Figure 4.

rNef-mediated inhibition of BFA-induced apoptosis in MDMs parallels cytoplasmic accumulation of eEF1A and is dependent on Exp-t. (a) rNef prevents BFA-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml) for 12 or 15 h or left untreated in the presence of increasing concentrations of rNef (0, 125, 750 ng/ml). Apoptosis was detected by annexin-V flow cytometric analysis. The histogram summarizes the survival of MDMs following treatment with BFA (10 μg/ml) for 12 or 15 h in the presence of increasing concentrations of rNef. The results represent means of three independent experiments. *P<0.05. (b) rNef prevents BFA-induced apoptosis, but neither TM-induced apoptosis nor TG-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml), TM (10 μg/ml) or TG (10 μg/ml) for 5 or 12 h or left untreated in the presence of rNef (1 μg/ml). Apoptosis was measured by the TUNEL assay. Results are representative of data observed in three independent experiments. The mock panel is shown in triplicate to the left of the BFA, TM and TG panels to facilitate the results reading. (c) The C-terminal extremity of Nef prevents BFA-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml) for 12 h or left untreated in the presence of WTNef, Nef1-60 or Nef55-206 (1 μg/ml). Apoptosis was measured by the TUNEL assay. Results are representative of data observed in two independent experiments. (d) Blockade of BFA-induced apoptosis in MDMs by rNef is dependent on Exp-t. MDM cultures were transfected with a scrambled control, Exp-t siRNA, Exp-1 siRNA, or Exp-5 siRNA for 48 h before treatment, with BFA (10 μg/ml) for 12 h or left untreated in the presence (1 μg/ml) or absence of rNef. Apoptosis was detected by flow cytometric analysis of annexin-V. The histogram shows the survival percentage of MDMs treated with mock, BFA, or BFA+Nef in the presence of exportin siRNAs. Results are representative of three independent experiments. *P<0.05. (e) Blockage of BFA-induced apoptosis in MDMs by rNef is dependent on eEF1A. MDMs were treated with control siRNA or eEF1A siRNA for 48 h before treatment, with BFA (10 μg/ml) for 12 h or left untreated in the presence (1 μg/ml) or absence of rNef. Apoptosis was measured by the TUNEL assay. Results are representative of two independent experiments. EF1A knockdown in MDMs was monitored by western blot (upper right panel). The histogram shows the percentage of TdT positive cells