Figure 5A . Co - depletion of ENSA and ARPP19 proteins in the MDA - MB - 231 cell line using specific siRNAs or control siRNAs against luciferase ( Luc ) . 72 h after transfection cells were starved for glucose for 1 h and re - stimulated with glucose for 15 min . Total cell extracts were collected and analyzed for the indicated antibodies . B . isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG - and HA - tagged versions of ENSA and ARPP19 phospho - mimetic mutants ( S67D and S62D ) or GFP as a control . Cells were treated with Dox to induce MASTL CRISPR / Cas9 - dependent knock - out , and 72 h later cells were treated as specified in A . The plot displays mean + SEM from 3 independent experiments . Significance determined by Student ' s t - test comparing ENSA / ARPP19 overexpression versus control cells ( ns , not significant ; * P < 0 . 05 ) . C . Control ( Dox − ) and MASTL knockout ( Dox + ) cells were treated with 5 µM fostriecin , 50 nM okadaic acid ( OA ) or vehicle ( Veh ) 10 min before insulin stimulation for 30 min . Total lysates were analyzed for the indicated antibodies and P - T308 - AKT signal was quantified . β - Actin was used as a loading control . The plot represents mean + SEM from 3 independent experiments , except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown . * P < 0 . 05 ; Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A. Co-depletion of ENSA and ARPP19 proteins in the MDA-MB-231 cell line using specific siRNAs or control siRNAs against luciferase (Luc). 72 h after transfection cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min. Total cell extracts were collected and analyzed for the indicated antibodies.B. isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG- and HA-tagged versions of ENSA and ARPP19 phospho-mimetic mutants (S67D and S62D) or GFP as a control. Cells were treated with Dox to induce MASTL CRISPR/Cas9-dependent knock-out, and 72 h later cells were treated as specified in A. The plot displays mean + SEM from 3 independent experiments. Significance determined by Student's t-test comparing ENSA/ARPP19 overexpression versus control cells (ns, not significant; *P<0.05).C. Control (Dox −) and MASTL knockout (Dox +) cells were treated with 5 µM fostriecin, 50 nM okadaic acid (OA) or vehicle (Veh) 10 min before insulin stimulation for 30 min. Total lysates were analyzed for the indicated antibodies and P-T308-AKT signal was quantified. β-Actin was used as a loading control. The plot represents mean + SEM from 3 independent experiments, except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown. *P<0.05; Student's t-test.


Figure 6A . MDA - MB - 231 cells were co - transduced with specific siRNAs against the PPP2R2A ( B55α ) and PPP2R2D ( B55δ ) transcripts ( B55αδ ) or against luciferase ( Luc ) as a control . Asynchronous cells were collected and blotted for the indicated antibodies . The histograms represent the quantifications of the specified phospho - residues . Plots show mean + SEM from 3 independent experiments . * P < 0 . 05 , Student ' s t - test . B . Immunodetection of the indicated antigens in control ( − ) and MASTL knockout ( + ) cells starved from glucose and re - stimulated with glucose for 15 min . C . siRNA - mediated genetic depletion of B55alpha and delta subunits ( B55αδ ) in control ( Dox − ) and MASTL - null ( Dox + ) cells . The phosphorylation status of the different proteins was scored in conditions of glucose re - addition . n = 3 independent experiments . The plot shows mean + SEM from 3 independent experiments . * P < 0 . 05 , 1 - way ANOVA . D . Immunoblot with the indicated antibodies in skeletal muscle ( gastrocnemius ) extracts . Mice were fasted overnight for 16 h , injected intraperitoneally with glucose ( 2 g / kg body ) , and sacrificed 30 min later for sample collection . Quantification of the relative fold change signal of total IRS1 , and phospho - S6K1 T389 ( normalized to total S6K1 level ; n = 6 Mastl ( + / + ) and 7 Mastl ( ∆ / ∆ ) ) . vinculin were used as a loading control . Data are mean + SEM ; * P < 0 . 05 , unpaired Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. MDA-MB-231 cells were co-transduced with specific siRNAs against the PPP2R2A (B55α) and PPP2R2D (B55δ) transcripts (B55αδ) or against luciferase (Luc) as a control. Asynchronous cells were collected and blotted for the indicated antibodies. The histograms represent the quantifications of the specified phospho-residues. Plots show mean + SEM from 3 independent experiments. *P<0.05, Student's t-test.B. Immunodetection of the indicated antigens in control (−) and MASTL knockout (+) cells starved from glucose and re-stimulated with glucose for 15 min.C. siRNA-mediated genetic depletion of B55alpha and delta subunits (B55αδ) in control (Dox −) and MASTL-null (Dox +) cells. The phosphorylation status of the different proteins was scored in conditions of glucose re-addition. n=3 independent experiments. The plot shows mean + SEM from 3 independent experiments. *P<0.05, 1-way ANOVA.D. Immunoblot with the indicated antibodies in skeletal muscle (gastrocnemius) extracts. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1, and phospho-S6K1 T389 (normalized to total S6K1 level; n=6 Mastl(+/+) and 7 Mastl(∆/∆)). vinculin were used as a loading control. Data are mean + SEM; *P<0.05, unpaired Student's t-test.


Figure 7A . MDA - MB - 231 cells were starved for 1 h and re - stimulated with glucose for 15 min and T194 phosphorylation was tested with a specific antibody . The plot shows the quantification of phospho - MASTL T194 ( n = 2 independent experiments ) . Data are mean + SEM from 3 independent experiments . ns , not significant ; Student ' s t - test . B . Cas9 - expressing control ( − ) cells or cells with CRIPSR / Cas9 - mediated TSC2 knockout ( sgTSC2 ) were treated with rapamycin ( Rap ) , Torin1 ( Tor ) , or vehicle as control ( V ) . MASTL - knockdown cells ( shMASTL ) were used as an additional control of the specificity of the antibody . Inhibitors were added 15 min before glucose re - stimulation for additional 15 min and total cell lysates were harvested for immunoblot . The plot shows the quantification of phospho - MASTL T194 as mean from 2 independent experiments . C . In vitro kinase assay using purified GST - human MASTL as a substrate , and recombinant mTORC1 and S6K1 kinases . Red arrowheads show the phosphorylation of MASTL in the presence of mTORC1 or S6K1 . Note the appearance of other phosporylation signals in mTORC1 and S6K1 reactions , independently of the presence of MASTL , which correspond to autophosphorylation of these kinases . Imperial staining shows the amount of GST - MASTL protein used as a substrate in each reaction . F . In vitro kinase activity of Mastl mutant isoforms . HEK - 293T cells were transiently transfected with the indicated GFP fusions of mouse Mastl mutants . Anti - GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 ( white circles ) or ENSA ( filled circles ) as a substrate . The graph shows the relative catalytic activity of the indicated mutants . Kinase activity of GFP - mMastl wild - type was set as 1 ( n = 3 independent experiments ) . Data are mean + SEM from 3 independent experiments . * * * P < 0 . 001 ; 1 - way ANOVA . G . isgMASTL MDA - MB - 231 cells were transduced with the indicated Mastl mutants or GFP alone as a control , and treated with doxycycline ( Dox ) to induce Mastl deletion . Cells were collected after glucose stimulation , and total lysates blotted for the indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7A. MDA-MB-231 cells were starved for 1 h and re-stimulated with glucose for 15 min and T194 phosphorylation was tested with a specific antibody. The plot shows the quantification of phospho-MASTL T194 (n=2 independent experiments). Data are mean + SEM from 3 independent experiments. ns, not significant; Student's t-test.B. Cas9-expressing control (−) cells or cells with CRIPSR/Cas9-mediated TSC2 knockout (sgTSC2) were treated with rapamycin (Rap), Torin1 (Tor), or vehicle as control (V). MASTL-knockdown cells (shMASTL) were used as an additional control of the specificity of the antibody. Inhibitors were added 15 min before glucose re-stimulation for additional 15 min and total cell lysates were harvested for immunoblot. The plot shows the quantification of phospho-MASTL T194 as mean from 2 independent experiments.C. In vitro kinase assay using purified GST-human MASTL as a substrate, and recombinant mTORC1 and S6K1 kinases. Red arrowheads show the phosphorylation of MASTL in the presence of mTORC1 or S6K1. Note the appearance of other phosporylation signals in mTORC1 and S6K1 reactions, independently of the presence of MASTL, which correspond to autophosphorylation of these kinases. Imperial staining shows the amount of GST-MASTL protein used as a substrate in each reaction.F. In vitro kinase activity of Mastl mutant isoforms. HEK-293T cells were transiently transfected with the indicated GFP fusions of mouse Mastl mutants. Anti-GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 (white circles) or ENSA (filled circles) as a substrate. The graph shows the relative catalytic activity of the indicated mutants. Kinase activity of GFP-mMastl wild-type was set as 1 (n=3 independent experiments). Data are mean + SEM from 3 independent experiments. ***P <0.001; 1-way ANOVA.G. isgMASTL MDA-MB-231 cells were transduced with the indicated Mastl mutants or GFP alone as a control, and treated with doxycycline (Dox) to induce Mastl deletion. Cells were collected after glucose stimulation, and total lysates blotted for the indicated antibodies.


