COCH suppresses neural differentiation of mouse ESCs.
A, R1 ESCs stably expressing GFP or COCH were cultured in normal KSR medium (ES) or N2B27 medium to allow neural differentiation for 5 days (ND). mRNA levels of the neural differentiation markers Sox1, Nes, and Tubb3 were determined by qRT-PCR. B, R1 ESCs stably expressing GFP or COCH were cultured in N2B27 medium for 5 days and were then subjected to anti-nestin immunostaining analysis. Scale bars: 50 μm. C, Coch knockdown R1 mouse ES cells (shCoch-1 and shCoch-2), and the control luciferase knockdown cells (shLuc) were cultured in normal KSR medium (ES) or N2B27 medium (ND) for 5 days, and then harvested for qRT-PCR analysis. D, Coch knockdown R1 mouse ESCs or control cells (shLuc) were cultured in N2B27 medium for 5 days, and then subjected to anti-nestin immunostaining analysis. Scale bars: 50 μm. E, Coch knockdown R1 mouse ESCs (shCoch-1 and shCoch-2) and control (shLuc) cells were cultured in N2B27 medium with or without BMP4 for 5 days and then harvested for qRT-PCR. BMP4 neural inhibition rate (+BMP4/−BMP4) was presented by the ratio of the corresponding mRNA levels of +BMP4 to −BMP4. Quantitation data are shown as mean ± S.E. (n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001).