Fig. 8

MDLS promoted the expression of Oct4 through the JAK/STAT3 and classic Wnt signaling pathway. A Left: the phosphorylation levels of STAT3 were detected by western blot analysis in P19 cells treated with MDLS for the indicated times. Right: quantitative analysis of Western blotting data by image J soft-ware. Levels of these proteins were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. B Left: the phosphorylation levels of Akt were detected by western blot analysis in P19 cells treated with MDLS for the indicated times. Right: quantitative analysis of Western blotting data by image J soft-ware. Levels of these proteins were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. C Left: the levels of β-Catenin in the nucleus were detected by western blot analysis in P19 cells treated with MDLS for the indicated times. Right: quantitative analysis of Western blotting data by image J soft-ware. Levels of these proteins were normalized to the corresponding Histone3 band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. D Ruxolitinib (JAK1 inhibitor) reversed the MDLS-induced up-regulation of Oct4. Left: P19 cells were treated with Ruxolitinib (2.5 μg/mL) and MDLS (5 μg/mL) or DMSO for 24 h. The expression of Oct4 was determined using Western blot analysis. Right: quantitative analysis of Western blotting data by image J soft-ware. Levels of these proteins were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. E Salinomycin (β-Catenin inhibitor) reversed the MDLS-induced up-regulation of Oct4. Top: P19 cells were treated with Salinomycin (0.0078125 μg/ml) and MDLS (5 μg/mL) or DMSO for 24 h. The expression of Oct4 was determined using Western blot analysis. Bottom: quantitative analysis of Western blotting data by image J soft-ware. Levels of these proteins were normalized to the corresponding GAPDH band expressed as relative fold changes in comparison to control samples. Values correspond to the mean ± S.E. of three independent experiments. F P19 cells were co-transfected with pGL3-Oct4p or pGL3-basic vector and pCMV-β-galactosidase plasmid and treated with Salinomycin, Ruxolitinib and MDLS for 24 h. DMSO was used as a negative control. Subsequently, the luciferase activity was measured and normalized to β-galactosidase activity. The results were expressed as the fold induction (over the activity of the negative control)