Expression pattern of SGCA in MPCs. (A) Western blot analysis showing SGCA expression in C2C12, wt or Sgca-null mouse MPCS in proliferating (p) or differentiating (d) conditions. Skeletal muscle tissue extract (sk muscle) was used as positive control. n=3. GAPDH was used as an internal control. (B) Western blot (WB) analysis of SGCA and myogenin (Myog) expression in differentiating wt MPCs. n=3. β-tubulin (βTub) was used as internal control. (C-C′′) Immunofluorescence staining for SGCA (red in C′) and myogenin (green in C′) expression during early stages of differentiation in wt MPCs (day 1 after starvation). Yellow arrows indicate double-positive cells. n=4. (D-D′′) Representative images of wt MPCs co-expressing SGCA (red in D′) and PAX7 (green in D′). Yellow arrows indicate PAX7⁺/SGCA⁺ wt MPCs. Scale bars: 100 μm.

Proliferative ability following cytokine treatment. (A) Total number of cells per clone from wt, Sgca-null and mdx mouse MPCs grown for 5 days in conditioned media containing bFGF (gray lines) or HGF (dashed lines). CTRL, growth curves of MPCs in non-conditioned media (black lines). n=5; ^*, P<0.01 for CTRL versus bFGF; ^**, P<0.01 for CTRL versus HGF; square, P<0.01 CTRL versus bFGF. Error bars indicate s.e.m. (B) WB for CycD expression in wt and Sgca-null MPCs stimulated with bFGF or HGF. ctrl, MPCs grown in non-conditioned media. GAPDH was used as an internal control. (C) Growth curves of wt MPC clones infected with shFGFR1 (gray line) or with scramble control shRNA (black dashed line) stimulated with bFGF. The shFGFR1 wt MPC curve strongly overlaps with that of Sgca-null MPCs grown with or without bFGF (compare gray line with Sgca-null gray line in A). n=3; ^*, P<0.01 for shFGFR1 versus scramble. (D) Growth curves of Sgca-null MPCs treated with YP-740 (black dashed line) or PMA (gray line). n=3; ^*, P<0.05 for Sgca null + bFGF versus PMA; ^**, P<0.01 for Sgca null + bFGF versus YP-740. (E) WB analysis for CycD expression in wt MPCs infected with shFGFR1 (lane 1) or with scramble (lane 2), and Sgca null treated with YP-740 (lane 3) or PMA (lane 4). n=3. (F) Human myoblasts isolated from healthy (30XY, left graph) or LGMD2D (32XY, right graph) patients were stimulated with bFGF (gray lines) or HGF (dashed lines) and the total number of cells was counted (n=5). Black lines, control growth curves of myoblasts in non-conditioned media. n=2 patient biopsies for LGMD2D and healthy myoblasts, respectively. ^*, P<0.05 for healthy control versus bFGF; ^**P<0.05 for healthy bFGF versus HGF; square, P<0.001 for LGMD2D bFGF versus HGF using two-way ANOVA test. (G) WB analysis for CycD expression in human myoblasts treated with bFGF and HGF. ctrl, human myoblasts grown in non-conditioned media. n=3.

SGCA directly cooperates with FGFR1. (A) qPCR for Fgfr1 and Fgfr4 in proliferating Sgca-null (white) and wt (black) MPCs. n=5; ^*, P<0.01 and ^**, P<0.01 for Sgca null versus wt. Error bars indicate s.e.m. (B) WB analysis for FGFR1 and syndecan 1 (Syn-1) expression in wt and Sgca-null MPCs in proliferating (p) and differentiating (d) conditions. n=3. (C-D′) Immunofluorescence staining for FGFR1 (green) and SGCA (red) expression in early differentiating MPCs (day 1 after starvation) showing a diverse cellular localization of FGFR1 in Sgca-null (C′) and wt (D′) MPCs. SGCA signal in D′ was slightly overexposed to highlight cell membrane borders. n=3. (E-E′) Immunofluorescence staining for FGFR1 (E, green) and SGCA (E′, red) showed protein colocalization in wt MPCs at an early step of differentiation (day 1 to 2) (E′, white arrows). Such colocalization was not observed in fibroblasts, which were found to be exclusively positive for FGFR1 (E′, yellow arrow). n=3. (F-F′) Confocal images of early differentiating MPCs (day 1) representing areas of partial colocalization (F′, white arrows) of FGFR1 (green in F) with SGCA (red in F′). n=3. (G-G′) A newly formed myotube and fibroblasts (G′, yellow arrows) show no colocalization of FGFR1 (green in G) and SGCA (red in G′) signal. n=3. (H) Surface protein biotinylation assay showing that FGFR1 is expressed both by wt (wt lysate) and Sgca-null (Sgca-null lysate) MPCs but can localize on the cell membrane together with SGCA only in wt MPCs (wt cell surface). No FGFR1 signal was detected in the surface protein fraction of Sgca-null MPCs (Sgca-null cell surface). β-tubulin was used as an internal control for the cell lysate fractions. n=3. (I) Immunoprecipitation analysis in 293T cells co-expressing SGCA and FGFR1, revealing an interaction of SGCA with FGFR1 (top, SGCA/Fgfr1). No signal was detected in 293T cells transfected only with vectors expressing Sgca (top, SGCA) or Fgfr1 (top, Fgfr1). n=3. (J) WB analysis of wt MPCs and muscle tissue extracts from wt and Sgca-null mice immunoprecipitated with SGCA polyclonal antibodies and detected with FGFR1 (top) and SGCA (bottom) monoclonal antibodies. FGFR1 co-immunoprecipitates with SGCA in samples derived from early differentiating wt MPCs (1d and 3d), skeletal muscle tissue extract (wt) and muscle crushed with cardiotoxin (wt + ctx). No signal was obtained from proliferating wt MPCs (0d) or Sgca-null skeletal muscle. n=3. Scale bars: 30 μm in C-D′; 50 μm in E-G′.

