Extended Data Fig. 5: Screening for COLV receptor candidates identifies CALCR.

a, Screening for the COLV receptor: satellite cells from Tg:Pax7-nGFP mice were incubated for ten days with COLV and candidate receptors were targeted with respective inhibitors: 7rh for DDR1 (sub-panels C, D), the broad-spectrum Integrin-binding competitor RGDS peptide (sub-panels E, F), Obtustatin for Integrin α1β1 (sub-panels G, H), TC-I 15 for Integrin α2β1 (sub-panels I, J). DMSO solvent was used as a control for TC-I 15 and 7rh (sub-panels A, B). Satellite cell differentiation was assayed by MyHC immunostaining. b, EdU (2 h pulse) and CALCR staining of GFP⁺ C2C12 cells isolated by FACS and transduced with Calcr-GFP or mock-GFP retrovirus and cultured for 24 h with COLI (top) or COLV (bottom). Quantification of EdU⁺ Calcr-transduced C2C12 cells or mock-GFP cells treated for 24h with COLV or with the controls, COLI and HOAc (n = 5 independent experiments, ≥250 cells counted, 2 wells per condition). There was no significant difference between HOAc and COLI treated samples (data not shown). c, Experimental scheme of tamoxifen administration to control (Ctr) (Calcr^+/+) and cKO (Calcr^flox/flox) mice. FACS plot of satellite cells from Pax7^CreERT2/+;Calcr^flox/flox;R26^stop-YFP and Pax7^CreERT2/+;Calcr^+/+;R26^stop-YFP mice. Cells sorted based on YFP expression. d, Control and Calcr cKO satellite cells isolated by FACS, fixed immediately after sorting and immunostained for CALCR to confirm the absence of CALCR protein from recombined cells. For control (upper panel), two fields from the same culture dish are shown, separated by a white line. Asterisk shows a non-recombined, CALCR⁺ cell in the cKO sample (lower panel). e, Quantification of PAX7⁺, Myogenin⁺ and EdU⁺ cells in Calcr-depleted satellite cells (Pax7^CT2/+;Calcr^flox/flox;R26^stop-YFP) isolated by FACS and treated for 32 h or 72 h with COLI or COLV (n = 3 mice, ≥250 cells counted, 2 wells per condition). f, Quantification of total PAX7⁺ (GFP), Myogenin⁺ and EdU⁺ myogenic cells isolated by FACS from Tg:Pax7-nGFP mice three days after cardiotoxin injury of tibialis anterior muscle, and incubated for 72 h in presence of COLI or COLV, or HOAc as a control, in the culture medium (n  = 3 mice, ≥200 cells counted). g, CALCR protein in freshly isolated satellite cells, or satellite cells cultured for 12 h, from Tg:Pax7-nGFP mice, demonstrating that CALCR protein is still present when satellite cells are treated with different collagens (see Extended Data Fig. 2). h, Induction of Calcr transcript expression by RT–qPCR of Tg:Pax7-nGFP satellite cells isolated by FACS and cultured for 72 h in the presence of COLI or COLV. Results are normalized to Tbp (n = 3 mice). i, Immunostainings for CALCR protein of Tg:Pax7-nGFP satellite cells cultured for 72 h in presence of COLI or COLV (n = 3 mice, ≥50 cells, 2 wells per condition). Data are mean ± s.d.; b, two-sided unpaired t-test; c–i, two-sided paired t-test. Scale bars, 25 μm (g), 50 μm (a, b, d, i).

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