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

Fig. 2.

Fig. 2.

NFIA controls proliferation and cell death. (A) NFIA promotes and shNFIA inhibits proliferation (BrdU uptake) of glioma cells freshly transduced with lentivirus expressing NFIA (red), shNFIA (blue), and controls. Means ± SD of 3 different experiments performed in 3 to 8 replicates; *P < .0001, **P < .05. (B) Knockdown of NFIA increases sub-G1 fraction of glioma cells. GBM1 or U87 glioma cells transduced with shNFIA or control analyzed by propidium iodide (PI) on day 3 after lentiviral infection; n = 3; *P < .0005, **P < .0001. (C) NFIA silencing increases caspase-3 activity. Caspase-3 activity was determined on day 3 after infection with shNFIA or shCont; n = 3; *P < .005, **P < .01. (D) Glioma cells infected with shNFIA or control shRNA were stained with SA-β-gal (x400) on day 3 of transduction. Quantification of SA-β-gal positive cells is on the right panel; n = 3, *P < .0001. Knockdown of NFIA is shown in panel E. (E) Loss of NFIA causes apoptosis evidenced by cleavage of PARP, caspase-8, and caspase-9. Whole cell lysates of glioma cells analyzed on day 3 after infection with shNFIA, vector control, or uninfected parental cells (immunoblotting). Shown is a representative experiment of 3 experiments. (F) Caspase inhibitor, Z-VAD, blocks shNFIA-induced PARP cleavage. Whole-cell lysates of shNFIA and control-infected U251 glioma cells 3 days after lentiviral infection and cultured with Z-VAD-fmk (20 μM) or vehicle (DMSO) for additional 24 and 48 hours were analyzed by immunoblotting. (G) *NFIA (protein coding domain only, resistant to the shNFIA) reverses shNFIA-induced PARP cleavage in U251 glioma cells. After 24 hour infection with shNFIA or control in U251, cells were reinfected with *NFIA for 48 hours and analyzed for PARP cleavage (left). Right panel: densitometric quantification; *P<.001, **P<.01. (H) NFIA protects glioma cells from etoposide-induced apoptosis. U251 glioma cells stably expressing NFIA or vector were treated with vehicle (DMSO) or etoposide (ETP, 1 μg/mL) in serum-free medium for 24 hours. Apoptosis was assessed using the Apo-Direct kit. Left panel: representative experiment of 3 experiments. The percentage of apoptotic cells (FITC-dUTP+ cells in top quadrants) is indicated for each condition. Right panel: means ± SD from 3 experiments; *P < .0001.