Figure 1. Linc-RAM enhances myogenic differentiation in a MRLN-independent manner.

(a) Linc-RAM was knocked down in C2C12 cells. Knockdown efficiency was examined by RT–qPCR. (b) The differentiation of Linc-RAM knockdown C2C12 cells was assayed by staining for MHC at 48 h in differentiation medium (DM). Scale bars, 50 μm. (c) MHC⁺ cells in b were counted. (d) MHC expression in (b) was detected by western blotting. β-actin served as a loading control. (e) Linc-RAM was overexpressed in C2C12 cells using a lentivirus system. The degree of Linc-RAM overexpression (fold increase) was determined by RT–qPCR. (f) The differentiation of C2C12 cells stably overexpressing Linc-RAM was examined by MHC staining at 48 h in DM. Scale bar, 50 μm. (g) Fusion index in f were calculated. (h) MHC expression in f was detected by western blotting. β-actin served as a loading control. (i) Schematic illustration of the plasmids for full-length Linc-RAM and two truncation mutants, Δ1 and Δ2; Δ1 contains exons 1 and 2, whereas Δ2 covers exons 2 and 3. MRLN peptide is indicated as blue line. (j) Differentiation of C2C12 cells transfected with the full length and truncated Δ1 and Δ2 was examined by staining for MHC after culturing in DM for 36 h. Scale bars, 50 μm. (k) MHC+ cells in j were counted and presented as positive cells per view. (l) MHC mRNA expression in j was detected by RT–qPCR. (m) Schematic illustration of the plasmids with WT Linc-RAM containing MRLN ORF and mutant Linc-RAM harbouring a frameshift for MRLN ORF. (n) Differentiation of C2C12 cells transfected with WT and mutant Linc-RAM was examined by staining for MHC after culturing in DM for 36 h. Scale bars, 100 μm. (o) MHC mRNA expression in n was detected by RT–qPCR. All images in the figure are representatives of three independent experiments. Values are means±s.e.m. of three independent experiments. The statistical significance of the difference between two means was calculated with the t-test. **P<0.01, ***P<0.001. NS stands for statistically non-significant.