Figure 3
From: Possible involvement of zinc transporter ZIP13 in myogenic differentiation
Characterization of myocytes differentiated from EDSSPD3-iPSCsMYOD harboring the ZIP13G64D mutation. Differentiations of H1-iPSCsMYOD and H2-iPSCsMYOD or EDSSPD3-P1-iPSCsMYOD and EDSSPD3-P2-iPSCsMYOD into myocytes were induced by hMyoD overexpression under the control of DOX as depicted in Supplementary Figure S5a. Myogenic differentiation using H1-iPSCsMYOD and EDSSPD3-P1-iPSCsMYOD or H2-iPSCsMYOD and EDSSPD3-P2-iPSCsMYOD was performed simultaneously, respectively. (a,b) IF staining of MYH in differentiated iPSCsMYOD from H1-iPSCsMYOD and EDSSPD3-P1-iPSCsMYOD (a) or H2-iPSCsMYOD and EDSSPD3-P2-iPSCsMYOD (b). The images of MYH—(green) and DAPI-stained (blue) differentiated cells as observed on Day 8. Scale bar, 500 µm and 100 µm for × 4 and × 20 magnified images, respectively. (c–h) Relative mRNA expression analysis for human ZIP13 (hZIP13) gene and myogenic differentiation markers in differentiated H1-iPSCsMYOD and EDSSPD3-P1-iPSCsMYOD (c–e) or H2-iPSCsMYOD and EDSSPD3-P2-iPSCsMYOD (f–h). mRNA expression of hZIP13 (c,f) (n = 9) was determined by RT-qPCR on Days 0, 3, 6, and 8, and expressed in relation to the levels in healthy-iPSCsMYOD on Day 0 (set as 1). mRNA expressions of myogenic differentiation markers hEndo-MYOD (d,g) and hMYOGENIN (e,h) (n = 9 each) were determined by RT-qPCR on Days 3, 6, and 8, and expressed in relation to the levels in healthy-iPSCsMYOD on Day 3 (set as 1). mRNA expression was normalized to that of human GAPDH. Data represent the mean ± SEM and are representative of three independent experiments. **P < 0.01 and ***P < 0.001 versus healthy-iPSCsMYOD on Days 0 or 3. †P < 0.05 and †††P < 0.001 versus EDSSPD3-iPSCsMYOD on Days 0 or 3. (i–n) Relative protein expression analysis for myogenic differentiation markers in differentiated H1-iPSCsMYOD and EDSSPD3-P1-iPSCsMYOD (i–k) and H2-iPSCsMYOD and EDSSPD3-P2-iPSCsMYOD (l–n). Protein levels of hMYOD and hMYOGENIN in cell lysates were analyzed by western blotting on Day 8 (i,l). β-ACTIN was used as an internal control. Signal intensities were measured using ImageJ software. Protein levels of hMYOD (j,m) and hMYOGENIN (k,n) (n = 5 each) were normalized to that of β-ACTIN and were expressed relative to the levels in healthy-iPSCsMYOD on Day 8 (set as 1). Data represent the mean ± SEM and are representative of five independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus healthy-iPSCsMYOD.
