Fig. 2: CEBPD is involved in intrinsic TMZ resistance of GSCs.

A Caspase 3/7 activity is decreased in TMZ-treated GSCs. U87MG cells were cultured in stem cell medium for 3 days to form spheroids and treated with 50 μM TMZ for 72 h. The caspase 3/7 activity was detected by the Caspase-Glo® 3/7 Reagent. B Attenuated CEBPD increases caspase 3/7 activity in TMZ-treated GSCs. Knockdown of CEBPD was conducted in U87MG stable cells with IPTG for 48 h, and then, the cells were cultured in stem cell medium for 3 days to form spheroids and treated with 50 μM TMZ for 72 h. The caspase 3/7 activity was determined by the Caspase-Glo® 3/7 Reagent. C U87MG cells were cultured in stem cell medium to form spheroids, and the spheroid size was increased at 3 and 6 days. D RT-qPCR analyses and (E) western blots were performed with total RNA and total cell lysates harvested from U87MG monolayer cells and U87MG-derived spheroids. F U87MG-CD133+ cells express high levels of CEBPD and stemness-related proteins. U87MG and U87MG-CD133+ cells were cultured in stem cell medium for 3 days to form spheroids and cell lysates were harvested for western blot analysis. Scale bar, 100 μm. The summary data are presented as the mean ± SEM; the numbers in the bars represent the sample sizes; one-way ANOVA and Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant, shLacZ: shβ-galactosidase, shCD: shCEBPD.