Fig. 5

Verification of the transformation mediated by RAS and p38 signaling pathways. a-d The morphology of SSCs after 5 passages were cultured in medium 1 (a), medium 10 (b), medium 11 (c) and medium 2 (d) for another 5 passages was exhibited. e The strategy to screen the key time point of SSCs transformation through culturing SSCs in medium 10, and replacing the medium with medium 11 at different passages. f-g The relative expression levels of Rac1, Hras, Kras, Snail and Gapdh in SSCs after addition of EGF into medium 1 (f), and expression of Stat3, Kras, Hras, Nras, Snail and Gapdh in SSCs after addition of LIF into medium 1 (g) were detected using RT-PCR. h The relative expression levels of Rac1, Hras, Kras, Smad3, Stat3, Nras, Snail and Gapdh in SSCs after EGF supplement into medium 2 were detected using RT-PCR. i A regulatory pattern of EGF and LIF on Klf4, Myc, p38, Snail and Smad3 was summarized. j-n The morphology of SSCs after 20 passages (j) were cultured in medium 1 (k), medium 2 (l), medium 2 plus BMS-582949 (m) and medium 1 plus BMS-582949 (n) for another 3 passages, respectively. o-p. Western blot determined the expression levels of NANOG, SOX2, MVH, PLZF and β-tubulin in SSCs of 20 passages after treatment of BMS-582949 (1. SSCs in medium 1, 2. SSCs in medium 2, 3. SSCs in medium 2 + BMS-582949, 4. SSCs in medium 1 + BMS-582949) (o), and the results were statistically analyzed (p). q The expression levels of Acvr1b, Bmpr1a, Bmpr1b, Bmpr2, Fgfr3, Fgfr4, Hras, Kras, Nodal, Nras, Rac1, Snail, Tgfr1, Tgfr2, Tgfr3 and Zeb2 in SSCs, In state cells, GSPCs and ES-like cells were detected using RT-PCR. Scale bar = 20 μm, data represent as mean ± SD *p < 0.05; **p < 0.01