Figure 4A . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . The top panel is a still of a neurite in mRFP , showing neurite morphology . The other panels show moving GFP - MT + TIP comets pointing in either an anterograde direction ( green arrowheads ) or retrograde direction ( blue arrowheads ) . P indicates the proximal direction and D the distal direction of the neurite . Timestamp in minutes : seconds given on bottom right . Scale bar = 5 µm . B . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( A ) , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Scale bar = 5 µm . C J . Example stills from a spinning - disk time - lapse recording of a neurite transfected with mRFP and GFP - MT + TIP . Red line denotes the location of LS . Scale bar = 5 µm . K . Kymographs and schematic representations of time - lapse recordings of the distal axon as shown in ( J ) following LS , for different time points ( day 7 ; day 13 ) and conditions ( control ; Centrinone - B ) . Red line and red arrowhead denote location and time of LS . Scale bar = 5 µm . L

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 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

Figure 4A. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. The top panel is a still of a neurite in mRFP, showing neurite morphology. The other panels show moving GFP-MT+TIP comets pointing in either an anterograde direction (green arrowheads) or retrograde direction (blue arrowheads). P indicates the proximal direction and D the distal direction of the neurite. Timestamp in minutes:seconds given on bottom right. Scale bar = 5 µm. B. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (A), for different time points (day 7; day 13) and conditions (control; Centrinone-B). Scale bar = 5 µm. C J. Example stills from a spinning-disk time-lapse recording of a neurite transfected with mRFP and GFP-MT+TIP. Red line denotes the location of LS. Scale bar = 5 µm. K. Kymographs and schematic representations of time-lapse recordings of the distal axon as shown in (J) following LS, for different time points (day 7; day 13) and conditions (control; Centrinone-B). Red line and red arrowhead denote location and time of LS. Scale bar = 5 µm. L


Figure 1 ( A ) Representative images showing the myogenic differentiation , as assessed by immunostaining for MyHC ( green ) , of human MuSCs ( hMuSCs ) isolated from muscle biopsies of DMD patients , either before the beginning of the clinical trial ( hMuSCs ( - ) ) or after 1 year of treatment with Givinostat ( hMuSCs Giv ( - ) ) . Cells were cultured alone or ( only for hMuSCs - ) in transwell co - culture with FAPs isolated from the same patients and exposed in vitro to Giv ( + ehFAPs Giv Vitro ) or control vehicle ( + ehFAPs ) . The upper panels represent myogenic differentiation of DMD - 1 patient ; bottom panels represent myogenic differentiation of DMD - 2 patient . Dose and timing of Giv treatment as described in Bettica et al . , 2016 ( 23 ) . Scale bar = 50 μm . ( C ) Representative images showing the myogenic differentiation of MuSCs isolated from 1 . 5 month old mdx mice , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with mdx FAPs , which were pretreated with DMSO or GW4869 ( GW 10mM added to FAPs 30 minutes before starting the co - culture with MuSCs ) . Scale bar = 50 μm . ( D ) Graph showing the fusion index of MuSCs in the conditions described in ( C ) , ( n = 5 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 ; Hash ( # ) indicates statistical analysis by Tukey test relative to MuSCs in co - cultured with FAPs not treated ( DMSO ) , # p < 0 . 05 ; § represents statistical analysis by 2Way Anova test ; § § § p < 0 , 001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM . ( E ) Representative images of PKH - 67 ( green ) and DAPI ( blue ) staining in a transwell co - culture between MuSCs and FAPs isolated from 1 . 5 month old mdx mice . FAPs exposed to PKH - 67 prior to the co - culture with MuSCs . Scale bar = 50 μm . ( F ) Graph showing the percentage of PKH - 67 positive MuSCs after the co - culture with FAPs , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test ; * * * * p < 0 . 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM . ( G ) Representative images of myogenic differentiation of mdx MuSCs , as assessed by immunostaining for MyHC ( green ) . MuSCs were cultured either alone ( - ) or incubated with EVs isolated from 1 . 5 month old mdx mice ( EVs FAPs ) treated with vehicle ( CTR ) or exposed to TSA in vivo ( TSA ) or in vitro ( TSA vitro ) . Scale bar = 50 μm . ( H ) Graph showing the fusion index of MuSCs in the condition described in ( G ) , ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by Tukey test on the % of n > 5 ( % of nuclei that were MyHC + inside myotubes containing more than 5 nuclei ) relative to MuSCs cultured alone ( - ) , * * p < 0 . 01 , * * * * p < 0 , 0001 ; Hash ( # ) indicates statistical analysis by Tukey test relative to EVs FAPs CTR , # # p < 0 . 01 , # # # p < 0 , 001 ; ns = not significant ; § represents statistical analysis by 2way Anova test , § § § § p < 0 , 0001 . Data information : Nuclei were counterstained with DAPI ( blue ) . All data correspond to the average ± SEM .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images showing the myogenic differentiation, as assessed by immunostaining for MyHC (green), of human MuSCs (hMuSCs) isolated from muscle biopsies of DMD patients, either before the beginning of the clinical trial (hMuSCs (-)) or after 1 year of treatment with Givinostat (hMuSCs Giv (-)). Cells were cultured alone or (only for hMuSCs -) in transwell co-culture with FAPs isolated from the same patients and exposed in vitro to Giv (+ehFAPs Giv Vitro) or control vehicle (+ehFAPs). The upper panels represent myogenic differentiation of DMD-1 patient; bottom panels represent myogenic differentiation of DMD-2 patient. Dose and timing of Giv treatment as described in Bettica et al., 2016 (23). Scale bar = 50 μm.(C) Representative images showing the myogenic differentiation of MuSCs isolated from 1.5 month old mdx mice, as assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with mdx FAPs, which were pretreated with DMSO or GW4869 (GW 10mM added to FAPs 30 minutes before starting the co-culture with MuSCs). Scale bar = 50 μm. (D) Graph showing the fusion index of MuSCs in the conditions described in (C), (n=5, biological replicates). Star (*) indicates statistical analysis by Tukey test relative to MuSCs cultured alone (-), ** p < 0.01; Hash (#) indicates statistical analysis by Tukey test relative to MuSCs in co-cultured with FAPs not treated (DMSO), # p<0.05; § represents statistical analysis by 2Way Anova test; §§§ p < 0,001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.(E) Representative images of PKH-67 (green) and DAPI (blue) staining in a transwell co-culture between MuSCs and FAPs isolated from 1.5 month old mdx mice. FAPs exposed to PKH-67 prior to the co-culture with MuSCs. Scale bar = 50 μm. (F) Graph showing the percentage of PKH-67 positive MuSCs after the co-culture with FAPs, (n=3, biological replicates). Star (*) indicates statistical analysis by t-test;**** p < 0.0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.(G) Representative images of myogenic differentiation of mdx MuSCs, as assessed by immunostaining for MyHC (green). MuSCs were cultured either alone (-) or incubated with EVs isolated from 1.5 month old mdx mice (EVs FAPs) treated with vehicle (CTR) or exposed to TSA in vivo (TSA) or in vitro (TSA vitro). Scale bar = 50 μm. (H) Graph showing the fusion index of MuSCs in the condition described in (G), (n=3, biological replicates). Star (*) indicates statistical analysis by Tukey test on the % of n>5 (% of nuclei that were MyHC+ inside myotubes containing more than 5 nuclei) relative to MuSCs cultured alone (-), **p <0.01, ****p<0,0001; Hash (#) indicates statistical analysis by Tukey test relative to EVs FAPs CTR, ##p <0.01, ###p<0,001; ns= not significant; § represents statistical analysis by 2way Anova test, §§§§p < 0,0001. Data information: Nuclei were counterstained with DAPI (blue). All data correspond to the average ± SEM.


