Fig. 4: TBX3 inhibits proliferation and promotes differentiation in RD cells.

a, b RD cells were transfected with pEF-TBX3 (TBX3) or pEF (EV) and three independent clones were characterized. mRNA expression of TBX3 (a) and TBX2 (b) was assayed by qRT-PCR. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus EV. c TBX3 inhibits proliferation. RD cells overexpressing TBX3 shown in (a) were assayed for proliferation by a cell counting assay. Error bars, S.E. and **p ≤ 0.01, ***p ≤ 0.001 versus EV. d Scratch assays were performed on RH30 cells overexpressing TBX3 (TBX3) or pEF (EV). Images were taken at ×100 magnification. Migration rate is quantitated in the right panel. Error bars, S.E. and *p ≤ 0.05, ***p ≤ 0.001 versus EV. e RD cells overexpressing TBX3 were assayed by immunofluorescence with antibodies against MyHC after 4 days differentiation (D4) DAPI was used to stain nuclei. Images were taken at ×100 and scale bars represent 100 μm. Nuclei per myofiber are quantitated in the right panel. f, g mRNA expression of TNNI2 (f) and MYOG (g) were examined in RD cells overexpressing TBX3 by qRT-PCR after 4 days of differentiation (D4). Error bars, S.E. and *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus EV. h Protein extracts from RD cells as in (f, g) were used for western blot assays with antibodies against MYOG. GAPDH was used as a loading control