( D ) Representative images of HE staining of the back tissues are shown on the left . The epidermal thickness measured using the scale in the microscope is shown on the right . ( E ) Representative images of Ki67 staining in back tissues are shown on the left . The number of Ki67 - positive cells per HPF in each mouse is shown on the right . ( F ) Representative images of HE staining of the ear tissues are shown on the left . The epidermis thickness measured using the scale in the microscope is shown on the right . ( G ) Representative images of Ki67 staining in ear tissues ( same specimen as in F ) are shown on the left . The number of Ki67 positive cells per HPF in each mouse is shown on the right . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent experiments . For back tissues , scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . For ear tissues , scale bar , 200 μm ( 100 × ) or 200 μm ( 200 × ) . Significant differences were tested using a two - tailed Student ' s t test Control , n = 3 , IMQ , n = 6 . * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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

(D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.


NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs . WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L - WT or NEDD4L - C943S mutants . cell signaling pathway activation induced by IL - 6 were detected by WB analyses Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent

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

NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. cell signaling pathway activation induced by IL-6 were detected by WB analyses Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


( G , H ) Primary keratinocytes were separated from the WT and Nedd4l KO mice . ( G ) WB analysis of the protein expression in IL - 6 - induced cell signaling pathway activation . ( H ) WB analysis of the proteins in the IL - 6 signaling pathway . Data information : Data are shown as the mean ± s . e . m . , and are representative of three independent

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary keratinocytes were separated from the WT and Nedd4l KO mice. (G) WB analysis of the protein expression in IL-6-induced cell signaling pathway activation. (H) WB analysis of the proteins in the IL-6 signaling pathway. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments


Six - to eight - weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment , the mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Representative images of Ki67 staining in back tissues are on the left . The number of Ki67 - positive cells per HPF is shown on the right . Data information : Data are shown as mean ± s . e . m . , and are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test C , control , n = 5 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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

Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


Six - to eight - weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment , the mice were injected intra - epidermally with 10 μg anti - GP130 antibody or with same dosage of isotype on every other day . Keratinocyte proliferation associated genes in the skin tissues are detected using real - time PCR analysis . Data information : Data are shown as mean ± s . e . m . , and are representative of two independent experiments . Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . Significant differences were tested using a two - tailed Student ' s t test D , control , n = 3 , IMQ , n = 6 . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 . NS , no significance .

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

Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.


( A ) Representative pictures of the expression of NEDD4L , GP130 and p - STAT3 in the epidermis of a normal skin section ( same specimen ) and sections from psoriasis patients . Isotype are IgG antibodies from same species served as negative control . ( B ) Semiquantitative scoring of NEDD4L , GP130 and p - STAT3 in the epidermis detected by IHC . Data information : Each symbol represents one patient Scale bar , 200 μm ( 100 × ) or 50 μm ( 400 × ) . All data are shown as the mean ± s . e . m . , and significant differences were analyzed using two - tailed Student ' s t test ( normal , n = 15 , psoriasis , n = 36 ) . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 .

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

(A) Representative pictures of the expression of NEDD4L, GP130 and p-STAT3 in the epidermis of a normal skin section (same specimen) and sections from psoriasis patients. Isotype are IgG antibodies from same species served as negative control. (B) Semiquantitative scoring of NEDD4L, GP130 and p-STAT3 in the epidermis detected by IHC. Data information: Each symbol represents one patient Scale bar, 200 μm (100×) or 50 μm (400×). All data are shown as the mean ± s.e.m., and significant differences were analyzed using two-tailed Student's t test (normal, n=15, psoriasis, n=36). * P＜0.05, ** P＜0.01, *** P＜0.001.


( F ) Full length Flag - GP130 , GP130 - aa 616 - 918 , GP130 - aa 616 - 889 , GP130 - aa 616 - 764 , GP130 - aa 616 - 734 were transfected with Myc - NEDD4L into HEK293T cells , and whole cell lysates were co - IP with ANTI - FLAG M2 magnetic beads , followed by WB with anti - Flag or anti - Myc antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Full length Flag-GP130, GP130-aa 616-918, GP130-aa 616-889, GP130-aa 616-764, GP130-aa 616-734 were transfected with Myc-NEDD4L into HEK293T cells, and whole cell lysates were co-IP with ANTI-FLAG M2 magnetic beads, followed by WB with anti-Flag or anti-Myc antibodies.


( C ) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L - WT , NEDD4L WW domain truncated mutant ( NEDD4L - ΔWW ) or its enzymatic activity mutant ( NEDD4L - C943S ) , the cells were followed by denaturation - IP with anti - GP130 antibody and WB analysis with anti - Ub antibodies .

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

(C) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies.


( E ) WB analysis of the polyubiquitination of GP130 in HEK293T cells co - transfected with full length Myc - NEDD4L , HA - Ub and Flag - GP130 or its lysine mutants ( K - R ) in the intracellular domain , followed by denaturation - IP with ANTI - FLAG M2 magnetic beads and WB analysis with anti - HA antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.


