DEV070706 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Total MPC number and comparison of isolation methods. (A) Overall MPC number obtained from wt (black bars) and Sgca-null (white bars) mice after a single round of isolation. Error bars indicate s.e.m. n=3, Asterisks indicate P<0.01 for wt versus Sgca null. (B) Direct comparison of PAX7⁺/MYF5⁺ (yellow bars), PAX7⁺/MYF5⁻ (green bars), PAX7⁻/MYF5⁺ (red bars) and PAX7⁻/MYF5⁻ (blue bars) MPC pools after enzymatic digestion (preplating) or FACS-based sorting (sorted) isolation. These results come from three independent experiments performed in triplicate per sample. n=3. (C-F) Representative images of wt (C,D) and Sgca-null (E,F) MPCs stained for PAX7 (green) and MYF5 (red). MPCs were isolated by a FACS-based sorting (C,E) or enzymatic digestion (D,F) procedure. Scale bar: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Molecular analysis of Sgca and Fgfr1 expression in MPCs. (A) qPCR analysis of proliferating and differentiating wt MPCs showing expression of Sgca at the mRNA level (black bars). No signal was detected from proliferating and differentiated Sgca-null MPCs (white bars). Skeletal muscle mRNA was used as positive control (blue bars) Error bars indicate s.e.m. *, P<0.01 wt prol versus Sgca-null prol. (B) Quantitative analysis of PAX7⁺/SGCA⁺ cells from freshly isolated wt MPCs. Error bars indicate s.e.m. α<0.05 PAX7⁺/SGCA⁺ versus PAX7⁻/SGCA⁺ MPCs. (C) RT-PCR analysis for Fgfr1 expression in proliferating wt MPCs infected with shRNA lentiviral particles targeting Fgfr1 (lane 1) or scrambled (lane 2). Ctrl +, 293T cells transfected with an Fgfr1-expressing vector. Gapdh was used as internal control. (D) WB analysis for FGFR1 expression in proliferating wt MPCs infected with shFGFR1 (lane 1) or scrambled (lane 2) lentiviral particles. Ctrl +, 293T cells transfected with an Fgfr1-expressing vector. GAPDH was used as internal control.

• Supplemental Figure S3 -

  Fig. S3. ERK1/2 phosphorylation analysis. (A) WB analysis for phosphoERK1/2 (pErk) and total ERK1/2 (tErk) in wt or Sgca-null MPCs in the proliferating (pwt and pSgca-null) or late differentiation (dwt and dSgca-null) state. (B) WB analysis for pErk and tErk in wt and Sgca-null MPCs in growth medium (Ctrl) and with the addition of HGF or bFGF. (C) WB analysis for pErk and tErk in wt MPCs infected with shFGFR1 (wt shFGFR1, lane 1) or scrambled (wt scramble, lane 2) lentiviral particles. The same analysis was performed on Sgca-null MPCs treated with YP-740 (lane 3) or PMA (lane 4). (D) WB analysis of pErk and tErk in human myoblasts from healthy (30XY) and LGMD2D patients (32XY) in growth medium (Ctrl) and with the addition of bFGF or HGF. GAPDH was used as loading control (see GAPDH panels in Fig. 3B,E,G).

• Supplemental Figure S4 -

  Fig. S4. Apoptotic rate and proliferative impairment of Sgca-null MPCs. (A) Percentage of pHH3⁺/MYF5⁺ in wt (black bars) and Sgca-null (white bars) MPCs cultured in proliferating conditions (Ctrl) and treated with bFGF or HGF. The experiments were performed in triplicate per sample. Error bars indicate s.e.m. Asterisks indicate α<0.01 for wt versus Sgca null. (B,C) Representative images of wt (B) and Sgca-null (C) MPCs cultured for 3 days in bFGF and stained for pHH3 (green) and MYF5 (red). Scale bars: 50 µm. (D) Caspase 9 concentration plot of wt (black squares) and Sgca-null (white dots) MPCs cultured in proliferation medium (Ctrl) and treated with bFGF or HGF. Values are presented as mean±s.e.m. Results are from three independent experiments performed in triplicate.

• Supplemental Figure S5 -

  Fig. S5. Data analysis for FGFR expression. (A) Gene card analysis on tibialis anterior from wt and Sgca-null mice showed upregulation (>3-fold) of both Fgfr1 and Fgfr4 in dystrophic mice as compared with wt. (B) Microarray analysis on differentiating and proliferating C2C12 cells showed that these cells express high levels of the fibroblast growth factor receptors 1 and 4 previously published by Biressi et al. (Biressi et al., 2007); accession numbers GSM124899, GSM124900, GSM124901, GSM124902, GSM124903, GSM124904. (C) WB analysis of FGFR4 in proliferating and differentiating wt and Sgca-null MPCs. (D) WB analysis of FGFR4 in cell lysates and surface protein fractions from wt and Sgca-null MPCs. GAPDH was used as internal control.