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Figure 4 The characterization of gene expression in induced C2C12 cells
(A) RT-PCR analysis of germ layer markers in C2C12 cells induced for 3 and 8 days. (B) Western blotting analysis of Mvh expression in C2C12 cells induced for 3 and 8 days. Mvh is anti-Mvh antibody; Act is anti-actin antibody which was used as an internal control. (C) Immunofluorescent images of RA-treated C2C12 cells. 1, anti-Mvh antibody; 2, PI staining; 3, overlaid image; 4, phase-contrast image; 5, anti-Prm1 antibody; 6, H342 staining; 7, overlaid image; 8 phase-contrast image; arrows indicate the protamine-positive cells. Magnification is at ×200. (D) RT-PCR analysis of premeiotic, meiotic and postmeiotic gene markers. (E) Determination of DNA content of C2C12 cells by flow cytometry analysis. (a) The haploid standard from mouse sperm cells; (b) non-induced C2C12 cells; (c and d) C2C12 cells treated with RA for 3 and 8 days, respectively. Abbreviations: T, mouse testicle tissue, used as the positive control; Gap, GAPDH used as internal control; Ctl-, non-template control; Ctl, non-induced C2C12 or 3T3 cells; Daz, deleted in azoospermia-like gene; Str, Stra8; Tex, testis-expressed gene 18; Rbm, RNA-binding motif gene on Y chromosome; Scp, synaptonemal complex protein 3; Acr, acrosin; Tp2, transition protein 2; Prm1, protamine 1; NF, neurofilament-68 for ectoderm markers; Fgf, fibroblast growth factor-5; Bra, Brachyury (T) gene for mesoderm marker.