Fig. 3.

Ablation of p73 prevents the nSREBP2-induced lytic cell death. A: Overexpression of nSREBP2 increased gene transcription of p53 pathway. B: QPCR analysis of p73 transcripts. Cells were treated with or without 0.3 μg/ml dox for 16 h, then RNA was harvested for QPCR analysis. The diagram below showed two transcripts of p73, TAp73 and ΔN p73. Data are presented as means ± SD. C: Western blotting analysis was used to detect the expression of p73 and p53 with the induction of nSREBP2. HeLa-pTet-nSREBP2 cells were treated with or without 0.3 μg/ml dox for indicated time, then harvested for Western blot analysis. D: QPCR analysis of nSREBP2 and p73 transcripts. HeLa-pTet-nSREBP2 cells were cultured in 10% FBS or sterol depletion medium (5% lipoprotein-deficient serum, 1 μM lovastatin, 10 μM mevalonate) for 24 h and then subjected to analysis. E: QPCR analysis of p73 siRNA knock-down efficiency. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. F: LDH release test. HeLa-pTet-nSREBP2 cells were transfected with indicated siRNA for 24 h, then treated with 0.3 μg/ml dox for 24 h. Lytic cell death was measured by LDH in the medium. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. G: The effect of p73 siRNA on nSREBP2 expression and caspase-9 cleavage. H: Immunoblotting analysis of WT and p73 knockout HeLa-pTet-nSREBP2 cells. I: LDH test identified the effect of p73 knockout on nSREBP2 induction. Data are presented as means ± SD. Statistical analyses, unpaired two-tailed Student’s t test. J: representative images of WT and p73 knockout HeLa-pTet-nSREBP2 cells. All cells were treated with 0.3 μg/ml dox for 36 h. Cells were preloaded with 10 μg/ml PI for 15 min and imaged at 63× magnification. Scale bar, 25 μm. ∗∗P ＜ 0.01, ∗∗∗P ＜ 0.001, ∗∗∗∗P ＜ 0.0001.