In vivo transplantation of dystrophic MPCs failed to reconstitute the myogenic compartment. (A-B′) MPCs isolated from wt (A,A′) and Sgca-null (B,B′) mice backcrossed with GFP transgenic mice. MPC purity was evaluated by double staining for MYOD (red in A,B) and GFP (green in A′,B′) expression. (C) Percentage MYOD⁺/GFP⁺ cells from wt (black) and Sgca-null (white) mice. n=200 clones. (D) qPCR for GFP expression at 2, 4 and 6 days following intramuscular injections of wt (black) and Sgca-null (white) GFP⁺ MPCs into the tibialis anterior of Sgca-null juvenile mice. Three mice were injected per time point for a total of nine mice per group. n=18 mice; ^*, P<0.01 and ^**, P<0.01 for Sgca null versus wt. (E) nlacZ⁺ MPCs (5×10⁵) were injected into the tibialis anterior of juvenile 3-week-old Sgca-null mice. Global cell death rate was calculated by counting TUNEL⁺ cells at three different time points (20 hours, 3 and 10 days). Three mice were injected per time point for a total of nine mice per group. n=18; ^*, P<0.01 and ^**, P<0.01 for Sgca-null versus wt. (F) Double staining for β-gal and TUNEL revealed the apoptotic rate of injected MPCs. Approximately 100 muscle sections were analyzed at each time point. n<100; ^*, P<0.01 for day 3 versus day 10; ^**, P<0.01 for day 3 versus 20 hours. Error bars (C-F) indicate s.e.m. (G-G′) Representative images of TUNEL⁺ Sgca-null donor cells. At day 3 after injection, massive cell death of dystrophic MPCs was observed. Apoptotic donor cells are β-gal⁺ (G′, red) and TUNEL⁺ (G′, green). (H) High-magnification images of donor MPCs (white arrows) following transplantation. In the bottom panel, a TUNEL⁺/β-gal⁺ Sgca-null MPC is situated close to an endogenous apoptotic cell (TUNEL⁺/β-gal^–, yellow arrow). Scale bars: 100 μm.

Genetic correction restores the myogenic potential of Sgca-null MPCs. (A,B) Phase contrast images of single clones from corrected (B) and non-corrected (A) Sgca-null MPCs. (C) Number of cells per clone of corrected (gray) and non-corrected (white) Sgca-null MPCs. Black bars indicate wt MPCs. n=5; ^*, P<0.01 for corrected versus non-corrected MPCs. (D,D′) MyHC (red) and V5 (green) staining of Sgca-null corrected cells. n=5. (E) Number of nuclei per myotube of corrected (gray) and non-corrected (white) Sgca-null MPCs. Black bars indicate wt MPCs. n=5 experiments performed in triplicate; ^*, P<0.01 for corrected versus non-corrected MPCs. (F) WB analysis of protein extracts from corrected Sgca-null and wt MPCs immunoprecipitated with SGCA polyclonal antibodies and detected with FGFR1 (top) and SGCA (middle) monoclonal antibodies. No SGCA signal was obtained from the immunodepleted (ID) fraction (bottom). n=3. (G) qPCR for GFP expression at 2, 4 and 6 days following intramuscular injections of wt (black), corrected Sgca-null (gray) and non-corrected Sgca-null (white) GFP⁺ MPCs into the tibialis anterior of Sgca-null juvenile mice. Three mice were injected per time point for a total of nine mice per group. n=18 mice; ^*, P<0.01 and ^**, P<0.01 for corrected versus non-corrected MPCs. Error bars (C,E,G) indicate s.e.m. Scale bars: 200 μm in A,B; 100 μm in D,D′.