Figure 1

ZIP13 is required for myogenic differentiation of C2C12 cells. (a) Schematic of the protocol for horse serum (HS)-induced myogenic differentiation of C2C12 cells. C2C12 cells were cultured for 24 h in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS); thereafter, the medium was changed to DMEM containing 2% HS and cultured for 5 days. The cells were harvested at the indicated time points for the indicated analyses. (b–d) Gene expression profiles of HS-induced mZip13 (b) and myogenic differentiation markers, mMyoD (c) and mMyogenin (d), (n = 3 each) analyzed by qPCR. C2C12 cells were harvested at the indicated time points. Data are presented as the mean ± standard error of mean (SEM) of three independent experiments. *P < 0.05 and **P < 0.01 relative to the data on Day 0. (e) The expression of Zip13 mRNA in Scramble 1 and two clones of Zip13-KD (clone 6 and 7) of C2C12 cells (n = 4 each), developed using the Zip13 shRNA plasmid was examined by qPCR. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01 relative to the values of Scramble C2C12 cells. (f–k) Scramble and two clones of Zip13-KD C2C12 cells (6 and 7) were differentiated into skeletal muscle cells by HS treatment as described in Fig. 1a. (f) Microscopic evaluation of the morphological changes in Scramble and Zip13-KD C2C12 cells (clone 7) on Days 0 and 3 of myogenic differentiation. The yellow arrows indicate myotube formation. Scale bar, 50 μm. (g–i) Gene expression profiles of mMyoD (g), mMyogenin (h), mMyf5 (i), and mMyh2 (j) (n = 4 each) in Zip13-KD C2C12 cells during myogenic differentiation was analyzed by qPCR at the indicated time points (Days 0–3). Data are presented as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus Scramble C2C12 cells. (k) MYH protein expression in Scramble and two clones of Zip13-KD C2C12 cells on Days 0 and 3 of myogenic differentiation were analyzed by western blotting. All data were collected from 3–4 independent experiments.