Figure 1A ATX gene expression from publicly available datasets in NASH patients ( n = 9 ) compared to healthy controls ( n = 7 ) ( * P < 0 . 05 ) ; cirrhosis patients ( n = 40 ) compared to healthy controls ( n = 19 ) ( * * * P < 0 . 001 ) . Bars represent the mean ± SEM , statistical analysis was performed by two - tailed student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 denotes significance versus respective healthy controls . B Relative ATX gene expression in the MCD - diet - fed ( n = 9 ) compared to control mice ( n = 9 ) ( * * * P < 0 . 001 ) ; CCl4 ( n = 8 ) compared to control mice ( n = 5 ) ( * P < 0 . 05 ) . Bars represent the mean ± SEM , statistical analysis was performed by two - tailed student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 denotes significance versus respective healthy controls . C Representative images ( scale = 50μm ) of ATX stained liver sections from human NASH ( n = 5 ) compared to healthy controls ( n = 4 ) ; human cirrhosis ( n = 5 ) compared to healthy controls ( n = 4 ) . D Representative images ( scale = 50μm ) of ATX stained liver sections ( using AEC chromogen denoted by red color staining or DAB chromogen denoted by brown color staining ; nuclei were stained blue with hematoxylin ) from MCD - diet - fed NASH ( n = 9 ) and CCl4 - induced liver fibrosis ( n = 8 ) mouse models compared to respective controls ( n = 9 or n = 5 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A ATX gene expression from publicly available datasets in NASH patients (n=9) compared to healthy controls (n=7) (*P < 0.05); cirrhosis patients (n=40) compared to healthy controls (n=19) (***P < 0.001). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.B Relative ATX gene expression in the MCD-diet-fed (n=9) compared to control mice (n=9) (***P < 0.001); CCl4 (n=8) compared to control mice (n=5) (*P < 0.05). Bars represent the mean ± SEM, statistical analysis was performed by two-tailed student's t-test. *P < 0.05, ***P < 0.001 denotes significance versus respective healthy controls.C Representative images (scale=50μm) of ATX stained liver sections from human NASH (n=5) compared to healthy controls (n=4); human cirrhosis (n=5) compared to healthy controls (n=4).D Representative images (scale=50μm) of ATX stained liver sections (using AEC chromogen denoted by red color staining or DAB chromogen denoted by brown color staining; nuclei were stained blue with hematoxylin) from MCD-diet-fed NASH (n=9) and CCl4-induced liver fibrosis (n=8) mouse models compared to respective controls (n=9 or n=5).


Figure 2A Structural binding of type I ATX inhibitor PF8380 to ATX ; Potency ( IC50 ) of PF8380 for inhibiting the catalysis of LPC to LPA . Bars represent the mean + SEM , n = 3 . B Structural binding of type IV ATX inhibitor Cpd17 to ATX ; Potency ( IC50 ) of Cpd17 for inhibiting the catalysis of LPC to LPA . Bars represent the mean + SEM , n = 3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A Structural binding of type I ATX inhibitor PF8380 to ATX; Potency (IC50) of PF8380 for inhibiting the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3. B Structural binding of type IV ATX inhibitor Cpd17 to ATX; Potency (IC50) of Cpd17 for inhibiting the catalysis of LPC to LPA. Bars represent the mean + SEM, n=3.


Figure 3A Representative images ( scale = 10µm ) and quantification of HepG2 cells stained with oil - red - O after 48 hours of incubation with or without 200μM palmitate with or without 1µM of PF8380 or Cpd17 . Bars represent the mean + SEM , n = 3 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Dunnett ' s multiple comparison test . # # P < 0 . 01 denotes significance versus control ( non - palmitate ) and * P < 0 . 05 denotes significance versus vehicle ( palmitate ) ; ns = non - significant . B Representative images ( at 0h and 24h ) ( scale = 50µm ) , and quantitative analysis ( after 24h ) of migration by control ( M0 ) and LPS - and IFNγ - induced M1 RAW264 . 7 macrophages treated with medium alone , PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent the mean + SEM , n = 4 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # P < 0 . 05 denotes significance versus M0 ( non - stimulated macrophages ) and * P < 0 . 05 denotes significance versus vehicle ( LPS - and IFNγ - induced M1 macrophages ) ; ns = non - significant . C Relative gene expression ( normalized with GAPDH ) for CCL2 and iNOS in control ( M0 ) and LPS - and IFNγ - induced M1 RAW 264 . 7 macrophages treated with medium alone , PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent the mean + SEM , n = 4 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # # # # P < 0 . 0001 denotes significance versus M0 ( non - stimulated macrophages ) and * P < 0 . 05 , * * P < 0 . 01 denotes significance versus vehicle ( LPS - and IFNγ - induced M1 macrophages ) ; ns = non - significant . D Relative gene expression ( normalized with 18s RNA ) for CCL2 and iNOS in control ( M0 ) and LPS - induced PMA - differentiated human THP1 macrophages treated with medium alone , PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent the mean + SEM , n = 4 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # # P < 0 . 01 , # # # P < 0 . 001 denotes significance versus M0 ( non - stimulated macrophages ) and * * P < 0 . 01 , * * * P < 0 . 001 denotes significance versus vehicle ( LPS - and IFNγ - induced M1 macrophages ) ; ns = non - significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A Representative images (scale=10µm) and quantification of HepG2 cells stained with oil-red-O after 48 hours of incubation with or without 200μM palmitate with or without 1µM of PF8380 or Cpd17. Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. ##P < 0.01 denotes significance versus control (non-palmitate) and *P < 0.05 denotes significance versus vehicle (palmitate); ns=non-significant.B Representative images (at 0h and 24h) (scale=50µm), and quantitative analysis (after 24h) of migration by control (M0) and LPS- and IFNγ-induced M1 RAW264.7 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.C Relative gene expression (normalized with GAPDH) for CCL2 and iNOS in control (M0) and LPS- and IFNγ-induced M1 RAW 264.7 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus M0 (non-stimulated macrophages) and *P < 0.05, **P < 0.01 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.D Relative gene expression (normalized with 18s RNA) for CCL2 and iNOS in control (M0) and LPS-induced PMA-differentiated human THP1 macrophages treated with medium alone, PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=4. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ##P < 0.01, ###P < 0.001 denotes significance versus M0 (non-stimulated macrophages) and **P < 0.01, ***P < 0.001 denotes significance versus vehicle (LPS- and IFNγ-induced M1 macrophages); ns=non-significant.


Figure 4A Collagen I and α - SMA stained images ( scale = 50µm ) of control and TGFβ - activated LX2 cells with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) ( n = 3 ) . B Relative Collagen I , α - SMA and PDGFβR gene expression ( normalized with GAPDH ) in control and TGFβ - activated LX2 cells with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent the mean + SEM , n = 3 . Statistical analysis was performed by two - tailed student ' s t - test . # P < 0 . 05 , # # # P < 0 . 001 denotes significance versus control ( non - TGFβ cells ) and * * P < 0 . 01 denotes significance versus vehicle ( TGFβ - activated cells ) ; ns = non - significant . C , D Representative images ( at 0h and 24h ) ( scale = 100µm ) ( C ) and quantitative analysis ( after 24h ) ( D ) of migration by control and TGFβ - activated LX2 cells with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent the mean + SEM , n = 3 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # # # # P < 0 . 0001 denotes significance versus control ( non - TGFβ cells ) and * * P < 0 . 01 denotes significance versus vehicle ( TGFβ - activated cells ) ; ns = non - significant . E , F Representative images ( E ) and quantitative analysis ( F ) showing 3D collagen gel contractility of control and TGFβ - activated LX2 cells with or without PF8380 ( 1μM ) or Cpd17 ( 1μM ) . Bars represent the mean + SEM , n = 3 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # # # # P < 0 . 0001 denotes significance versus control ( non - TGFβ cells ) and * * P < 0 . 01 , * * * * P < 0 . 0001 denotes significance versus vehicle ( TGFβ - activated cells ) .

