Figure 6
The FSH pathway transduces effects of ING5 on stem cell properties. (a) Sphere formation assay for cells treated with calcium modulators and FSHR blocking antibody (Anti-FSHR) at the indicated concentrations (n=3, *P<0.05 and **P<0.01 compared to iPB-ctr/DMSO; #P<0.05 and ##P<0.01 compared to iPB-ING5/DMSO). (b) IPA downstream function analysis indicates the FSH pathway is elevated by ING5. Genes positively correlated with this function were listed with fold changes. (c) RT-qPCR of genes related to hormone and steroidogenesis functions. (n=3, *P<0.05, **P<0.01) (d) The expression levels of FSHB and FSHR genes in BT 189 cells before and after differentiation for 1–5 days. (e) Immunostaining for ING5 and FSHR in iPB cells. Scale bar=100 μm. (f) Flow cytometry analysis of CD133 positive cells in BT 189 cells treated with FSHR neutralizing antibody or IgG control, gated by isotype control. (g) Immunofluorescence of Nestin and Tubb3 shows inhibition of the FSH pathway induces neuronal differentiation. Scale bar=200 μm. (h) FSH recombinant protein treatment at indicated concentrations increases sphere formation rates in shRNA cell lines (n=3, *P<0.05 and **P<0.01 compared to untreated shR-ctr; #P<0.05 compared to untreated shR-ING cells). (i) FSH recombinant protein treatment induces sphere-forming abilities in iPB-ctr cells but not in ING5 overexpressing cells (n=3, **P<0.01). (j) Sphere formation rates for cells treated with Anti-FSHR or BAPTA alone, and the combination of both (n=3, *P<0.05). (k) FSH treatment at 5 ng/ml for 3 days induces the expression of OCT4 and Nestin in BT 189 cells.