Figure 2 ( A ) Graph showing the relative expression of Drosha in FAPs after Drosha down - regulation by siRNA ( n = 4 , biological replicates ) . Values correspond to the average ± SEM . Star ( * ) indicates statistical analysis by t - test relative to FAPs TSA treated and transfected with scramble ( Scr ) ; * * p < 0 . 01 . ( B ) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC ( green ) . MuSCs were cultured alone ( - ) or in transwell co - culture with FAPs isolated from mdx mice treated either with vehicle ( + FAPs CTR ) or TSA ( 0 . 6 mg / kg / day for 15 days by i . p . ) ( + FAPs TSA ) , and transfected with scramble ( Scr siRNA ) or Drosha siRNA ( Drosha siRNA ) prior to co - culture with MuSCs . Scale bar = 50 μm . ( E ) Representative images of miR - 206 - 3p ( violet ) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages : 1 year ( n = 2 ) , 4 years ( n = 3 ) and 9 years old ( n = 2 ) . ( F ) Graph representing miR - 206 - 3p violet area quantification relative to ( E ) . ( G ) Representative images of immunofluorescence for eMyHC ( red ) and Laminin ( green ) in conditions described in ( E ) . ( H ) Graph showing the percentage of eMyHC positive fibers of condition described in ( G ) . ( I ) Representative images of Masson ' s Trichrome staining in conditions described in ( E ) . ( J ) Graph showing the fibrotic area quantification of conditions described in ( I ) .

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

Figure 2(A) Graph showing the relative expression of Drosha in FAPs after Drosha down-regulation by siRNA (n=4, biological replicates). Values correspond to the average ± SEM. Star (*) indicates statistical analysis by t-test relative to FAPs TSA treated and transfected with scramble (Scr); **p<0.01.(B) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). MuSCs were cultured alone (-) or in transwell co-culture with FAPs isolated from mdx mice treated either with vehicle (+FAPs CTR) or TSA (0.6 mg/kg/day for 15 days by i.p.) (+FAPs TSA), and transfected with scramble (Scr siRNA) or Drosha siRNA (Drosha siRNA) prior to co-culture with MuSCs. Scale bar = 50 μm.(E) Representative images of miR-206-3p (violet) immunohistochemistry in vastus medialis bioptic samples of Control and DMD patients at different ages: 1 year (n=2), 4 years (n=3) and 9 years old (n=2). (F) Graph representing miR-206-3p violet area quantification relative to (E). (G) Representative images of immunofluorescence for eMyHC (red) and Laminin (green) in conditions described in (E). (H) Graph showing the percentage of eMyHC positive fibers of condition described in (G). (I) Representative images of Masson's Trichrome staining in conditions described in (E). (J) Graph showing the fibrotic area quantification of conditions described in (I).


Figure 3 ( A ) Representative images of Sca - 1 ( green ) , Laminin ( white ) , DAPI ( blue ) immunofluorescence , and miR - 206 - 3p ( violet ) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior ( TA ) of young mdx mice ( CTR ) and after TSA treatment ( TSA ) ( 0 . 6 mg / kg / day for 15 days by i . p . ) ( n = 6 ) . Scale bar = 25 μm . ( C ) Graph representing the miR - 206 - 3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice ( CTR ) , mdx young and old mice ( control - CTR - and TSA treated ) ( n = 4 ) . Star ( * ) indicates Tukey analysis compared to wild type mice ( CTR ) , * * * * p < 0 . 0001 ; hash ( # ) indicates Tukey analysis compared to mdx young CTR , # # # # p < 0 . 0001 . § represents statistical analysis by Anova test . § § § § p < 0 , 0001 . ( D ) Representative images of CD90 ( green ) , Dapi ( blue ) and miR - 206 - 3p ( violet ) immunohistochemistry in brachial biceps bioptic samples of DMD patient before ( CTR ) and after Givinostat treatment ( GIV ) . Scale bar = 50 μm . ( F ) Graph showing the relative expression of miR - 206 - 3p in EVs isolated from a human population of muscle resident cells enriched in FAPs ( ehFAPs ) isolated from biopsies of two DMD patients ( DMD - 1 and DMD - 2 ) before ( CTR ) and after ( GIV ) Givinostat treatment , Two technical replicates are shown .

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 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 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

Figure 3(A) Representative images of Sca-1 (green), Laminin (white), DAPI (blue) immunofluorescence, and miR-206-3p (violet) immunohistochemistry on 10 uM sequencial cryosections of Tibialis Anterior (TA) of young mdx mice (CTR) and after TSA treatment (TSA) (0.6 mg/kg/day for 15 days by i.p.) (n=6). Scale bar = 25 μm.(C) Graph representing the miR-206-3p relative expression in EVs isolated from muscle interstitium of tibialis anterior from wild type mice (CTR), mdx young and old mice (control -CTR- and TSA treated) (n=4). Star (*) indicates Tukey analysis compared to wild type mice (CTR), ****p < 0.0001; hash (#) indicates Tukey analysis compared to mdx young CTR, #### p<0.0001. § represents statistical analysis by Anova test. §§§§ p<0,0001.(D) Representative images of CD90 (green), Dapi (blue) and miR-206-3p (violet) immunohistochemistry in brachial biceps bioptic samples of DMD patient before (CTR) and after Givinostat treatment (GIV). Scale bar = 50 μm.(F) Graph showing the relative expression of miR-206-3p in EVs isolated from a human population of muscle resident cells enriched in FAPs (ehFAPs) isolated from biopsies of two DMD patients (DMD-1 and DMD-2) before (CTR) and after (GIV) Givinostat treatment, Two technical replicates are shown.


