentations. D. Edge-to-edge interactions form cylindrical structures. Data information: Scale bars: 50 nm.


A - C cation concentrations used are indicated for each experiment ; AU , arbitrary units .

A-C The cation concentrations used are indicated for each experiment; AU, arbitrary units.


A . Spike - binding antibodies in serum . Binding by antibodies of classes M , G , A is detected with secondary antibodies conjugated to different fluorochromes , results are shown on left , middle , right plots , respectively . Binding ( geometric mean - GMFI - of corresponding secondary antibody ) to TE expressing SARS - CoV - 2 spike is plotted on vertical axis , binding to untransfected on horizontal axis . Each point represents a value from one donor . Samples from donors with no known exposure to SARS - CoV - 2 antigens ( n = 86 ) are plotted with blue circles , recently infected donors ( n = 34 ) with red circles , vaccinated donors ( n = 14 ) with green diamonds . Red circles with black triangles correspond to donors from whose B monoclonal antibodies were isolated ( n = 5 , P values are derived from a two - way of variance followed by Tukey ' s to compare specific binding , i . e . , ( binding to spike - expressing ) / ( binding to untransfected ) between conditions ( convalescent or vaccinated against unexposed ) within each antibody class . experiment was independently repeated three times , results shown come from third replicate .

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 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 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

A. Spike-binding antibodies in serum. Binding by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding (geometric mean fluorescence intensity - GMFI- of corresponding secondary antibody) to TE cells expressing SARS-CoV-2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to SARS-CoV-2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated (n = 5, P values are derived from a two-way analysis of variance followed by Tukey's test to compare the specific binding, i.e., (binding to spike-expressing cells)/(binding to untransfected cells) between conditions (convalescent or vaccinated against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.


B . Change in antibody binding following vaccination against SARS - CoV - 2 . Binding of antibodies from sera assessed in experiment shown in ( A ) , is shown for two samples of serum from each of 7 donors . Data are plotted as in ( A ) with binding to spike - expressing on vertical axis , to untransfected on horizontal , using blue symbols for samples from before vaccination , red symbols for samples from 15 - 29 days after first vaccination ( but before any second vaccination ) . Black lines connect points corresponding to pre / post pairs of samples from each donor . Left plot shows results for IgM right plot for IgG . C . Comparison of post - vaccination increase in binding to untransfected between IgG IgM . increase in binding to untransfected TE 0 between pre - post - vaccination samples is shown on left with blue symbols for IgG on right with black symbols for IgM . horizontal at y = 0 is expected result when no increased binding is observed . P value is derived from a two - , Wilcoxon matched pairs signed rank .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Change in antibody binding following vaccination against SARS-CoV-2. Binding of antibodies from sera assessed in the experiment shown in (A), is shown for two samples of serum from each of 7 donors. Data are plotted as in (A) with binding to spike-expressing cells on the vertical axis, and to untransfected cells on the horizontal, using blue symbols for samples from before vaccination, and red symbols for samples from 15-29 days after the first vaccination (but before any second vaccination). Black lines connect points corresponding to pre/post pairs of samples from each donor. Left plot shows results for IgM and right plot for IgG. C. Comparison of post-vaccination increase in binding to untransfected cells between IgG and IgM. The increase in binding to untransfected TE 0 cells between pre- and post-vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM. The horizontal line at y=0 is the expected result when no increased binding is observed. P value is derived from a two-tailed, Wilcoxon matched pairs signed rank test.


D . Comparison of spike - specific IgM , as measured by flow cytometry or ELISA . left two " FC " are results of flow cytometry , left verical axis shows of serum IgM binding to spike - expressing , vs binding to non - expressing control , as plottted in A . right two columns show spike - RBD - specific IgM as measured by an IgM - capture ELISA , right vertical axis shows optical densities ( OD ) . P values are calculated by two - way of variance , followed by Sidak ' s multiple comparisons . entire experiment was repeated three times , data shown are derived from third replicate .

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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Comparison of spike-specific IgM, as measured by flow cytometry or ELISA. The left two "FC" are the results of flow cytometry, and the left verical axis shows the ratio of serum IgM binding to spike-expressing cells, vs binding to non-expressing control cells, as plottted in A. The right two columns show spike-RBD-specific IgM as measured by an IgM-capture ELISA, and the right vertical axis shows the optical densities (OD). P values are calculated by two-way analysis of variance, followed by Sidak's multiple comparisons test. The entire experiment was repeated three times, and the data shown are derived from the third replicate.


of blood B . B from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent expressing spike - mCherry for 3 hours , then retrieved labeled with antibodies measured by flow cytometry . ( A ) UMAP algorithm ( 5000 randomly selected / sample ) was used to depict major B subsets clustered according to marker expression .

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

Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. (A) UMAP algorithm (5000 randomly selected cells/sample) was used to depict the major B cell subsets clustered according to marker expression.


of blood B . B from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent expressing spike - mCherry for 3 hours , then retrieved labeled with antibodies measured by flow cytometry . FlowSOM - B subpopulations are overlaid as a color dimension , colors of clusters are shown on left of ( B ) . B . showing mean population expression of markers used for UMAP visualization FlowSOM - clustering . Colors shown in legend on left are also used in UMAP representation in ( A ) .

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

Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. FlowSOM-based B cell subpopulations are overlaid as a color dimension, and the colors of the clusters are shown on the left of heatmap (B). B. Heatmap showing mean population expression levels of markers used for UMAP visualization and FlowSOM-clustering. Colors shown in the legend on the left are also used in the UMAP representation in (A).


of blood B . B from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent expressing spike - mCherry for 3 hours , then retrieved labeled with antibodies measured by flow cytometry . C . Gating strategy to define CD69 - high , mCherry - high , spike - capturing B ( i . e . , antigen capture activated , MACAC , red box ) . Dot plot shows CD69 mCherry for B from one of three donors gated by forward side scatter , negative for dye used to mark antigen donor .

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

Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. C. Gating strategy to define CD69-high, mCherry-high, spike-capturing B cells (i.e., membrane antigen capture activated cells, MACAC, red box). Dot plot shows CD69 and mCherry fluorescence for B cells from one of three donors gated by forward and side scatter, and negative for the dye used to mark the antigen donor cells.


of blood B . B from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads , exposed to adherent expressing spike - mCherry for 3 hours , then retrieved labeled with antibodies measured by flow cytometry . D . Relative fractions of different B subpopulations , from three donors , as shown in A - B , in B compared to within MACAC B . P values are on two - t - between groups . Correlation coefficients ( r ) were calculated from z - statistic of Wilcoxon - Mann - Whitney . A black horizontal represents median . Boxplots represent interquartile range ( IQR ) . Whiskers extend to farthest data point within a maximum of 1 . 5 × IQR . Every point represents one donor .

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

Phenotypes of blood B cells. B cells from 15 ml of peripheral blood from each of three convalescent donors were isolated by negative selection with magnetic beads, exposed to adherent cells expressing spike-mCherry for 3 hours, then retrieved and labeled with fluorescent antibodies and measured by flow cytometry. D. Relative fractions of different B cell subpopulations, from three donors, as shown in A-B, in all B cells compared to within MACAC B cells. P values are based on two-tailed t-tests between the groups. Correlation coefficients (r) were calculated from the z-statistic of the Wilcoxon-Mann-Whitney test. A black horizontal line represents the median. Boxplots represent the interquartile range (IQR). Whiskers extend to the farthest data point within a maximum of 1.5× IQR. Every point represents one donor.