O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A Collagen I and α-SMA stained images (scale=50µm) of control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM) (n=3).B Relative Collagen I, α-SMA and PDGFβR gene expression (normalized with GAPDH) in control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by two-tailed student's t-test. #P < 0.05, ###P < 0.001 denotes significance versus control (non-TGFβ cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.C,D Representative images (at 0h and 24h) (scale=100µm) (C) and quantitative analysis (after 24h) (D) of migration by control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ cells) and **P < 0.01 denotes significance versus vehicle (TGFβ-activated cells); ns=non-significant.E,F Representative images (E) and quantitative analysis (F) showing 3D collagen gel contractility of control and TGFβ-activated LX2 cells with or without PF8380 (1μM) or Cpd17 (1μM). Bars represent the mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (non-TGFβ cells) and **P < 0.01, ****P < 0.0001 denotes significance versus vehicle (TGFβ-activated cells).


Figure 5B Top panel shows time - course stimulation to LPA and uninhibited or inhibited LPC - treated ATX followed by RhoA activation , measured as a YFP / CFP fluorescent ratio . Bottom panel depicts quantitation of the burst response to the stimulants . Bars represent the Mean + SEM , n = 3 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # P < 0 . 05 denotes significance versus control ( non - stimulated cells ) ; * P < 0 . 05 , * * * P < 0 . 001 denotes significance versus ATX + LPC treated cells . C Representative images and quantification showing Western - blot analysis for p - Akt and p - ERK normalized with β - actin in control , LPC , LPA , ATX , ATX + LPC , ATX + LPC + PF8380 , and ATX + LPC + Cpd17 stimulated LX2 cells . Bars represent the Mean + SEM , n = 3 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # P < 0 . 05 denotes significance versus control ( non - stimulated cells ) ; * P < 0 . 05 denotes significance versus ATX + LPC treated cells . D Representative images and quantification showing Western - blot analysis for LPAR1 on the cell surface and in lysate , normalized with β - actin in lysate , in LPC , ATX + LPC , ATX + LPC + PF8380 , and ATX + LPC + Cpd17 stimulated LX2 cells . Bars represent the Mean + SEM , n = 3 . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # P < 0 . 05 denotes significance versus control ( non - stimulated cells ) ; * P < 0 . 05 denotes significance versus ATX + LPC treated cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B Top panel shows time-course stimulation to LPA and uninhibited or inhibited LPC-treated ATX followed by RhoA activation, measured as a YFP/CFP fluorescent ratio. Bottom panel depicts quantitation of the burst response to the stimulants. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05, ***P <0.001 denotes significance versus ATX+LPC treated cells.C Representative images and quantification showing Western-blot analysis for p-Akt and p-ERK normalized with β-actin in control, LPC, LPA, ATX, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17 stimulated LX2 cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC treated cells.D Representative images and quantification showing Western-blot analysis for LPAR1 on the cell surface and in lysate, normalized with β-actin in lysate, in LPC, ATX+LPC, ATX+LPC+PF8380, and ATX+LPC+Cpd17 stimulated LX2 cells. Bars represent the Mean + SEM, n=3. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05 denotes significance versus control (non-stimulated cells); *P < 0.05 denotes significance versus ATX+LPC treated cells.


Figure 6B Liver weight to total body weight ratio from control ( n = 5 ) , CCl4 ( n = 5 ) and CCl4 + Cpd17 ( n = 4 ) mice . Bars represent the Mean + SEM . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # # # # P < 0 . 0001 denotes significance versus control ( healthy mice ) ; * * * P < 0 . 001 denotes significance versus CCl4 mice . C Serum ALT levels from control ( n = 5 ) , CCl4 ( n = 5 ) and CCl4 + Cpd17 ( n = 4 ) mice . Bars represent the Mean + SEM . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # # # P < 0 . 001 denotes significance versus control ( healthy mice ) ; P = 0 . 26 denotes significance versus CCl4 mice . D , E Representative images ( scale = 100μm ) ( D ) and quantitative analysis ( E ) of liver sections from control ( n = 5 ) , CCl4 ( n = 5 ) and CCl4 + Cpd17 ( n = 4 ) mice and stained with H & E , collagen - I and F4 / 80 . Bars represent the Mean + SEM . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # P < 0 . 05 , # # # P < 0 . 001 denotes significance versus control ( healthy mice ) ; P = 0 . 098 or P = 0 . 08 denotes significance versus CCl4 mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6B Liver weight to total body weight ratio from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ####P < 0.0001 denotes significance versus control (healthy mice); ***P < 0.001 denotes significance versus CCl4 mice.C Serum ALT levels from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. ###P < 0.001 denotes significance versus control (healthy mice); P = 0.26 denotes significance versus CCl4 mice.D,E Representative images (scale=100μm) (D) and quantitative analysis (E) of liver sections from control (n=5), CCl4 (n=5) and CCl4+Cpd17 (n=4) mice and stained with H&E, collagen-I and F4/80. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); P = 0.098 or P = 0.08 denotes significance versus CCl4 mice.


Figure 7D , Representative images ( scale = 100μm ) ( D ) of liver sections from control ( n = 5 ) , MCD ( n = 6 ) and MCD + Cpd17 ( n = 6 ) mice and stained with H & E , oil - red - O and Collagen I . quantitative analysis ( E ) of liver sections from control ( n = 5 ) , MCD ( n = 6 ) and MCD + Cpd17 ( n = 6 ) mice stained with oil - red - O and Collagen I . Bars represent the Mean + SEM . Statistical analysis was performed by one - way analysis of variance ( ANOVA ) with Bonferroni post - hoc test . # P < 0 . 05 , # # # P < 0 . 001 denotes significance versus control ( healthy mice ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 denotes significance versus MCD - diet fed mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7D, Representative images (scale=100μm) (D) of liver sections from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice and stained with H&E, oil-red-O and Collagen I.quantitative analysis (E) of liver sections from control (n=5), MCD (n=6) and MCD+Cpd17 (n=6) mice stained with oil-red-O and Collagen I. Bars represent the Mean + SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. #P < 0.05, ###P < 0.001 denotes significance versus control (healthy mice); *P < 0.05, **P < 0.01, ***P < 0.001 denotes significance versus MCD-diet fed mice.


Figure 1WT and CKO mice aged 4 weeks were selected and divided into 4 groups , and fed a normal diet until to 3 months or 18 months , respectively ( n = 10 ， there were 10 mice in each group , and 6 mice in each group were selected for detection at the end of the experiment ) . H Representative images of H & E staining in distal femora , scale bar , 50µm . I Representative images of TRAP staining in distal femora , scale bar , 50µm . J , K Quantification of number of osteoblasts ( J ) and number of osteoclasts ( K ) in distal femora ( n = 6 ) . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 and * * * P ＜ 0 . 001 ( Student ' s t test ) . WT and CKO mice aged 4 weeks were selected and divided into 4 groups , and fed a normal diet until to 3 months or 18 months , respectively ( n = 10 ， there were 10 mice in each group , and 6 mice in each group were selected for detection at the end of the experiment ) . L , M Representative images of Ocn + ( red ) and DAPI ( blue ) immunostaining ( L ) and quantification of Ocn + cells on trabecular ( M ) , scale bar , 50µm ( n = 6 ) . N , O Representative images of TRAP ( red ) and DAPI ( blue ) immunostaining ( N ) and quantification of TRAP cells on trabecular ( O ) , scale bar , 50µm ( n = 6 ) . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 and * * * P ＜ 0 . 001 ( Student ' s t test ) . WT and CKO mice aged 4 weeks were selected and divided into 4 groups , and fed a normal diet until to 3 months or 18 months , respectively ( n = 10 ， there were 10 mice in each group , and 6 mice in each group were selected for detection at the end of the experiment ) . R , S The serum levels of CTX ( R ) and PINP ( S ) ( n = 6 ) . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 and * * * P ＜ 0 . 001 ( Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H Representative images of H&E staining in distal femora, scale bar, 50µm. I Representative images of TRAP staining in distal femora, scale bar, 50µm. J, K Quantification of number of osteoblasts (J) and number of osteoclasts (K) in distal femora (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). L, M Representative images of Ocn+ (red) and DAPI (blue) immunostaining (L) and quantification of Ocn+ cells on trabecular (M), scale bar, 50µm (n=6). N, O Representative images of TRAP (red) and DAPI (blue) immunostaining (N) and quantification of TRAP cells on trabecular (O), scale bar, 50µm (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).WT and CKO mice aged 4 weeks were selected and divided into 4 groups, and fed a normal diet until to 3 months or 18 months, respectively (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). R, S The serum levels of CTX (R) and PINP (S) (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).