Figure 4 ( A ) Flow cytometry analysis of mdx muscles injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA . The left panel shows the percentage of PKH - 67 positive cells in whole muscle . The right panel shows the percentage of the PKH - 67 positive MuSCs . ( B ) Representative images of PKH - 67 ( green ) and DAPI ( blue ) staining in MuSCs freshly isolated from muscles previously injected with PKH67 - labeled EVs . Scale bar = 25 μm . Data information : Nuclei were counterstained with DAPI ( blue ) . ( C ) Graph showing the relative expression of miR - 206 - 3p in MuSCs isolated as described in ( A and B ) and in MuSCs from muscles injected with PKH67 - labeled EVs isolated from FAPs of 1 . 5 month old mdx mice exposed to TSA , and then transfected or not with Drosha siRNA ( siRNA Drosha ) ( n = 4 ) . Star ( * ) means significance relative to MuSCs that not uptake PKH67 - labled EVs , * * p < 0 . 01 . Hash ( # ) means significance compared to MuSCs that uptake EVs - FAPs TSA , # # p < 0 . 01 . § indicates Anova analysis , § § p < 0 , 01 . ( D ) Graph showing the expression levels of miR - 206 - 3p and miR - 145 - 5p in FAPs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test relative to FAPs - ; * p < 0 . 05 , * * p < 0 . 01 ( E ) Graph showing the expression levels of miR - 206 - 3p and miR - 145 - 5p in FAPs - derived EVs treated or not with TSA after antagomiR treatment ( n = 3 , biological replicates ) . Star ( * ) indicates statistical analysis by t - test relative to FAPs - ; * p < 0 . 05 . Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( F ) Representative images of immunofluorescence for embryonic myosin heavy chain ( eMyHC - red ) and laminin ( Lam - cyan ) stainings . Scale bar = 50 μm . ( G ) Graph showing the quantification of cross - sectional area ( CSA ) . ( H ) Graph showing the quantification of muscle regeneration ( eMyHC ) . Data information : Nuclei were counterstained with DAPI ( blue ) . Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( I ) Representative images of Masson ' s Trichrome staining . Scale bar = 50 μm . ( J ) Graph showing the quantifications of muscle fiber area fraction ( MFAF ) . ( K ) Graph showing the quantifications of fibrotic area . Data information : Nuclei were counterstained with DAPI ( blue ) . Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1 . 5 months old mdx mice treated daily for 21 days with intra - peritoneal injection of vehicle ( CTR ) or TSA , or once a week with intramuscular injections ( Tibialis Anterior ) of EVs derived from FAPs ( EVs - FAPs ) exposed or not to TSA in vivo ( EVs - FAPs CTR - , EVs - FAPs TSA - ) EVs derived from FAPs control transfected with the antagomiR - 206 ( EVs - FAPs CTR A - 206 ) and of EVs - FAPs TSA transfected with antagomiR - 206 and antagomiR - 145 ( EVs - FAPs TSA A - 206 , EVs - FAPs TSA A - 145 ) . EVs were injected every seven days and sacrificed after 21 days of treatment ( n = 5 for CTR , TSA EVs - FAPs CTR and EVs - FAPs TSA while for AntagomiRs n = 3 , biological replicates ) . ( L ) Representative images of myeloperoxidase staining ( MPO - red ) . Scale bar = 50 μm . ( M ) Graph showing the quantifications of inflammation ( MPO ) . Data information : Nuclei were counterstained with DAPI ( blue ) .

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

Figure 4(A) Flow cytometry analysis of mdx muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA. The left panel shows the percentage of PKH-67 positive cells in whole muscle. The right panel shows the percentage of the PKH-67 positive MuSCs.(B) Representative images of PKH-67 (green) and DAPI (blue) staining in MuSCs freshly isolated from muscles previously injected with PKH67-labeled EVs. Scale bar = 25 μm. Data information: Nuclei were counterstained with DAPI (blue).(C) Graph showing the relative expression of miR-206-3p in MuSCs isolated as described in (A and B) and in MuSCs from muscles injected with PKH67-labeled EVs isolated from FAPs of 1.5 month old mdx mice exposed to TSA, and then transfected or not with Drosha siRNA (siRNA Drosha) (n=4). Star (*) means significance relative to MuSCs that not uptake PKH67-labled EVs, **p < 0.01. Hash (#) means significance compared to MuSCs that uptake EVs-FAPs TSA, ##p < 0.01. § indicates Anova analysis, §§p<0,01.(D) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05, **p<0.01(E) Graph showing the expression levels of miR-206-3p and miR-145-5p in FAPs-derived EVs treated or not with TSA after antagomiR treatment (n=3, biological replicates). Star (*) indicates statistical analysis by t-test relative to FAPs -; *p<0.05.Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (F) Representative images of immunofluorescence for embryonic myosin heavy chain (eMyHC-red) and laminin (Lam-cyan) stainings. Scale bar = 50 μm. (G) Graph showing the quantification of cross-sectional area (CSA). (H) Graph showing the quantification of muscle regeneration (eMyHC). Data information: Nuclei were counterstained with DAPI (blue).Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (I) Representative images of Masson's Trichrome staining. Scale bar = 50 μm. (J) Graph showing the quantifications of muscle fiber area fraction (MFAF). (K) Graph showing the quantifications of fibrotic area. Data information: Nuclei were counterstained with DAPI (blue).Stainings and relative measurements on Tibialis Anterior muscle transversal sections of 1.5 months old mdx mice treated daily for 21 days with intra-peritoneal injection of vehicle (CTR) or TSA, or once a week with intramuscular injections (Tibialis Anterior) of EVs derived from FAPs (EVs-FAPs) exposed or not to TSA in vivo (EVs-FAPs CTR -, EVs-FAPs TSA -) EVs derived from FAPs control transfected with the antagomiR-206 (EVs-FAPs CTR A-206) and of EVs-FAPs TSA transfected with antagomiR-206 and antagomiR-145 (EVs-FAPs TSA A-206, EVs-FAPs TSA A-145). EVs were injected every seven days and sacrificed after 21 days of treatment (n=5 for CTR, TSA EVs-FAPs CTR and EVs-FAPs TSA while for AntagomiRs n=3, biological replicates). (L) Representative images of myeloperoxidase staining (MPO-red). Scale bar = 50 μm. (M) Graph showing the quantifications of inflammation (MPO). Data information: Nuclei were counterstained with DAPI (blue).


Figure 5A ) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC ( green ) . Nuclei were counterstained with DAPI ( blue ) . MuSCs were cultured alone ( - ) or with EVs ( EVs FAPs ) isolated from FAPs in vivo exposed or not to TSA ( CTR and TSA ) and transfected or not with Antagomir - 206 ( - and A - 206 ) . ( n = 7 for all samples , but antagomir ' s samples n = 3 , biological replicates ) . Scale bar = 50 μm . ( C ) Graph relative to Notch3 expression in MuSCs isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) , and in EVs derived from FAPs ( EVs - FAPs ) isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) ( n = 3 ) . Star ( * ) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice ( CTR ) ; * p < 0 , 05 ; . hash ( # ) means significance compared to EVs - FAPs CTR , # # p < 0 , 01 . § indicates significance by Anova test ; § § p > 0 , 01 . ( D ) Graph relative to Notch1 expression in MuSCs isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) , and in EVs derived from FAPs ( EVs - FAPs ) isolated from 1 . 5 month old mdx mice treated either with vehicle ( CTR ) or TSA ( TSA ) n = 3 . Star ( * ) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice ( CTR ) ; * p < 0 , 05 ; . hash ( # ) means significance compared to EVs - FAPs CTR ; * p < 0 . 05 . § indicates significance by Anova test ; § § p > 0 , 01 . ( E ) Representative images showing myofibers cultured alone ( Ctr ) or with extracellular vesicles ( F ) From left to right : representative images of myofibers cultured for 24 hours alone ( - ) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 ( MEDIA - FAPs ) or extracellular vesicles ( EVs - FAPs ) collected from FAPs exposed or not to TSA in vivo ( CTR and TSA ) and transfected with antagomiR - 206 ( TSA A - 206 ) . Myofibers have been stained for EdU ( Cyan ) , immunofluorescence with Pax7 ( green ) , MyoD ( red ) . Nuclei were counterstained with DAPI ( blue ) . ( n = 3 ) . Scale bar = 25 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 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 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 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 5A) Representative images of myogenic differentiation of MuSCs assessed by immunostaining for MyHC (green). Nuclei were counterstained with DAPI (blue). MuSCs were cultured alone (-) or with EVs (EVs FAPs) isolated from FAPs in vivo exposed or not to TSA (CTR and TSA) and transfected or not with Antagomir-206 (- and A-206). (n=7 for all samples, but antagomir's samples n=3, biological replicates). Scale bar =50 μm.(C) Graph relative to Notch3 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) (n=3). Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR, ##p<0,01. § indicates significance by Anova test; §§ p>0,01.(D) Graph relative to Notch1 expression in MuSCs isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA), and in EVs derived from FAPs (EVs-FAPs) isolated from 1.5 month old mdx mice treated either with vehicle (CTR) or TSA (TSA) n=3. Star (*) indicates statistical analysis by Tukey test relative to mdx vehicle treated mdx mice (CTR); *p<0,05;. hash (#) means significance compared to EVs-FAPs CTR; *p < 0.05. § indicates significance by Anova test; §§ p>0,01.(E) Representative images showing myofibers cultured alone (Ctr) or with extracellular vesicles(F) From left to right: representative images of myofibers cultured for 24 hours alone (-) or with conditioned media of FAPs that were previously exposed or not to TSA and GW4869 (MEDIA-FAPs) or extracellular vesicles (EVs-FAPs) collected from FAPs exposed or not to TSA in vivo (CTR and TSA) and transfected with antagomiR-206 (TSA A-206). Myofibers have been stained for EdU(Cyan), immunofluorescence with Pax7 (green), MyoD (red). Nuclei were counterstained with DAPI (blue). (n=3). Scale bar = 25 μm.