( A - D ) The serial dilutions of post - second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2 - Line19F - FFL virions . The serum samples were serially diluted , and the detection of antibodies were measured by optical density ( OD ) at 490 nm . End - point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control ( PBS immunized animal ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal).


( A ) Lung RSV titers ( PFU / gram of lung tissue ) were determined in individual lungs ( n = 5 mice per group ) collected at 5 days post - RSV infection . Results were determined and 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RSV titers (PFU/gram of lung tissue) were determined in individual lungs (n = 5 mice per group) collected at 5 days post-RSV infection. Results were determined and 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.


( A ) Serum samples collected from individual mouse ( M 1 - 5 ) immunized with F 110 - 136 peptide were tested for antibody binding against F - p27 ( 110 - 136 ) peptide or F 1 - 34 peptide ( control ) in ELISA .

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

(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA.


( B ) A549 cells were infected with RSV ( MOI = 0 . 1 ) for 16 h , and fixed . Cells were treated with mock control rabbit sera ( left panels ) , or rabbit anti - sera against F ( center panels ) , or against F - p27 ( 110 - 136 ) peptide ( right panels ) , followed by Alexa 594 conjugated anti - Rabbit IgG ( red ) . Nuclei are stained with DAPI ( blue ) . Scale bar = 10 µm . The number of cells positive for RSV - F ( middle panel ) and RSV - p27 ( right panel ) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the ' merge ' panel . The experiments were performed twice and variation between the two independent expeiremnts was < 6 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.


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 % .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) and in primary 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 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 - and Mfn2 - KO MEFs ( mean ± SE ; n = 3 , with 3 replicates / experiment ) .

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

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 cells , which was blocked upon the addition of 15 μM exogenous PtdEtn ( mean ± SD ; n = 3 , with 3 replicates / experiment ) .

O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 ] .

O O O O O O O O O O O O O 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 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 [ - ] ) , 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 ) .

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 cells , 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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 - and 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 , and lipid droplets were visualized ( example at left ) and counted ( right ) . Note significantly more lipid droplets in ApoE4 - treated cells ( mean ± SD , 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 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of sparsely cultured RPE cells transfected with the indicated siRNAs . Green , YAP . Blue , DAPI . Scale bar , 10 μm .

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

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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 cells were transfected with the indicated siRNAs , fractionated into cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins .

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

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

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 . 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 ) .

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 cytosolic and nuclear extracts , and analyzed by Western blotting for the indicated proteins .

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

O O O O O O O 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. 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 cells were analyzed by Western blotting for the indicated proteins .

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 the indicated proteins .

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


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

O O O O O O O O O O O O O O O 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 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 .

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 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 .

O O O O O O O O O O O O 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 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 the cells described in ( E ) ( n = 3 ) . Error bars indicate the S . E . M . ( * p < 0 . 05 , paired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O 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 . 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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 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 cells were transfected with the indicated combinations of DNAs , cultured for 24 hours , and 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 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 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 .

O O O O O O O O O O O O O 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 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 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 .

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

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

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


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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . 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 .

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

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


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 .

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

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


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 .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of soft agar assay results using cells 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 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 ) .

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 cells were transfected with the indicated DNAs and 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 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 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 .

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 and selected with 400 μg / ml of Zeocin for 4 days . Harvested cells were analyzed by Western blotting for the indicated proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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


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

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


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

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


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 .

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 the value for a given embryo .

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

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

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

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


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 .

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 levels detected by RNAscope on Lgr5 - DTReGFP proximal intestine .

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


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 ) .

O O O O O 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

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 and quantification of the number of traced clones per recombined surface at PN18 ( ileum ) . Each symbol indicates the value for a given mouse .

O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 and Rspo4 genes was analyzed on E15 , E16 . 5 and adult stages by RNAscope . Arrows evidence expressing cells .

O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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).


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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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

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


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

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

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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 staining of the 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 analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; 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 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 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

O O O O O O O O O O O O O O O O O O O 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 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 Heatmap of representative genes associated with BMP2 signaling pathway


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

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

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

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

O O O O O 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 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 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

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

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 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

O O O O O O O O O O O O O O O O O O O O O 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 staining 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 analysis of bone volume versus total tissue volume ( BV / TV ) . Results are shown as mean ± SEM ; 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 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 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

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

N 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 analysis of newly formed bone volume versus original bone volume ( % ) . Results are shown as mean ± SEM ; 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

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


B Immunoblot analysis showing decreased FLAG - Smad1 after USP34 knockdown

B Immunoblot analysis showing decreased FLAG-Smad1 after USP34 knockdown


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

O O O O O O O O O O O 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 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 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

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

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

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 , and Prx1 - Cre ( - ) indicates those from Usp34f / f control

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

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

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 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 ,

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 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

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

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 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

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

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

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

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

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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ,

O O O O O O 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 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 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

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

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 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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

A, B, Western 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 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 .

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

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 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 .

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

A, Western 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 ) 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 .

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

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 ) 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 .

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

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 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O 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 TEM . 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 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

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 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 ) .

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

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 ) 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)