Skip to main content
. 2014 Jan 31;21(5):735–747. doi: 10.1038/cdd.2013.200

Figure 4.

Figure 4

Smac mimetic-induced astrocytic differentiation is blocked in IκBα-SR cells. (a) GBM10 cells were transduced with IκBα-SR or vector control and were analyzed for expression of IκBα by western blotting. α-tubulin expression served as a loading control. (b) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. Expression levels of p100 and p52 were analyzed by western blotting. Expression of GAPDH served as loading control. (c) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. Expression levels of p100, p52, p65 and p50 were analyzed in cytoplasmic (C) and nuclear (N) fractions by western blotting. α-tubulin served as purity and loading control for cytoplasmic fractions and lamin A/C for nuclear fractions. (d and e) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. Cell morphology was analyzed by phase-contrast microscope; scale bar, 500 μm. A representative experiment of three experiments is shown (d). Cell elongation was quantified by measuring cell length and width and by calculating cell elongation index (length/width); fold increase in cell elongation is shown (e). (f) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. GFAP promoter activity was determined by luciferase assay. (g) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. GFAP mRNA levels were analyzed by quantitative RT-PCR and fold increase in GFAP mRNA levels is shown. (h) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. Cells were then fixed and stained with anti-GFAP antibody and counterstained with DAPI. Representative pictures of three independent experiments are shown. Scale bar, 500 μm. (i and j) GBM10 cells stably expressing IκBα-SR or vector control were treated for 7 days with 1 μM BV6 or DMSO. Cells were dissociated and stained with anti-GFAP antibody and isotype control for FACs analysis (i). The percentage of CD133-positive cells is presented (j). Mean+S.E.M. of three independent experiments is shown. In (e–g) mean+S.E.M. of three independent experiments performed in duplicate (f) or triplicate (e and g) is shown; *P<0.05; **P<0.01; n.s., not significant