Figure 2 ( A ) Colocalization analysis of KIF3A / B / KAP3 with APC by immunostaining of hippocampal primary neurons . ( D ) Pull - down analysis of the binding of APCARM with KIF3 / KAP3 .

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Figure 2(A) Colocalization analysis of KIF3A/B/KAP3 with APC by immunostaining of hippocampal primary neurons.(D) Pull-down analysis of the binding of APCARM with KIF3/KAP3.


Figure 3 ( D ) XL - MS analysis of the interaction sites between KIF3 / KAP3 and APCARM . Two crosslinked site pairs were found to correspond to the possible KIF3 / KAP3 - APCARM binding region marked by asterisks in C . Cartoon diagrams , sequence alignment and LC - MS data are shown with crosslinked sites highlighted .

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Figure 3(D) XL-MS analysis of the interaction sites between KIF3/KAP3 and APCARM. Two crosslinked site pairs were found to correspond to the possible KIF3/KAP3-APCARM binding region marked by asterisks in C. Cartoon diagrams, sequence alignment and LC-MS data are shown with crosslinked sites highlighted.


Figure 4 ( A ) HS - AFM imaging of KIF3 / KAP3 - APC particles . ( B ) Time lapse HS - AFM images of a representative ABK - APCARM particle with a continuous tapping force . ( C ) Representative HS - AFM images of ABK - APCARM illustrating the dissociation dynamic . Continuous tapping of AFM triggered the dissociation of APCARM from ABK . Red arrows indicate the binding interface between ABK and ABK - APCARM . ( D ) Statistical analysis of the AFM - based ABK and ABK - APCARM particle conformations . 200 frames extracted from three independent AFM movies of ABK and ABK - APCARM were used for the analysis . Distance from the coiled coil distal tip to the docking domain ( length of the protrusion as shown by the dotted lines ) was calculated and shown as violin plots . Data derived from each of the three movies were indicated by circles , triangles and rectangles , respectively . Based on the distal - center distance , particle conformations were classified into three forms , L ( cargo - locked globular form ) , T ( Transitional curved form ) and D ( cargo - docked elongated form ) . Unpaired t test was used to evaluate the statistical significance . The solid and dotted lines indicate the median and quartiles , respectively . The AFM images shown here are re - used in the figure 5A .

O O B-PANEL_START 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 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

Figure 4(A) HS-AFM imaging of KIF3/KAP3-APC particles.(B) Time lapse HS-AFM images of a representative ABK-APCARM particle with a continuous tapping force.(C) Representative HS-AFM images of ABK-APCARM illustrating the dissociation dynamic. Continuous tapping of AFM triggered the dissociation of APCARM from ABK. Red arrows indicate the binding interface between ABK and ABK-APCARM.(D) Statistical analysis of the AFM-based ABK and ABK-APCARM particle conformations. 200 frames extracted from three independent AFM movies of ABK and ABK-APCARM were used for the analysis. Distance from the coiled coil distal tip to the docking domain (length of the protrusion as shown by the dotted lines) was calculated and shown as violin plots. Data derived from each of the three movies were indicated by circles, triangles and rectangles, respectively. Based on the distal-center distance, particle conformations were classified into three forms, L (cargo-locked globular form), T (Transitional curved form) and D (cargo-docked elongated form). Unpaired t test was used to evaluate the statistical significance. The solid and dotted lines indicate the median and quartiles, respectively. The AFM images shown here are re-used in the figure 5A.


figf1 ( b ) SAR405 inhibits the function of Vps34 in the GFP - FYVE cellular assay . HeLa cells stably transfected with GFP - FYVE were incubated with DMSO ( left ) or with 1 μM SAR405 ( right ) for 2 h . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . Fluorescence was analyzed using an imaging cytometer ( X40 ) . One representative image out of three independent experiments is depicted . Scale bars , 20 μm . ( c ) SAR405 does not prevent phosphorylation of Akt and S6 . U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations . The effect of compounds on phosphorylated Akt ( p - Akt ) Ser473 and Thr308 residues , total Akt and on phosphorylated S6 ( Ser240 and Ser244 residues ) and total S6 protein was measured using western blotting . One representative experiment from at least two independent experiments is shown . Full - length blots are shown in Supplementary Figure 6 .

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figf1(b) SAR405 inhibits the function of Vps34 in the GFP-FYVE cellular assay. HeLa cells stably transfected with GFP-FYVE were incubated with DMSO (left) or with 1 μM SAR405 (right) for 2 h. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). Fluorescence was analyzed using an imaging cytometer (X40). One representative image out of three independent experiments is depicted. Scale bars, 20 μm.(c) SAR405 does not prevent phosphorylation of Akt and S6. U87MG cells were treated with SAR405 or PI103 for 2 h at the indicated concentrations. The effect of compounds on phosphorylated Akt (p-Akt) Ser473 and Thr308 residues, total Akt and on phosphorylated S6 (Ser240 and Ser244 residues) and total S6 protein was measured using western blotting. One representative experiment from at least two independent experiments is shown. Full-length blots are shown in Supplementary Figure 6.


figf3 ( a ) SAR405 induces late endosome - lysosome swelling . RKO cells were treated with DMSO ( upper panels ) or with 10 μM SAR405 for 16 h ( lower panels ) . The late endosomes - lysosomes compartment was assessed using Lysotracker green ( left ) or Alexa 488 - conjugated anti - Lamp1 antibodies ( right ) , as indicated . Nuclei were stained using Hoechst 33342 ( blue ) . One representative image chosen from three independent experiments , showing fluorescence imaged using a Zeiss microscope ( X40 ) , is shown for each condition . Arrows indicate accumulation of swollen late endosome - lysosomes . Scale bars , 20 μm . ( b ) SAR405 triggers accumulation of p62 . RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h . The effect on the p62 biomarker was analyzed by western blotting . GAPDH was used as a loading control . The results are representative of at least two independent experiments . Full - length blots are shown in Supplementary Figure 14 . ( c ) SAR405 affects the maturation of cathepsin D ( cat D ) . RKO cells were treated at the indicated concentrations of SAR405 for 24 h . The different forms of cathepsin D ( PPCD , pre - pro - catD ) were analyzed by western blotting . GAPDH was used as a loading control . The results are representative of at least two independent experiments . Full - length blots are shown in Supplementary Figure 16 .