Figure 2BMT was conducted on 12 - month - old CKO and WT mice with normal diet , and then the recipients from each group were performed for analysis 3 months after interventions ( n = 10 ， there were 10 mice in each group , and 6 mice in each group were selected for detection at the end of the experiment ) . H Representative images of H & E staining ( upper panel ) , TRAP staining ( middle panel ) and calcein double - labeling ( lower panel ) in distal femora , scale bar , 100 µm for H & E staining . 50 µm for TRAP staining , 50μm for calcein double - labeling . I , J Quantification of number for osteoblasts ( I ) and number of osteoclasts ( J ) in distal femora ( n = 6 ) . K Quantification of bone formation rate per bone surface ( BFR / BS ) ( n = 6 ) . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 and * * * P ＜ 0 . 001 ( Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2BMT was conducted on 12-month-old CKO and WT mice with normal diet, and then the recipients from each group were performed for analysis 3 months after interventions (n=10，there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H Representative images of H&E staining (upper panel), TRAP staining (middle panel) and calcein double-labeling (lower panel) in distal femora, scale bar, 100 µm for H&E staining. 50 µm for TRAP staining, 50μm for calcein double-labeling. I, J Quantification of number for osteoblasts (I) and number of osteoclasts (J) in distal femora (n=6). K Quantification of bone formation rate per bone surface (BFR/BS) (n=6). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01 and ***P＜0.001 (Student's t test).


Figure 3in situ myeloid specific MYDGF overexpression in bone marrow was performed on CKO mice aged 9 months . Micro - CT analysis and histomorphometric analysis of distal femora were performed after 3 months intervention . H Representative images of H & E in distal femora , scale bar , 100 µm . I Quantification of number for osteoblasts in distal femora ( n = 6 ) . J Representative images of TRAP staining in distal femora , scale bar , 50µm K Quantification of number for osteoclasts in distal femora ( n = 6 ) . L Representative images of calcein double - labeling , scale bar , 50μm . M Quantification of bone formation rate per bone surface ( BFR / BS ) ( n = 6 ) . Data information : Results are shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3in situ myeloid specific MYDGF overexpression in bone marrow was performed on CKO mice aged 9 months. Micro-CT analysis and histomorphometric analysis of distal femora were performed after 3 months intervention. H Representative images of H&E in distal femora, scale bar, 100 µm. I Quantification of number for osteoblasts in distal femora (n=6). J Representative images of TRAP staining in distal femora, scale bar, 50µm K Quantification of number for osteoclasts in distal femora (n=6). L Representative images of calcein double-labeling, scale bar, 50μm. M Quantification of bone formation rate per bone surface (BFR/BS) (n=6). Data information: Results are shown as mean ±SEM. *P<0.05, **P<0.01, ***P<0.001 (Student's t test).


Figure 4A - C Primary BMMs from WT and CKO mice were induced with M - CSF ( 100 ng / ml ) and RANKL ( 50 ng / ml ) for osteoclast differentiation . Representative TRAP staining images ( A ) , scale bar , 200μm . Quantification of osteoclast number ( B ) percentage of osteoclast area ( C ) . Results are shown as mean ± SEM , * * * P < 0 . 001 ( Student ' s t test ) . Data information : Each experiment was repeated 5 times . D - F BMMs with treatment of conditioned medium of BMCs from WT or CKO mice for 7 days of osteoclast differentiation with or without RANKL ( 50 ng / ml ) . M - CSF ( 100 ng / ml ) was present in all settings . Representative TRAP staining images , upper panel represent cultured without RANKL , lower panel represent cultured with RANKL ( 50 ng / ml ; D ) , scale bar , 200μm . Quantification of osteoclast number of TRAP - positive cells ( E ) and percentage of osteoclast area ( F ) . Results are shown as mean ± SEM , * P < 0 . 05 ( Student ' s t test ) . Data information : Each experiment was repeated 5 times . N - P BMMs were co - cultured with osteoclasts in presence of 1 , 25 ( OH ) 2D3 and PGE2 with conditioned medium of BMCs from WT or CKO mice for 5 days . Representative images of osteoclasts co - cultured with osteoblasts , scale bar , 200μm ( N ) . Quantification of osteoclast number ( O ) and percentage of osteoclast area ( P ) . Results are shown as mean ± SEM , * P < 0 . 05 ( Student ' s t test ) . Data information : Each experiment was repeated 5 times .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A-C Primary BMMs from WT and CKO mice were induced with M-CSF (100 ng/ml) and RANKL (50 ng/ml) for osteoclast differentiation. Representative TRAP staining images (A), scale bar, 200μm. Quantification of osteoclast number (B) percentage of osteoclast area (C). Results are shown as mean ± SEM, ***P<0.001 (Student's t test). Data information: Each experiment was repeated 5 times.D-F BMMs with treatment of conditioned medium of BMCs from WT or CKO mice for 7 days of osteoclast differentiation with or without RANKL (50 ng/ml). M-CSF (100 ng/ml) was present in all settings. Representative TRAP staining images, upper panel represent cultured without RANKL, lower panel represent cultured with RANKL (50 ng/ml; D), scale bar, 200μm. Quantification of osteoclast number of TRAP-positive cells (E) and percentage of osteoclast area (F). Results are shown as mean ± SEM, *P<0.05 (Student's t test). Data information: Each experiment was repeated 5 times.N-P BMMs were co-cultured with osteoclasts in presence of 1,25(OH)2D3 and PGE2 with conditioned medium of BMCs from WT or CKO mice for 5 days. Representative images of osteoclasts co-cultured with osteoblasts, scale bar, 200μm (N). Quantification of osteoclast number (O) and percentage of osteoclast area (P). Results are shown as mean ± SEM, *P < 0.05 (Student's t test). Data information: Each experiment was repeated 5 times.


Figure 5G - I Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP staining assay at 7 days or ARS staining assay at 14 days . Representative images of ALP staining and ARS staining of osteoblasts , scale bar , 100μm ( G ) . Quantitative analysis of ALP activity ( H ) and calcium mineralization ( I ) . Data information : Results are shown as mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) . Each experiment was repeated 5 times . J - L Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of 0ng / ml ( vehicle ) or 100ng / ml rMYDGF . Representative images of ALP staining and ARS staining of osteoblasts , scale bar , 100μm ( J ) . Quantitative analysis of ALP activity ( K ) and calcium mineralization ( L ) . Data information : Results are shown as mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) . Each experiment was repeated 5 times .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5G-I Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of conditioned medium of BMCs from WT or CKO mice for ALP staining assay at 7 days or ARS staining assay at 14 days. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (G). Quantitative analysis of ALP activity (H) and calcium mineralization (I). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.J-L Primary calvarial osteoblasts cultured in osteogenesis induction medium with treatment of 0ng/ml (vehicle) or 100ng/ml rMYDGF. Representative images of ALP staining and ARS staining of osteoblasts, scale bar, 100μm (J). Quantitative analysis of ALP activity (K) and calcium mineralization (L). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001(Student's t test). Each experiment was repeated 5 times.


Figure 6Bone defects models were conducted on young ( 3 - month - old ) and aged ( 18 - month - old ) mice , AAV - GFP or AAV - MYDGF intra - marrow injection once 2 weeks before bone defects surgery on young and aged mice , and the defects area was analyzed after 2 weeks ( n = 10 , there were 10 mice in each group , and 5 mice in each group were selected for detection at the end of the experiment ) . C , D Quantitative micro - CT analysis of BMD ( C ) and BV / TV ( D ) of the regenerated bone in calvarial defects ( n = 5 ) . Data information : Results are shown as mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) . Bone defects models were conducted on young ( 3 - month - old ) and aged ( 18 - month - old ) mice , AAV - GFP or AAV - MYDGF intra - marrow injection once 2 weeks before bone defects surgery on young and aged mice , and the defects area was analyzed after 2 weeks ( n = 10 , there were 10 mice in each group , and 5 mice in each group were selected for detection at the end of the experiment ) . I , J Quantitative micro - CT analysis of BMD ( I ) and BV / TV ( J ) of the regenerated bone in calvarial defects ( n = 5 ) . Data information : Results are shown as mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Bone defects models were conducted on young (3-month-old) and aged (18-month-old) mice, AAV-GFP or AAV-MYDGF intra-marrow injection once 2 weeks before bone defects surgery on young and aged mice, and the defects area was analyzed after 2 weeks (n=10, there were 10 mice in each group, and 5 mice in each group were selected for detection at the end of the experiment). C, D Quantitative micro-CT analysis of BMD (C) and BV/TV (D) of the regenerated bone in calvarial defects (n=5). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 (Student's t test).Bone defects models were conducted on young (3-month-old) and aged (18-month-old) mice, AAV-GFP or AAV-MYDGF intra-marrow injection once 2 weeks before bone defects surgery on young and aged mice, and the defects area was analyzed after 2 weeks (n=10, there were 10 mice in each group, and 5 mice in each group were selected for detection at the end of the experiment). I, J Quantitative micro-CT analysis of BMD (I) and BV/TV (J) of the regenerated bone in calvarial defects (n=5). Data information: Results are shown as mean ± SEM, *P<0.05, **P<0.01, ***P<0.001 (Student's t test).


