Figure 5

ING5 maintains high intracellular calcium levels to promote BTIC self-renewal. (a, b) Live cell calcium imaging in iPB (left panels) and shRNA (right panels) cell lines. Fluo3-AM (7 μM) was loaded for 1 h before imaging. Scale bar=400 μm. (c, d) Flow cytometry analysis of Fluo3-AM fluorescence intensity in shRNA (c) and iPB (d) cell populations, gated by calcium chelator BAPTA-AM treatment control. (e) Sphere formation rate in shRNA cell lines treated with Ionomycin, CPA, BAPTA-AM, Nifedipine or SKF96365 at the indicated concentrations for 12 days (n=3, **P<0.01 compared to shR-ctr/DMSO, ^#P<0.05 and ^##P<0.01 compared to shR-ING5/DMSO). (f) Flow cytometry analysis of CD133 positive cells in BTIC 189 cells treated with calcium inducers Ionomycin (20 nM) or CPA (100 nM) for 2 days, gated by isotype control. (g) The effect of Ionomycin treatment on levels of phosphorylated AKT and ERK1/2 in the PI3K and MEK pathways, respectively. Cells were treated with 10 nM Ionomycin for 48 h. (h) Sphere formation rate in iPB cells treated with KN-93 or Cyclosporin A at the indicated concentrations. n=3, *P<0.05 compared to iPB-ctr/DMSO and ^#P<0.05 compared to iPB-ING5/DMSO.