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figf3(a) SAR405 induces late endosome-lysosome swelling. RKO cells were treated with DMSO (upper panels) or with 10 μM SAR405 for 16 h (lower panels). The late endosomes-lysosomes compartment was assessed using Lysotracker green (left) or Alexa 488-conjugated anti-Lamp1 antibodies (right), as indicated. Nuclei were stained using Hoechst 33342 (blue). One representative image chosen from three independent experiments, showing fluorescence imaged using a Zeiss microscope (X40), is shown for each condition. Arrows indicate accumulation of swollen late endosome-lysosomes. Scale bars, 20 μm.(b) SAR405 triggers accumulation of p62. RKO cells were treated with the indicated concentration of Vps34 inhibitor for 24 h. The effect on the p62 biomarker was analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 14.(c) SAR405 affects the maturation of cathepsin D (cat D). RKO cells were treated at the indicated concentrations of SAR405 for 24 h. The different forms of cathepsin D (PPCD, pre-pro-catD) were analyzed by western blotting. GAPDH was used as a loading control. The results are representative of at least two independent experiments. Full-length blots are shown in Supplementary Figure 16.


figf4 ( a ) GFP - LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . One representative image , acquired with an iCyte imaging cytometer , for three independent experiments is depicted ( X40 ) . White arrows indicate GFP - LC3 spots , which correspond to the autophagosomes . Scale bars , 10 μm . ( b ) Histograms show the percentage of positive cells and GFP intensity , measured using an imaging cytometer for different compound concentrations on GFP - LC3 HeLa cells . Black and gray histograms correspond to fed and starved conditions , respectively . For GFP intensity , the values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments . ( c ) Wild - type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine ( HCQ ) , as indicated . Cells were treated at the same time with SAR405 at different concentrations . LC3 - II was measured by western blotting , and GAPDH was used as a loading control . One representative experiment for three independent experiments is shown . Full - length blots are shown in Supplementary Figure 18 . ( d ) Histograms show the LC3 - II / GAPDH ratio for each condition in the two cell lines . Black and gray histograms correspond to fed condition and EBSS conditions , respectively . The addition of hydroxychloroquine at 10 μM is indicated . Values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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figf4(a) GFP-LC3 HeLa cells were cultured in fed conditions or starved using EBSS plus 10 μM hydroxychloroquine and treated with Vps34 inhibitor. Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image, acquired with an iCyte imaging cytometer, for three independent experiments is depicted (X40). White arrows indicate GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells and GFP intensity, measured using an imaging cytometer for different compound concentrations on GFP-LC3 HeLa cells. Black and gray histograms correspond to fed and starved conditions, respectively. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.(c) Wild-type HeLa and H1299 cell lines were cultured either in fed or starved conditions with EBSS and 10 μM hydroxychloroquine (HCQ), as indicated. Cells were treated at the same time with SAR405 at different concentrations. LC3-II was measured by western blotting, and GAPDH was used as a loading control. One representative experiment for three independent experiments is shown. Full-length blots are shown in Supplementary Figure 18. (d) Histograms show the LC3-II/GAPDH ratio for each condition in the two cell lines. Black and gray histograms correspond to fed condition and EBSS conditions, respectively. The addition of hydroxychloroquine at 10 μM is indicated. Values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


figf5 ( a ) GFP - LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO ( left ) or with 1 μM SAR405 ( right ) . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . One representative image of cells out of three independent experiments , acquired using an iCyte imaging cytometer , is shown ( X40 ) . White arrows indicate the GFP - LC3 spots , which correspond to the autophagosomes . Scale bars , 10 μm . ( b ) Histograms show the percentage of positive cells ( top ) and the GFP intensity ( bottom ) , as measured using an imaging cytometer . Treatment of GFP - LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated . For GFP intensity , the values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments . ( c ) The concentration needed to reach 40 % inhibition ( IC40 ) of cell proliferation is indicated . The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines . The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405 . The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼ 0 . 5 , corresponding to compounds that are in equipotent proportion in the mixture ( Supplementary Fig . 20 ) . The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments .

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figf5(a) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right). Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image of cells out of three independent experiments, acquired using an iCyte imaging cytometer, is shown (X40). White arrows indicate the GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells (top) and the GFP intensity (bottom), as measured using an imaging cytometer. Treatment of GFP-LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.(c) The concentration needed to reach 40% inhibition (IC40) of cell proliferation is indicated. The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines. The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405. The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼0.5, corresponding to compounds that are in equipotent proportion in the mixture (Supplementary Fig. 20). The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments.


Figure 2Figure 2 . SPOP localizes to nuclear speckles or other nuclear bodies . Confocal microscopy images of fixed NIH 3T3 cells transiently expressing ( A ) full length HA - SPOP , ( B ) GFP - Gli31455 , and ( C ) HA - SPOP + GFP - Gli31 - 455 are shown . DAPI was used to stain the nucleus , SPOP localization was identified using a anti - HA antibody , nuclear speckle localization was identified using a anti - SC - 35 antibody , and Gli31 - 455 localization was identified via GFP fluorescence . SPOP co - localizes with a marker for nuclear speckles or with the substrate Gli31455 . The areas with overlapping HA - and GFP signal contain 75 % of the punctate HA signal , and 100 % of the punctate GFP signal .

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Figure 2Figure 2. SPOP localizes to nuclear speckles or other nuclear bodies. Confocal microscopy images of fixed NIH 3T3 cells transiently expressing (A) full length HA-SPOP, (B) GFP-Gli31455, and (C) HA-SPOP+GFP-Gli31-455 are shown. DAPI was used to stain the nucleus, SPOP localization was identified using a anti-HA antibody, nuclear speckle localization was identified using a anti-SC-35 antibody, and Gli31-455 localization was identified via GFP fluorescence. SPOP co-localizes with a marker for nuclear speckles or with the substrate Gli31455. The areas with overlapping HA- and GFP signal contain 75% of the punctate HA signal, and 100% of the punctate GFP signal.