Figure 7OVX mice and 18 - month - old male mice serve as the model of osteoporosis , AAV - MYDGF or AAV - GFP were injected into bone marrow cavity every 3 weeks for 12 weeks ( n = 10 , there were 10 mice in each group , and 6 mice in each group were selected for detection at the end of the experiment ) . Micro - CT reconstruction , H & E staining of trabecular bone from distal femurs in Sham + GFP , OVX + GFP and OVX + MYDGF groups ( n = 6 ) . Representative images of micro - CT reconstruction , scale bar , 2mm ( A ) . Representative images of H & E staining in distal femora , scale bar , 500μm for upper panel , 100μm for lower panel ( D ) . OVX mice and 18 - month - old male mice serve as the model of osteoporosis , AAV - MYDGF or AAV - GFP were injected into bone marrow cavity every 3 weeks for 12 weeks ( n = 10 , there were 10 mice in each group , and 6 mice in each group were selected for detection at the end of the experiment ) . H & E staining of trabecular bone from distal femurs in aged mice with AAV - GFP or AAV - MYDGF ( n = 6 ) . Representative images of H & E staining in distal femora , scale bar , 500μm for upper panel , 100μm for lower panel ( J ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7OVX mice and 18-month-old male mice serve as the model of osteoporosis, AAV-MYDGF or AAV-GFP were injected into bone marrow cavity every 3 weeks for 12 weeks (n=10, there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). Micro-CT reconstruction, H&E staining of trabecular bone from distal femurs in Sham +GFP, OVX +GFP and OVX + MYDGF groups (n=6). Representative images of micro-CT reconstruction, scale bar, 2mm (A). Representative images of H&E staining in distal femora, scale bar, 500μm for upper panel,100μm for lower panel (D).OVX mice and 18-month-old male mice serve as the model of osteoporosis, AAV-MYDGF or AAV-GFP were injected into bone marrow cavity every 3 weeks for 12 weeks (n=10, there were 10 mice in each group, and 6 mice in each group were selected for detection at the end of the experiment). H&E staining of trabecular bone from distal femurs in aged mice with AAV-GFP or AAV-MYDGF (n=6). Representative images of H&E staining in distal femora, scale bar, 500μm for upper panel,100μm for lower panel (J).


Figure 8A qRT - PCR analysis for Nfatc1 , Ctsk , c - Fos , Mmp9 and Acp5 mRNA expression in BMMs cultured in osteoclastogenesis induction medium for 48 hours . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 ( Student ' s t test ) . Representative WB images of IKBα , P65 , PKCβ treated with or without rMYDGF for RANKL - induced osteoclastogenesisM BMMs were treated with control , RANKL or MG132 for osteoclastogenesis , then with or without rMYDGF for 24 hours . IgG , P65 and histone antibodies were used to ChIP and RT - PCR was performed to determine Nfatc1 promoters . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 ( Student ' s t test ) . O , P Quantification of osteoclast number ( O ) and percentage of osteoclast area ( P ) . Data information : Results are shown as mean ± SEM . * P ＜ 0 . 05 , * * P ＜ 0 . 01 , * * * P ＜ 0 . 001 ( Student ' s t test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8A qRT-PCR analysis for Nfatc1, Ctsk, c-Fos, Mmp9 and Acp5 mRNA expression in BMMs cultured in osteoclastogenesis induction medium for 48 hours. Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).Representative WB images of IKBα, P65, PKCβ treated with or without rMYDGF for RANKL -induced osteoclastogenesisM BMMs were treated with control, RANKL or MG132 for osteoclastogenesis, then with or without rMYDGF for 24 hours. IgG, P65 and histone antibodies were used to ChIP and RT-PCR was performed to determine Nfatc1 promoters. Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).O, P Quantification of osteoclast number (O) and percentage of osteoclast area (P). Data information: Results are shown as mean ± SEM. *P＜0.05, **P＜0.01, ***P＜0.001 (Student's t test).


Figure 9Representative WB images of Smad2 / 3 , mTOR , STAT3 ( S ) , STAT3 ( Y ) , MAPK1 / 3 and JAK1 / 2 proteins expressionH The mRNA levels of Runx2 in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated interventions . I , J Quantitative analysis of ALP activity ( I ) and calcium mineralization ( J ) in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated intervention . Data information : Results are shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) . O , P Representative WB diagram ( O ) and the protein levels of STAT3 ( S ) and MAPK1 / 3 expression ( P ) . Data information : Results are shown as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t test ) .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9Representative WB images of Smad2/3, mTOR, STAT3(S), STAT3(Y), MAPK1/3 and JAK1/2 proteins expressionH The mRNA levels of Runx2 in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated interventions. I, J Quantitative analysis of ALP activity (I) and calcium mineralization (J) in BMSCs cultured in osteogenesis medium induction for 48 hours with indicated intervention. Data information: Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001(Student's t test).O, P Representative WB diagram (O) and the protein levels of STAT3(S) and MAPK1/3 expression (P). Data information: Results are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001(Student's t test).


Figure 1A Phase contrast images of Mabs cultured into PF hydrogels ( 5 days ) : cells formed a thick three - dimensional myotube network with numerous thick myofibres ( arrows ) . Scale bar : 50 μm . B Confocalimmunofluorescence analysis with an antibody against myosin heavy chain ( MyHC ) ( red ) and SYTOX green which labels nuclei ( green ) , showing multi - nucleated , mature musclefibres ( arrows ) developed from Mabs in PF ( 5 days ) . Scale bar : 50 μm . C , D Western blot and densitometry analysis of different protein extracts of Mabs differentiated inside PF constructs ( PF ) showing expression levels comparable to adult TA ( Ctrl ) . Group of n = 6 of Mabs differentiated inside PF and host TA has been tested , densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle markers is presented as means ± standard error .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A Phase contrast images of Mabs cultured into PF hydrogels (5 days): cells formed a thick three-dimensional myotube network with numerous thick myofibres (arrows). Scale bar: 50 μm.B Confocalimmunofluorescence analysis with an antibody against myosin heavy chain (MyHC) (red) and SYTOX green which labels nuclei (green), showing multi-nucleated, mature musclefibres (arrows) developed from Mabs in PF (5 days). Scale bar: 50 μm.C, D Western blot and densitometry analysis of different protein extracts of Mabs differentiated inside PF constructs (PF) showing expression levels comparable to adult TA (Ctrl). Group of n = 6 of Mabs differentiated inside PF and host TA has been tested, densitometric analysis from n = 3 different Western blots gauging the level of expression of muscle markers is presented as means ± standard error.


Figure 2A Gross morphology of the PF implant containing Mabs - nLacZ / PlGF immediately after implantation on the surface of underlying TA ( arrow ) . B , C Artificial muscles ( arrows ) developed within 8 weeks over the host TA surface ( arrowheads ) , encapsulated by the host perimysium ( B ) and released after tendon resection of the TA ( C ) . D The isolated artificial muscle from the TA surface ( arrow ) is comparable in size to the underlying host TA ( arrowhead ) . E Stereomicroscopy images revealing blood vessel formation in the artificial muscle . Scale bar : 100 μm . F X - Gal and H & amp ; amp ; E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink , injected into the femoral artery immediately before sacrifice . Scale bar : 20 μm . G Western blot analysis of protein extracts from artificial muscles ( PF ) and host TA ( Ctrl ) reveals remarkable expression of muscle - and vessel - specific proteins ( n = 5 ) . I - L X - Gal and H & amp ; amp ; E staining of artificial muscle sections at 4 ( I , K ) and 8 weeks ( J , L ) after implantation , showing donor origin of muscle cells ( nLacZ + nuclei ) and correlation between implant duration and myofibre maturation ( I , J images are the results of the collage of several photos ) . Scale bar : ( I , J ) 200 μm , ( K , L ) 100 μm . M - P Immunofluorescence analysis on adjacent sections revealing laminin organization ( green ) and myosin heavy chain ( MyHC ) expression ( red ) at 4 weeks ( M ) and 8 weeks ( N ) after grafting ( M , N displayed items are obtained from a multi - photo collage ) ; DAPI staining ( blue ) shows centrally located nuclei in developing fibres ( O ) and peripheral nuclei in mature myofibres ( P ) . Scale bar : ( M , N ) 200 μm , ( O , P ) 50 μm . Q Confocal isosurface image of neurofilament ( green ) and bungarotoxin ( red ) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque . Scale bar : 10 μm . R Immunofluorescence against smooth muscle actin ( blue ) , laminin ( green ) and VE - Cad ( red ) superimposed on phase contrast image shows blood vessels ( arrows ) adjacent to the myofibres in the artificial muscle section . Scale bar : 20 μm . S X - Gal staining on mature artificial muscle ( arrowhead ) and the underneath host TA ( arrow ) revealing no tendon development at the extremity of the artificial tissue ; implanted LacZ - positive Mabs are present within the host TA . Scale bar : 200 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A Gross morphology of the PF implant containing Mabs-nLacZ/PlGF immediately after implantation on the surface of underlying TA (arrow).B, C Artificial muscles (arrows) developed within 8 weeks over the host TA surface (arrowheads), encapsulated by the host perimysium (B) and released after tendon resection of the TA (C).D The isolated artificial muscle from the TA surface (arrow) is comparable in size to the underlying host TA (arrowhead).E Stereomicroscopy images revealing blood vessel formation in the artificial muscle. Scale bar: 100 μm.F X-Gal and H&amp;E staining on artificial muscle section revealing vessel connection with host vasculature by Indian ink, injected into the femoral artery immediately before sacrifice. Scale bar: 20 μm.G Western blot analysis of protein extracts from artificial muscles (PF) and host TA (Ctrl) reveals remarkable expression of muscle- and vessel-specific proteins (n = 5).I-L X-Gal and H&amp;E staining of artificial muscle sections at 4 (I, K) and 8 weeks (J, L) after implantation, showing donor origin of muscle cells (nLacZ+ nuclei) and correlation between implant duration and myofibre maturation (I, J images are the results of the collage of several photos). Scale bar: (I, J) 200 μm, (K, L) 100 μm.M-P Immunofluorescence analysis on adjacent sections revealing laminin organization (green) and myosin heavy chain (MyHC) expression (red) at 4 weeks (M) and 8 weeks (N) after grafting (M, N displayed items are obtained from a multi-photo collage); DAPI staining (blue) shows centrally located nuclei in developing fibres (O) and peripheral nuclei in mature myofibres (P). Scale bar: (M, N) 200 μm, (O, P) 50 μm.Q Confocal isosurface image of neurofilament (green) and bungarotoxin (red) immunofluorescence in the artificial muscle section shows a mature neuromuscular plaque. Scale bar: 10 μm.R Immunofluorescence against smooth muscle actin (blue), laminin (green) and VE-Cad (red) superimposed on phase contrast image shows blood vessels (arrows) adjacent to the myofibres in the artificial muscle section. Scale bar: 20 μm.S X-Gal staining on mature artificial muscle (arrowhead) and the underneath host TA (arrow) revealing no tendon development at the extremity of the artificial tissue; implanted LacZ-positive Mabs are present within the host TA. Scale bar: 200 μm.


