Figure 2. A20 sensitizes cells to oxidative-stress-mediated cell death.

(A) HeLa cells were transfected with A20 (open bars) or vector control (closed bars). At 48 h after transfection, cells were treated with H₂O₂ or TNF-α (50 ng/ml)/CHX (1 μg/ml) as indicated for 16 h. Living cells were analysed by Crystal Violet staining. Results are means±S.D. A20 expression was shown by immunoblotting (α-FLAG, the lower band is non-specific). (B) HeLa cells were transfected with vector control (pSuper) or A20 RNAi (pSuper.A20 RNAi Seq.1 or Seq.2) for 48 h, and were then treated with H₂O₂ (500 nM, 4 h). A20 was immunoprecipitated and immunoblotted (α-A20) or RNA was isolated and RT-PCR was performed. β-Actin expression served as a control (immunoblot: α-β-actin, or RT-PCR). (C) HeLa cells were transfected with vector control (pSuper, closed bars) or A20 RNAi (pSuper.A20 RNAi Seq.1, open bars) for 48 h, and were then treated with increasing concentrations of H₂O₂ or with TNF-α, as indicated. Living cells were analysed by Crystal Violet staining. Results are means±S.D.