Figure 3 ( A - B ) NIH 3T3 cells were ( A ) co - transfected with constructs expressing full length SPOP and GFP - Gli31455 or ( B ) transfected with a construct expressing SC - 35 - GFP , and GFP fluorescence was monitored in live cells . ( A ) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching ( at 0 s ) , recovery , and nuclear body fusion events . The arrow in each panel points to the body that was photobleached and subsequently fuses with another body . These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery . ( B ) Snapshots at indicated timepoints show a nuclear speckle fusion event . Additional images are shown in Fig EV1 . ( C ) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec . Data from 45 individual cells and FRAP events were corrected for background , normalized to the minimum and maximum intensity , and the mean is shown with error bars representing the standard error of the mean . The mean characteristic recovery time is indicated ± SEM . ( D - E ) Nuclear bodies are close to spherical . ( D ) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA - SPOP ( 151 cells ) ( D ) , or GFP - Gli31 - 455 and HA - SPOP ( 155 cells ) ( E ) were calculated as described previously ( Brangwynne et al , 2011 ) . Representative individual images of cells are shown as insets in each panel .

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Figure 3(A-B) NIH 3T3 cells were (A) co-transfected with constructs expressing full length SPOP and GFP-Gli31455 or (B) transfected with a construct expressing SC-35-GFP, and GFP fluorescence was monitored in live cells. (A) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. (B) Snapshots at indicated timepoints show a nuclear speckle fusion event. Additional images are shown in Fig EV1.(C) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM.(D-E) Nuclear bodies are close to spherical. (D) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA-SPOP (151 cells) (D), or GFP-Gli31-455 and HA-SPOP (155 cells) (E) were calculated as described previously (Brangwynne et al, 2011). Representative individual images of cells are shown as insets in each panel.


Figure 4 ( A ) SEC chromatograms of given loading concentrations of SPOP28 - 359 are shown . The concentrations were normalized to the monomer molecular weight , i . e . , identical concentrations of dimeric SPOP ΔBACK ( see Fig EV2 ) and oligomeric SPOP28 - 359 contain identical numbers of protomers . ( B ) SEC chromatograms for SPOP constructs defective in self - association in one or both oligomerization domains are shown . Proteins were injected at the same loading concentration ( 533 µM ) and the elution volume of globular molecular weight standards is noted above the graph . ( C ) SEC chromatograms of SPOP28 - 359 ( 200 µM ) , SPOP mutBACK ( 718 µM ) , and mixtures of the two ( ratios given in the Figure , SPOP at 200 µM ) are shown . The elution volume of globular molecular weight standards is noted above the graph . ( D ) Constructs for expressing full - length HA - SPOP or HA - SPOP mutants capable of oligomerization through only one ( or neither ) of the self - association domains were transfected into NIH 3T3 cells . DAPI was used to stain the nucleus and SPOP localization was identified using an anti - HA antibody . Experiments were performed at least twice on four biological samples . Multiple cells were examined and representative cells are shown . For additional images see Appendix Fig S3 . Scale bar represents 5 µm .

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Figure 4(A) SEC chromatograms of given loading concentrations of SPOP28-359 are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP28-359 contain identical numbers of protomers. (B) SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. (C) SEC chromatograms of SPOP28-359 (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph.(D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.


Figure 5 ( A ) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide . All SPOP proteins bound substrate with an affinity similar to that of SPOP28 - 359 ( 4 - 8 µM , see Table 2 ) . Experimental data are shown as circles ; the solid lines are fits to eq . 2 ( Roehrl et al , 2004 ) . ( B ) In vitro crosslinking assays were performed for SPOP28 - 359 and each mutant at 30 μM protein with the amide - specific BS3 crosslinker . Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non - specific crosslinking of protein species . ( C ) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP , SPOP mutBACK , SPOP mutBTB , or SPOP mutBTB - BACK . SPOP1 , SPOP2 and SPOPn identify SPOP monomers , dimers and larger species , respectively . For loading levels see Appendix Fig S4 .

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Figure 5(A) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP28-359 (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl et al, 2004).(B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.


Figure 6 ( A ) SV - AUC data for 0 . 2 nM AF488 - SPOP ΔBACK . Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation ( circles ) . The data were subjected to standard c ( s ) analysis in SEDFIT ( Schuck , 2000 ) and the results of this fit are shown ( solid lines , rainbow color scheme ) . ( B ) Sedimentation coefficient distributions for a selection of values from the dilution series are shown . ( C ) The isotherm of weighted - average s - values vs . concentration ( black circles ) , was fit to a dimer self - association model and reveals a KD of 1 . 11 nM , [ 95 % confidence interval 0 . 88 , 1 . 40 nM ] ( solid line ) . ( D ) The BACK domain dimerizes with micromolar affinity . Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP mutBTB ( black circles ) was fitted to a self - association model ( see text for details ) for BACK - BACK dimerization ( black line ) yielding a KD of 36 ± 3 µM ( average and standard deviation from 3 independent experiments ) . Fits to the monomer / dimer equilibrium model and to alternative monomer / trimer , tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines , respectively . Residuals for all models are depicted in the lower panel .

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Figure 6(A) SV-AUC data for 0.2 nM AF488-SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c(s) analysis in SEDFIT (Schuck, 2000) and the results of this fit are shown (solid lines, rainbow color scheme).(B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown.(C) The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a KD of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line).(D) The BACK domain dimerizes with micromolar affinity. Experimental weight-average molar mass (Mw) from CG-MALS for SPOP mutBTB (black circles) was fitted to a self-association model (see text for details) for BACK-BACK dimerization (black line) yielding a KD of 36 ± 3 µM (average and standard deviation from 3 independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer/trimer, tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines, respectively. Residuals for all models are depicted in the lower panel.


Figure 7 ( B ) Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP28 - 359 ( orange circles ) was fitted to an isodesmic self - association model in which SPOP dimers are the self - associating unit ( orange line ) . The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ( ( SPOP2 ) 11 ) . The fits from three independent experiments yielded a KD2 of 2 . 4 ± 0 . 4 µM . Lines for fits of the data to self - association models that assume formation of individual oligomeric species instead of isodesmic self - association are shown for reference ( gray lines ) . ( C ) Experimental weight - average molar mass ( Mw ) from CG - MALS data ( circles ) and fits ( solid lines ) are depicted for each SPOP construct assayed , showing that a progressive increase in SPOP oligomer size is observed only when both self - association domains are interaction - competent . This Figure is comprised of data shown in Figures 6D , 7B , and Appendix Fig S5A for direct comparison .

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Figure 7(B) Experimental weight-average molar mass (Mw) from CG-MALS for SPOP28-359 (orange circles) was fitted to an isodesmic self-association model in which SPOP dimers are the self-associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ((SPOP2)11). The fits from three independent experiments yielded a KD2 of 2.4 ± 0.4 µM. Lines for fits of the data to self-association models that assume formation of individual oligomeric species instead of isodesmic self-association are shown for reference (gray lines).(C) Experimental weight-average molar mass (Mw) from CG-MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self-association domains are interaction-competent. This Figure is comprised of data shown in Figures 6D, 7B, and Appendix Fig S5A for direct comparison.


Figure 8 ( A ) Native MS spectrum of 30 µM SPOP28 - 359 confirms that SPOP self - associates through addition of dimers . The increase in mass indicates that the dimer is the stable building block within the assemblies . ( C ) Concentration - dependence of the SPOP oligomer size distribution ( mole fraction ) from isodesmic modeling of CG - MALS data .

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Figure 8(A) Native MS spectrum of 30 µM SPOP28-359 confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies.(C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.