Figure 3A Electron microscopy with Bluo - Gal staining reveals a LacZ - positive donor nucleus ( arrow ) peripheral to transversely sectioned sarcomeres of the artificial muscle . Scale bar : 1 μm . B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization . Scale bar : 0 . 5 μm . C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre . Scale bar : 10 μm . D X - Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ - positive donor nuclei . Scale bar : 100 μm . E Immunofluorescence analysis of LacZ - positive nuclei ( green ) and dystrophin ( red ) in an individual muscle fibre after functional analysis . Scale bar : 10 μm . F The same fibre shown in ( E ) is counterstained with DAPI to show that the interstitial nuclei ( blue ) are of donor origin . Scale bar : 10 μm . G - I Specific force ( Po / CSA ) ( G ) , maximum shortening velocity ( Vo ) ( H ) , and cross - sectional area ( CSA ) ( I ) of single fibres ( n = 153 ) from artificial muscles and control ( CTRL ) muscles ( n = 103 ) . J Silver - stained SDS - PAGE ( 8 % ) gel revealing myosin heavy chain ( MyHC ) - 2B isoform expression in two single fibres of artificial muscles ( lanes 1 , 2 ) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in ( K ) ( lane 3 ) . K Comassie blue - stained SDS - PAGE ( 8 % ) of bulk samples of two artificial muscles ( lanes 1 , 2 ) and of a bulk control muscle sample ( lane 3 ) , as in ( J ) , showing different MyHC isoform expression .

O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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 3A Electron microscopy with Bluo-Gal staining reveals a LacZ-positive donor nucleus (arrow) peripheral to transversely sectioned sarcomeres of the artificial muscle. Scale bar: 1 μm.B Longitudinal EM section of an artificial muscle showing mature sarcomere myofibril organization. Scale bar: 0.5 μm.C A single fibre from an artificial muscle showing a striation pattern typical of a mature myofibre. Scale bar: 10 μm.D X-Gal staining of freshly dissected artificial muscle bundle at 8 weeks showing abundant LacZ-positive donor nuclei. Scale bar: 100 μm.E Immunofluorescence analysis of LacZ-positive nuclei (green) and dystrophin (red) in an individual muscle fibre after functional analysis. Scale bar: 10 μm.F The same fibre shown in (E) is counterstained with DAPI to show that the interstitial nuclei (blue) are of donor origin. Scale bar: 10 μm.G-I Specific force (Po/CSA) (G), maximum shortening velocity (Vo) (H), and cross-sectional area (CSA) (I) of single fibres (n = 153) from artificial muscles and control (CTRL) muscles (n = 103).J Silver-stained SDS-PAGE (8%) gel revealing myosin heavy chain (MyHC)-2B isoform expression in two single fibres of artificial muscles (lanes 1, 2) and a bulk muscle control sample from soleus muscle containing all four adult MyHC isoforms indicated in (K) (lane 3).K Comassie blue-stained SDS-PAGE (8%) of bulk samples of two artificial muscles (lanes 1, 2) and of a bulk control muscle sample (lane 3), as in (J), showing different MyHC isoform expression.


