Down-regulation of PMLRARα enhances Fas-mediated apoptosis in transgenic mouse APL cells and human APL NB4 cells. (A) Transgenic mouse APL cells were treated with vehicle or 1μM arsenic trioxide (As2O3) for 24 hours. Whole cell lysates were analyzed for protein expression with anti-PML and anti–α-tubulin antibodies (left). Cells were then incubated with or without 50 ng/mL FasL for additional 24 hours, and stained with PI to analyze apoptosis by flow cytometry. The data represented are the means ± SD of 3 independent experiments (right). (B) Human APL NB4 cells were transduced with scrambled shRNA or 4 different PML/PMLRARα-targeting shRNAs as described in “Experiments using shRNA.” After selection with puromycin, cells were analyzed for protein expression with anti-RARα, anti-PML, and anti–β-actin antibodies to confirm down-regulation of targets. (C) NB4 clone 3 and clone 4 cells with knockdown of PML and PMLRARα expression were incubated with indicated concentrations of FasL or CH-11 antibody for 48 hours. The cells were then stained with PI and analyzed for apoptosis by flow cytometry. The data represent the means ± SD of 3 independent experiments.
