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. 2013 Dec 4;16(2):191–203. doi: 10.1093/neuonc/not167

Fig. 4.

Fig. 4.

Growth properties induced by NFIA is mediated by repression of p21. (A) NFIA represses p21 transcription. Normalized p21 mRNA relative to GAPDH in U87 cells transduced with increasing NFIA plasmid as described in Figure 3B (RT-PCR). P < .0001 by 1-way ANOVA; n = 3. (B) GAPDH-normalized p21 mRNA in U251 and GBM1 cells stably expressing NFIA or vector control; qPCR; *P < .0001. (C) Overexpression of NFIA regulates phosphorylation of cell-cycle dependent proteins, CDK2 (T160) and Rb (S780). Western blot of whole cell lysates of 18 hour serum-starved (ie, synchronized) glioma cells stably expressing NFIA or vector control. (D and E) Overexpression of p21 inhibits NFIA-induced cell growth and proliferation. Cell growth (D) or BrdU incorporation (E) were measured in U87 (p53-wild-type) or U251 (p53-mutant) cells stably expressing NFIA or vector that was transfected with p21 or empty-vector plasmid for 48 h. (assessed as in Figs 1D and 2A). Representative protein expression and densitometric analysis are shown in Figure S6A and B. *P < .01, **P < .0005 (D); *P = .001, **P < .0001 (E). (F) NFIA represses p21 promoter activity. Top: wild-type and mutant NFIA binding sites (161 bp) in the p21 promoter luciferase reporter. Left: luciferase activity was measured 48 hours after transfection of p21 luciferase reporter into U87 (p53-wild-type; top panels) or LN18 (p53-mutant; bottom panels) cells stably expressing NFIA or vector or 3 days post lentiviral transduction with shNFIA or controls; *P < .01, **P = .001; n = 3. Right: Relative luciferase activity of pGL3 wild-type (wt) or mutant (mt) p21 promoter transfected into U87 or LN18 cells expressing shNFIA or shCont similar to the left panel; see also Figure S6C for a schema; *P = .0001, **P < .05 (U87); *P < .005, **P < .001 (LN18).