Figure 4A , B Gross morphology of the TA injury at 6 months after massive muscle ablation , revealing the new artificial TA regeneration ( arrow ) when grafted with PF - embedded Mabs - PlGF ( B ) and absence of TA muscle ( arrow ) when treated with empty PF as a control ( A ) . C PF - embedded Mabs - PlGF - derived artificial TA ( right ) showing comparable size to the wild - type normal TA ( left ) . D - O Immunofluorescence against myosin heavy chain ( MyHC ) ( red ) and laminin ( green ) on mice TA cross sections at early ( 10 days ) ( D - I ) and later stage ( 6 months ) ( J - O ) after massive removal of the muscle ( D , G , J , M are the results of a collage of several photos ) . TA regeneration at early stages showed very few residual myofibres after implantation of acellular PF in place of the ablated TA ( white box in D ) , an enlarged view ( E ) , and DAPI - counterstained view ( F ) reveals the infiltration of undifferentiated mono - nucleated cells . Conversely , a PF - embedded Mabs - PlGF graft ( G ) demonstrated early - stage regeneration with several MyHC - positive ( red ) regenerating myofibres surrounded by laminin ( green ) ( H ) , most of these ( arrows ) being centre - nucleated ( DAPI labelled in I ) . At the later stages , acellular PF implanted mice ( J ) showed a very small portion of muscle tissue likely derived from residual myofibres from TA removal , higher magnification ( K ) revealed very few mature myofibres with peripheral nuclei ( DAPI labelled in L ) . Contrary , the PF - embedded Mabs - PlGF treatment ( M ) showed a complete replacement of the ablated TA with the recovered native structure of MyHC - positive myofibres surrounded by laminin ( N ) , some of these ( arrows ) still being centre nucleate ( DAPI labelled in O ) . Scale bar : ( D , G , J , M ) 500 μm , ( E , F , H , I , K , L , N , O ) 100 μm . P - R Functional analysis demonstrated the effective ability of the PF and Mabs - PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury . ( P ) The fore - hind limb grip test diagram reveals functional grab force recovery for mice treated with PF - embedded Mabs - PlGF ( TA ABL - PF + MABS ) after only 4 weeks post - TA injury , being comparable with not - ablated wild - type ( WT ) mice and significantly higher than control ablated mice ( TA ABL - CONT ) or mice implanted with void PF ( TA ABL - PF CONT ) ( n = 10 mice per group ) . ( Q , R ) The treadmill analysis of the treated mice relative to wild - type not - ablated ( wt ) shows both exhaustion time and distance indicative of a full recovery of motor functionality already at 3 weeks after TA ablation with an increase in time and distance run for mice treated with PF - embedded Mabs - PlGF , while control mice ( TA ABL - CONT or TA ABL - PF CONT ) showed a much lower resistance ( n = 10 mice per group ) . The values shown in the diagrams are expressed as means ± standard error and statistical significance was tested using Student ' s t - test ( P < 0 . 05 was considered significant ) . S - Z Morphological analysis demonstrated the effective ability of the PF and Mabs - PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury . H & amp ; amp ; E staining of TA injury in mice by cross section at an early ( 10 days ) ( S - V ) and late ( 6 months ) ( W - Z ) time point after ablation ( in S , U , W , Y displayed items are obtained from a multi - photo collage ) . At the early time point , the acellular PF - treated mice revealed few mature muscle fibres that survive the massive ablation ( box in S ) signs of continued muscle degeneration ( arrows in T ) , whereas PF - embedded Mabs implanted mice showed remarkable muscle regeneration indicated by centre - nucleated myofibres ( arrows in V ) . At later stages , the control mice grafted with acellular PF showed an almost devoid TA ( W ) with very few muscle fibres ( X ) , in contrast to PF - embedded Mabs , which exhibited a complete muscle replacement generating an artificial TA ( Y ) , enlarged view ( Z ) proved the complete and mature organization of the newly formed muscle showing capillary and blood vessel around myofibres ( arrows ) . Scale bar : ( S , U , W , Y ) 500 μm , ( T , V , X , Z ) 50 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A, B Gross morphology of the TA injury at 6 months after massive muscle ablation, revealing the new artificial TA regeneration (arrow) when grafted with PF-embedded Mabs-PlGF (B) and absence of TA muscle (arrow) when treated with empty PF as a control (A).C PF-embedded Mabs-PlGF-derived artificial TA (right) showing comparable size to the wild-type normal TA (left).D-O Immunofluorescence against myosin heavy chain (MyHC) (red) and laminin (green) on mice TA cross sections at early (10 days) (D-I) and later stage (6 months) (J-O) after massive removal of the muscle (D, G, J, M are the results of a collage of several photos). TA regeneration at early stages showed very few residual myofibres after implantation of acellular PF in place of the ablated TA (white box in D), an enlarged view (E), and DAPI-counterstained view (F) reveals the infiltration of undifferentiated mono-nucleated cells. Conversely, a PF-embedded Mabs-PlGF graft (G) demonstrated early-stage regeneration with several MyHC-positive (red) regenerating myofibres surrounded by laminin (green) (H), most of these (arrows) being centre-nucleated (DAPI labelled in I). At the later stages, acellular PF implanted mice (J) showed a very small portion of muscle tissue likely derived from residual myofibres from TA removal, higher magnification (K) revealed very few mature myofibres with peripheral nuclei (DAPI labelled in L). Contrary, the PF-embedded Mabs-PlGF treatment (M) showed a complete replacement of the ablated TA with the recovered native structure of MyHC-positive myofibres surrounded by laminin (N), some of these (arrows) still being centre nucleate (DAPI labelled in O). Scale bar: (D, G, J, M) 500 μm, (E, F, H, I, K, L, N, O) 100 μm.P-R Functional analysis demonstrated the effective ability of the PF and Mabs-PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury. (P) The fore-hind limb grip test diagram reveals functional grab force recovery for mice treated with PF-embedded Mabs-PlGF (TA ABL-PF+MABS) after only 4 weeks post-TA injury, being comparable with not-ablated wild-type (WT) mice and significantly higher than control ablated mice (TA ABL-CONT) or mice implanted with void PF (TA ABL-PF CONT) (n = 10 mice per group). (Q, R) The treadmill analysis of the treated mice relative to wild-type not-ablated (wt) shows both exhaustion time and distance indicative of a full recovery of motor functionality already at 3 weeks after TA ablation with an increase in time and distance run for mice treated with PF-embedded Mabs-PlGF, while control mice (TA ABL-CONT or TA ABL-PF CONT) showed a much lower resistance (n = 10 mice per group). The values shown in the diagrams are expressed as means ± standard error and statistical significance was tested using Student's t-test (P < 0.05 was considered significant).S-Z Morphological analysis demonstrated the effective ability of the PF and Mabs-PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury. H&amp;E staining of TA injury in mice by cross section at an early (10 days) (S-V) and late (6 months) (W-Z) time point after ablation (in S, U, W, Y displayed items are obtained from a multi-photo collage). At the early time point, the acellular PF-treated mice revealed few mature muscle fibres that survive the massive ablation (box in S) signs of continued muscle degeneration (arrows in T), whereas PF-embedded Mabs implanted mice showed remarkable muscle regeneration indicated by centre-nucleated myofibres (arrows in V). At later stages, the control mice grafted with acellular PF showed an almost devoid TA (W) with very few muscle fibres (X), in contrast to PF-embedded Mabs, which exhibited a complete muscle replacement generating an artificial TA (Y), enlarged view (Z) proved the complete and mature organization of the newly formed muscle showing capillary and blood vessel around myofibres (arrows). Scale bar: (S, U, W, Y) 500 μm, (T, V, X, Z) 50 μm.


Figure 1 ( A ) Still images of time - lapse imaging of H3 . 3 - mCherry and H3 . 2 - EGFP proteins in wild - type zygotes ( n = 17 ) . ( B ) IF of DAXX and ATRX in zygotes at different pronuclear ( PN ) stages ( PN0 , n = 8 ; PN2 , n = 6 ; PN5 , n = 33 ) . Data information : All panels : images of paternal and maternal pronuclei at given stage / condition are from same zygote . ( C ) Boxplot displaying signal intensity of DAXX at pat - and mat - PCH in mouse zygotes at PN5 stage , relative to mean signal at pat - PCH . In boxplots , the enter lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by R software ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 3 times . * * * P < 0 . 001 , n = 28 , paired t - test . ( D ) IF of DAXX and ATRX in PN5 zygotes upon siRNA - mediated knockdown of Daxx ( n = 14 ) or with control siRNAs ( n = 8 ) . Data information : All panels : images of paternal and maternal pronuclei at given stage / condition are from same zygote . Yellow dashed circles represent the contours of the pronuclei . All scale bars , 10µm . ( E ) IF of DAXX and ATRX in control ( n = 15 ) and Atrxm - z + ( n = 14 ) PN5 zygotes . Data information : All panels : images of paternal and maternal pronuclei at given stage / condition are from same zygote . Yellow dashed circles represent the contours of the pronuclei . All scale bars , 10µm .

O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1(A) Still images of time-lapse imaging of H3.3-mCherry and H3.2-EGFP proteins in wild-type zygotes (n=17).(B) IF of DAXX and ATRX in zygotes at different pronuclear (PN) stages (PN0, n=8; PN2, n=6; PN5, n= 33). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote.(C) Boxplot displaying signal intensity of DAXX at pat- and mat-PCH in mouse zygotes at PN5 stage, relative to mean signal at pat-PCH. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, n= 28, paired t-test.(D) IF of DAXX and ATRX in PN5 zygotes upon siRNA-mediated knockdown of Daxx (n=14) or with control siRNAs (n=8). Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.(E) IF of DAXX and ATRX in control (n=15) and Atrxm-z+ (n=14) PN5 zygotes. Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.


Figure 2 ( A ) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m - z + ( n = 5 ) , Ezh2m - z + ( n = 3 ) and Ezh1m - z + ; Ezh2m - z + ( n = 4 ) late zygotes . Yellow dashed circles represent the contours of the pronuclei . Data information : All scale bars , 10µm . ( B ) IF of DAXX and RNF2 in PN0 zygotes ( n = 10 ) . Data information : All scale bars , 10µm . ( C ) IF of DAXX in wild type ( n = 30 ) , Ring1m - z + ( n = 12 ) , Rnf2m - z + ( n = 11 ) and Ring1m - z + ; Rnf2m - z + ( n = 12 ) paternal pronuclei of PN5 zygotes . Data information : All scale bars , 10µm . ( D ) Anti - Flag co - IP of DAXXMycHis , RNF2 and H3K27me3 with CBX23 × Flag from extracts of HEK293 cells in presence or absence of benzonase , followed by immuno - blot analyses for indicated antigens .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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 2(A) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m-z+ (n=5), Ezh2m-z+ (n=3) and Ezh1m-z+; Ezh2m-z+ (n=4) late zygotes. Yellow dashed circles represent the contours of the pronuclei. Data information: All scale bars, 10µm.(B) IF of DAXX and RNF2 in PN0 zygotes (n=10). Data information: All scale bars, 10µm.(C) IF of DAXX in wild type (n=30), Ring1m-z+ (n=12), Rnf2m-z+ (n=11) and Ring1m-z+; Rnf2m-z+ (n=12) paternal pronuclei of PN5 zygotes. Data information: All scale bars, 10µm.(D) Anti-Flag co-IP of DAXXMycHis, RNF2 and H3K27me3 with CBX23×Flag from extracts of HEK293 cells in presence or absence of benzonase, followed by immuno-blot analyses for indicated antigens.