Figure 9Figure 9 . The self - association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo . UAS transgenes were expressed under the control of an epithelial driver C765 - Gal4 in Drosophila melanogaster . ( A ) UAS vector served as a control and yielded a normal wing . Longitudinal veins are denoted by numbers 1 - 5 . Expression of ( B ) SPOP WT resulted in a modest Hh loss - of function phenotype with fusion of LV3 and LV4 ( arrow ) . ( C ) mutBTB acts in a dominant - negative manner , as evidenced by a modest Hh gain - of function phenotype with expansion of the LV3 - LV4 intervein space . Expression of ( D ) mutBACK and ( E ) mutBTB - BACK does not induce a wing patterning defect . ~ 50 animals each were analyzed from two independent crosses .

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Figure 9Figure 9. The self-association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo. UAS transgenes were expressed under the control of an epithelial driver C765-Gal4 in Drosophila melanogaster. (A) UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1-5. Expression of (B) SPOP WT resulted in a modest Hh loss-of function phenotype with fusion of LV3 and LV4 (arrow). (C) mutBTB acts in a dominant-negative manner, as evidenced by a modest Hh gain-of function phenotype with expansion of the LV3-LV4 intervein space. Expression of (D) mutBACK and (E) mutBTB-BACK does not induce a wing patterning defect. ~50 animals each were analyzed from two independent crosses.


Figure 1A , Reconstructed MSOT images ( 800 nm ) without and with linear unmixing data in the neck and the upper and lower abdominal areas , showing the ROIs for the liver , kidney , iBAT , rpWAT , aorta , and SV . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value neck / upper abdominal / lower abdominal : Hb : 2 . 5 / 3 . 5 / 0 . 9 ; HbO2 : 2 . 9 / 4 . 9 / 2 . 5 ; Lipid : 24000 / 19000 / 13000 ) Scale bar : 4 mm .

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Figure 1A, Reconstructed MSOT images (800 nm) without and with linear unmixing data in the neck and the upper and lower abdominal areas, showing the ROIs for the liver, kidney, iBAT, rpWAT, aorta, and SV. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm.


Figure 2A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , and lipid from chow and HFD - fed mice . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value chow / HFD : Hb : 1 . 3 / 0 . 6 ; HbO2 : 1 . 8 / 0 . 9 ; Lipid : 27000 / 27000 ) . Scale bar : 4 mm . D , Tissue oxygenation ( sO2 ) and total blood volume ( TBV ) results from liver , kidney , iBAT , and rpWAT , Each dot represents data from one animal , in total 8 animals ( n = 8 ) . Data represent the mean ( + / - 95 % confidence ) . The unpaired t test was used to verify the statistical significance . Data information : In the figure , A . U . = arbitrary units .

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Figure 2A, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value chow/HFD: Hb: 1.3/0.6; HbO2: 1.8/0.9; Lipid: 27000/27000). Scale bar: 4 mm.D, Tissue oxygenation (sO2) and total blood volume (TBV) results from liver, kidney, iBAT, and rpWAT, Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units.


Figure 3A , Oil - red - O staining of control ( healthy ) and steatotic liver . Scale bar : 100 µm . B , Reconstructed MSOT image ( 800 nm ) with linear unmixing and difference value of lipid in lower abdominal section . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid , jet for 700 nm - 930 nm difference . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : Hb : 1 . 1 / 0 . 5 ; HbO2 : 1 . 9 / 0 . 8 ; Lipid : 21000 / 24000 ; 700 - 900nm : 3000 / 3000 ) Scale bar : 4 mm .

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Figure 3A, Oil-red-O staining of control (healthy) and steatotic liver. Scale bar: 100 µm.B, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of lipid in lower abdominal section. Unmixing result: blue for Hb, red for HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5; HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.


Figure 4A , HE staining of livers with grade 0 - 3 steatosis . Scale bar : 100 µm . E , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , and lipid from chow and HFD - fed mice . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value chow 1 month / chow 2 month / chow 3 month / HFD 1 month / HFD 2 month / HFD 3 month : Hb : 2 . 5 / 1 . 9 / 2 . 9 / 2 . 7 / 1 . 8 / 1 . 3 ; HbO2 : 2 . 8 / 2 . 6 / 2 . 8 / 3 . 1 / 2 . 1 / 1 . 9 ; Lipid : 27000 / 19000 / 29000 / 32000 / 26000 / 27000 ) . Scale bar : 4 mm . F , Longitudinal track of lipid in mouse livers . Each line represents data from one animal . Each data point is from a single measurement of each animal at each time point . All animals have grade 3 steatosis at the endpoint confirmed by histology .

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Figure 4A, HE staining of livers with grade 0 - 3 steatosis. Scale bar: 100 µm.E, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm.F, Longitudinal track of lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each time point. All animals have grade 3 steatosis at the endpoint confirmed by histology.


Figure 5A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of ICG from mice with and without steatosis . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : 0 . 07 / 0 . 02 ) Scale bar : 4 mm . E , Representative fluorescence microscopy and HE staining images of control and steatotic liver . Scale bar : 100 µm .

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Figure 5A, Reconstructed MSOT image (800 nm) with linear unmixing data of ICG from mice with and without steatosis. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: 0.07/0.02) Scale bar: 4 mm.E, Representative fluorescence microscopy and HE staining images of control and steatotic liver. Scale bar: 100 µm.


Figure 2A FBN2 mRNA levels in multiple human tissues . Human tissue cDNAs were used as templates for PCR of FBN2 , followed by agarose gel analyses . GAPDH levels served as loading controls ( biological replicates , n = 2 ) . B FBN2 mRNA levels in mouse tissues based on quantitative RT - PCR . Data were normalized based on levels in adipose tissues . C Comparison of FBN2 transcript levels between human and mouse placentas . Common primers for FBN2 and GAPDH in both species were used for RT - PCR analyses ( biological replicates , n = 3 ) . D Immuno - histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy . During early pregnancy , placensin immunostaining was found in cytotrophoblasts ( c ) , whereas syncytiotrophoblasts ( s ) showed weak staining . At 38 weeks of pregnancy , strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts ( arrows ) . Staining using E - cadherin antibodies confirmed the identity of cytotrophoblasts ( Inset showing higher magnification E - cadherin ( E - Cad ) staining ) . Lower panels depict sections stained with nonimmune IgG as negative controls . Scale bars , 100 μm . E Immunoblotting of placensin in human placental villi ( term pregnancy ) . Samples from three individual patients were processed with or without N - glycosidase pre - treatment to reveal untreated and deglycosylated placensin . F For prokaryotic cell expression , a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6 - histidine tags and sub - cloned into the PGEX - 6P - 1 vector for expression in E . coli . Left panel : Coomassie blue staining of proteins in the SDS - PAGE gel before and after digestion with the PreScission protease . 1 : Flow through ; 2 : Washes ; 3 : Before PreScission treatment ; 4 : After PreScission treatment . Right panel : Immunoblotting using placensin antibodies . G Immunoblotting of recombinant placensin generated in E . coli . and CHO cells . For CHO cell expression , placensin cDNA sequence was subcloned into an adenoviral vector pAV [ Exp ] - CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later . Recombinant placensin proteins secreted from CHO cells ( b ) showed higher molecular weight than those from bacteria ( a ) . Following treatment with N - glycosidase to remove N - linked carbohydrate side chains , prokaryotic - and eukaryotic cell - derived placensin proteins showed similar sizes .

