Fig. 6: Overexpression and knockdown of EP2 in Hu5/KD3 cells.

a Experimental design of overexpression of EP2 in Hu5/KD3 cells. b Western blotting for EP2 (overexpression: OE1-3) and GAPDH (loading control) 3 days after transfection of the control vector (CMV-DsRed) or CMV-EP2-ires DsRed vectors. Relative expression levels of EP2 (after normalization to GAPDH) are shown below. c Representative image of myotubes (MF20, red) formed by Hu5/KD3 cells after transfection of the control vector (CMV-DsRed) or CMV-EP2-ires DsRed vectors. Scale bar, 100 µm. d Fusion index of (c). n = 4 samples/group. Dunnett’s multiple comparisons test. e RT-qPCR for EP2 (PTGER2) of nontarget (control) short-hairpin (sh) RNA plasmid-transfected and EP2 shRNA plasmid-transfected Hu5/KD3 cells. Plasmids were introduced into Hu5/KD3 cells by electroporation. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test, n = 3 samples/group. f Immunoblot analysis for EP2 and GAPDH in nontarget and shEP2 plasmid-transfected Hu5/KD3 cells. g EP2 signals were normalized to GAPDH. h Representative immunostaining of myotubes formed by nontarget shRNA plasmid or EP2 shRNA plasmid-transfected Hu5/KD3 cells with anti-myosin heavy chain antibody, MF20 (red) and nuclei (DAPI, blue). Scale bar = 200 μm. i Fusion index of Hu5/KD3 cells four days after transfection of shRNA plasmids. Data are shown as mean ± S.D. and analyzed by unpaired two-tailed Student’s t-test, n = 4 independent experiments. Each dot is an average of 3 views/sample. Both p-value and the effect sizes of Pearson’s r (r) are shown. j Full, uncropped images of western blots in b and f are shown in Supplementary Fig. 10.