Figure 3 ( C ) Occupancy ( % of IP - ed DNA / input DNA ) of GAL4DBDCBX2 , RNF2 , DAXXMycHis , H3 . 3 , and H3 proteins at 5 × UAS site as measured by ChIP - qPCR of chromatin from HEK2935xUAS cells . Data are means ± SEM ( n = 3 ) . ( D ) Immunoblots for the indicated proteins in HEK2935xUAS cells ( E ) ChIP - seq analysis of HEK2935xUAS cells expressing full length GAL4DBDCBX2 . 1 and DaxxMycHis showing CBX2 . 1 dependent recruitment of DAXX to promoters of CCND2 and RUNX1 genes . ( F ) ChIP - seq analysis of GAL4DBDCBX2 . 1 occupancy in HEK2935xUAS cells expressing DAXXMycHis ( X - axis ) or GAL4DBDCBX2 . 1 and DAXXMycHis ( Y - axis ) , showing ( log2 ) enrichment for GAL4DBDCBX2 . 1 at many TSSs ( ± 1 kb ) in cells expressing the CBX2 . 1 protein ( Y - axis ) . ( G ) ChIP - seq analysis of GAL4DBDCBX2 . 1 versus DAXXMycHis occupancy in HEK2935xUAS cells expressing GAL4DBDCBX2 . 1 and DAXXMycHis , revealing very comparable enrichment levels ( log2 ) of both proteins at TSSs . TSSs highlighted in red show enrichment with anti - Myc antibody independently of GAL4DBDCBX2 . 1 occupancy . ( H ) ChIP - seq analysis of DAXXMycHis occupancy in HEK2935xUAS cells expressing DAXXMycHis ( X - axis ) versus GAL4DBDCBX2 . 1 and DAXXMycHis ( Y - axis ) showing strong enrichment ( log2 ) for DAXXMycHis at many TSSs of genes , but only when both proteins are expressed ( Y - axis ) , indicating that CBX2 . 1 targets DAXX to chromatin . TSSs highlighted in red show enrichment with anti - Myc antibody independently of GAL4DBDCBX2 . 1 occupancy .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(C) Occupancy (% of IP-ed DNA / input DNA) of GAL4DBDCBX2, RNF2, DAXXMycHis, H3.3, and H3 proteins at 5×UAS site as measured by ChIP-qPCR of chromatin from HEK2935xUAS cells. Data are means ± SEM (n = 3).(D) Immunoblots for the indicated proteins in HEK2935xUAS cells(E) ChIP-seq analysis of HEK2935xUAS cells expressing full length GAL4DBDCBX2.1 and DaxxMycHis showing CBX2.1 dependent recruitment of DAXX to promoters of CCND2 and RUNX1 genes.(F) ChIP-seq analysis of GAL4DBDCBX2.1 occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) or GAL4DBDCBX2.1 and DAXXMycHis (Y-axis), showing (log2) enrichment for GAL4DBDCBX2.1 at many TSSs (± 1 kb) in cells expressing the CBX2.1 protein (Y-axis).(G) ChIP-seq analysis of GAL4DBDCBX2.1 versus DAXXMycHis occupancy in HEK2935xUAS cells expressing GAL4DBDCBX2.1 and DAXXMycHis, revealing very comparable enrichment levels (log2) of both proteins at TSSs. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.(H) ChIP-seq analysis of DAXXMycHis occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) versus GAL4DBDCBX2.1 and DAXXMycHis (Y-axis) showing strong enrichment (log2) for DAXXMycHis at many TSSs of genes, but only when both proteins are expressed (Y-axis), indicating that CBX2.1 targets DAXX to chromatin. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.


Figure 4 ( A ) IF of CBX2 and SUMO2 / 3 in PN0 ( n = 10 ) and PN5 ( n = 12 ) zygotes . Data information : Scale bars , 10µm . ( B ) ChIP - qPCR analyses of HEK2935xUAS cells co - expressing GAL4DBDCBX2 . 1 either with DAXXMycHis or DAXXΔSIM1 / 2MycHis . Data are means ± SEM ( n = 3 ) . ( C ) IF of EGFP and DAXX in Daxxm - z + PN5 zygotes micro - injected with Daxx - EGFP or DaxxΔSIM1 / 2 - EGFP mRNA ( 100ng / µl ) . Data information : Scale bars , 10µm . ( D ) Left : Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm - z + zygotes micro - injected with Daxx - EGFP or DaxxΔSIM1 / 2 - EGFP mRNA . Right : Boxplot displaying signal intensity of EGFP at euchromatin in same samples . In boxplots , the enter lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by R software ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 3 times . * * * P < 0 . 001 , N . S . : no statistical difference , P > 0 . 05 , t - test . ( F ) Ni - pulldown of 6xHisSUMO1 / 2 / 3 - conjugated proteins from HEK293 extracts expressing 6xHis - tagged SUMO1 , SUMO2 or SUMO3 in combination with CBX2 - EGFP . CBX2 and SUMO - conjugated CBX2 were detected with an anti - GFP antibody . ( G ) Ni - pulldown of 6xHis - SUMO2 conjugated proteins from HEK293 extracts transiently co - expressing 6xHis - SUMO2 with CBX2 - EGFP or CBX24KR - EGFP . Detection of CBX2 ( H ) IF of DAXX and EGFP in wild - type PN5 zygotes , micro - injected with Cbx2 or Cbx24KR - EGFP mRNA ( 100ng / µl ) . Data information : Scale bars , 10µm . ( I ) Left : Boxplot displaying signal intensity of DAXX at PCH relative to mean intensity at pat - PCH in wild - type zygotes micro - injected with Cbx2 or Cbx24KR - EGFP mRNA . Right : Absolute intensities of EGFP in same samples . In boxplots , the enter lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by R software ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 times . * * * P < 0 . 001 , t - test .

O O B-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(A) IF of CBX2 and SUMO2/3 in PN0 (n=10) and PN5 (n=12) zygotes. Data information: Scale bars, 10µm.(B) ChIP-qPCR analyses of HEK2935xUAS cells co-expressing GAL4DBDCBX2.1 either with DAXXMycHis or DAXXΔSIM1/2MycHis. Data are means ± SEM (n = 3).(C) IF of EGFP and DAXX in Daxxm-z+ PN5 zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm.(D) Left: Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm-z+ zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA. Right: Boxplot displaying signal intensity of EGFP at euchromatin in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, N.S.: no statistical difference, P>0.05, t-test.(F) Ni-pulldown of 6xHisSUMO1/2/3-conjugated proteins from HEK293 extracts expressing 6xHis-tagged SUMO1, SUMO2 or SUMO3 in combination with CBX2-EGFP. CBX2 and SUMO-conjugated CBX2 were detected with an anti-GFP antibody.(G) Ni-pulldown of 6xHis-SUMO2 conjugated proteins from HEK293 extracts transiently co-expressing 6xHis-SUMO2 with CBX2-EGFP or CBX24KR-EGFP. Detection of CBX2(H) IF of DAXX and EGFP in wild-type PN5 zygotes, micro-injected with Cbx2 or Cbx24KR-EGFP mRNA (100ng/µl). Data information: Scale bars, 10µm.(I) Left: Boxplot displaying signal intensity of DAXX at PCH relative to mean intensity at pat-PCH in wild-type zygotes micro-injected with Cbx2 or Cbx24KR-EGFP mRNA. Right: Absolute intensities of EGFP in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test.


Figure 5 ( A ) IF of DAXX and ATRX in PN5 stage pronuclei of wild - type ( n = 19 ) , Hp1βm - z + ( n = 10 ) and Suv39h2m - z + ( n = 9 ) zygotes . Data information : Panels Paternal and maternal pronuclei of a given condition originate from same zygotes . All scale bars 10µm . ( B ) Boxplots displaying signal intensities of DAXX or ATRX at pat - and mat - PCH in wild - type ( n = 5 ) and Hp1βm - z + ( n = 10 ) mouse zygotes at PN5 . In boxplots , the enter lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by R software ; whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Datapoints are indicated by circles . Experiments were replicated 2 times . * P < 0 . 05 , * * P < 0 . 01 , t - test . ( C ) IF of DAXX in PN5 stage pronuclei of zygotes expressing CBX2 - EGFP ( n = 9 ) or CBX2F12A - EGFP ( n = 17 ) . mRNAs ( 100ng / µl ) were injected into mouse PN2 stage zygotes . Yellow dashed circles represent the contours of the pronuclei . . Data information : Panels C : Paternal and maternal pronuclei of a given condition originate from same zygotes . All scale bars 10µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(A) IF of DAXX and ATRX in PN5 stage pronuclei of wild-type (n=19), Hp1βm-z+ (n=10) and Suv39h2m-z+ (n=9) zygotes. Data information: Panels Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.(B) Boxplots displaying signal intensities of DAXX or ATRX at pat- and mat-PCH in wild-type (n=5) and Hp1βm-z+ (n=10) mouse zygotes at PN5. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. *P<0.05, **P<0.01, t-test.(C) IF of DAXX in PN5 stage pronuclei of zygotes expressing CBX2-EGFP (n=9) or CBX2F12A-EGFP (n=17). mRNAs (100ng/µl) were injected into mouse PN2 stage zygotes. Yellow dashed circles represent the contours of the pronuclei.. Data information: Panels C: Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.