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Figure 2A FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n=2).B FBN2 mRNA levels in mouse tissues based on quantitative RT-PCR. Data were normalized based on levels in adipose tissues.C Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT-PCR analyses (biological replicates, n=3) .D Immuno-histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E-cadherin antibodies confirmed the identity of cytotrophoblasts (Inset showing higher magnification E-cadherin (E-Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm.E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin.F For prokaryotic cell expression, a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6-histidine tags and sub-cloned into the PGEX-6P-1 vector for expression in E. coli. Left panel: Coomassie blue staining of proteins in the SDS-PAGE gel before and after digestion with the PreScission protease. 1: Flow through; 2: Washes; 3: Before PreScission treatment; 4: After PreScission treatment. Right panel: Immunoblotting using placensin antibodies.G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.


Figure 3A Prokaryotic cell - derived placensin stimulated cAMP production ( at 10 min . ) , and PKA activity ( at 30 min . ) by primary mouse hepatocytes and hepatocyte - derived AML12 cells ( biological replicates , n = 5 ) . B Placensin stimulation of glucose secretion ( at 5h ) and key gluconeogenesis gene transcripts ( at 1h ) in both cell types ( biological replicates , n = 4 ) . Transcript levels for PEPCK and G6Pase were determined using quantitative RT - PCR . Treatment with glucagon ( 10 nM ) served as positive controls . C Liver from adult female mice was perfused in situ with placensin ( 1 . 0 ug / pulse ) or PBS before measurement of hepatic glucose output from inferior vena cava ( biological replicates , n = 3 ) . D Adult female mice were injected intraperitoneally with placensin ( 30 ug / injection ) with blood sampling at different time points for glucose levels ( biological replicates , n = 5 ) .

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Figure 3A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5).B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls.C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3).D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).


Figure 4A Left panel : Immunoblotting of placensin content in HTR - 8 / SVneo , but not BeWo , JAR , and JEG3 , cells . Right panel : Time - dependent secretion of placensin by HTR - 8 / SVneo , but not JAR , cells monitored using an ELISA ( biological replicates , n = 4 - 5 ) . B Knockdown of FBN2 in HTR - 8 / SVneo cells led to decreases in FBN2 transcripts and placensin secretion . Cells were treated with FBN2 siRNA ( SiFBN2 ; 20 nM ) or control siRNA ( SiC ; 20 nM ) for 6h . Following media change , cells and conditioned media were collected at 48h after incubation . Placensin transcripts and proteins were determined by RT - PCR and ELISA ( biological replicates , n = 8 ) , respectively . Lower panel : immunoblotting of secreted placensin by HTR - 8 / Svneo . C Placensin stimulation of cAMP production and PKA activities by HTR - 8 / SVneo cells . Cells were treated with placensin for 10 min . before cAMP measurement ( biological replicates , n = 5 ) . For PKA activities , cells were treated with placensin ( 50 ng / ml ) for 30 min . Treatment with forskolin ( 10 uM ) served as positive controls . D Placensin stimulation of transcript levels for MMP9 but not MMP2 ( biological replicates , n = 12 ) . Cells were treated with placensin for 24h before RT - PCR analyses of transcript levels . E Placensin stimulation of MMP9 , but not MMP2 , activities without affecting cell proliferation . After 24h of treatment with placensin , media were collected for zymographic and densitometric analyses as well as determination of total cell numbers ( biological replicates , n = 4 ) . F Placensin stimulation of HTR - 8 / SVneo cell invasion . After placensin treatment for 24h , cell invasiveness was determined using the Transwell assay ( left panel : micrographs of cells ; right panel : fold changes in number of migrated cells , biological replicates , n = 4 ) . Cells were incubated with mitomycin C to rule out effects on cell proliferation . Scale bars : 100μm . C : control .

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Figure 4A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5).B Knockdown of FBN2 in HTR-8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6h. Following media change, cells and conditioned media were collected at 48h after incubation. Placensin transcripts and proteins were determined by RT-PCR and ELISA (biological replicates, n=8), respectively. Lower panel: immunoblotting of secreted placensin by HTR-8/Svneo.C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls.D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels.E Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n=4).F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.


Figure 5A Immunoblotting of serum placensin from patients at second trimester ( 1 - 3 ) , term pregnancy ( 4 - 6 ) using specific placensin antibodies . B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients . Numbers in parentheses represent number of patients .

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Figure 5A Immunoblotting of serum placensin from patients at second trimester (1-3), term pregnancy (4-6) using specific placensin antibodies.B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.


Figure 1stoichiometry ' s for B56α measured by ITC . Global direct fitting shown for one experiment ( reverse ) . Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value . The experiment was done in both direct ( B56 in cell ) and reverse ( B56 in syringe ) with similar results . C ) Time from Nuclear envelope breakdown ( NEBD ) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell . Only cells with similar expression levels of the various B56 inhibitor constructs were analysed . Median time is indicated by red line . A representative result from at least three independent experiments is shown . At least 25 cells were counted per condition in the experiment shown . A Mann - Whitney U ‐ test was used for statistical analysis ( ns : non ‐ significant , * * * P ≤ 0 . 001 ) . D ) Volcano plot representing mass spectrometry identified proteins co - purifying with B56 inhibitor versus control inhibitor from HeLa cells . PP2A - B56 subunits co - purifying with the B56 inhibitor are indicated . E ) and F ) Competition assay in HeLa cells stably expressing RFP - tagged B56 inhibitor ( LxxIxE ) or control inhibitor ( AxxAxA ) . YFP - B56α was transfected into and subsequently purified from these cell lines . Loss of binding of indicated proteins determined by either mass spectrometry ( pink - B56 SLIM containing protein and known B56 interactor ; blue - known B56 interactor , green - B56 SLIM containing protein ) ( E ) or Western blotting ( F ) .

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Figure 1stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results.C) Time from Nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analysed. Median time is indicated by red line. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann-Whitney U‐test was used for statistical analysis (ns: non‐significant, ***P ≤ 0.001).D) Volcano plot representing mass spectrometry identified proteins co-purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A-B56 subunits co-purifying with the B56 inhibitor are indicated.E) and F) Competition assay in HeLa cells stably expressing RFP-tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP-B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink - B56 SLIM containing protein and known B56 interactor; blue - known B56 interactor, green - B56 SLIM containing protein) (E) or Western blotting (F).


Figure 2A - B ) Schematic of synchronisation protocol for G1 / S ( A ) or mitotic ( B ) arrested cells and accompanying volcano plot of phosphorylation sites quantified . The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey . Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink .

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Figure 2A-B) Schematic of synchronisation protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey. Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink.


Figure 3A ) Michaelis - Menten kinetic parameters of purified PP2A - B56α and PP2A - B55α holoenzymes were determined against the indicated phospho - peptides . Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments ) B - D ) In vitro dephosphorylation by the PP2A - B55α and PP2A - B56α holoenzymes of panels of phosphorylated peptides as indicated . Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments )

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Figure 3A) Michaelis-Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)B-D) In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)