Expression of PMLRARα blocks Fas-mediated apoptosis. (A) ZnSO4 (0, 100 and 200μM) was used to induce PMLRARα expression in U937/PR9 cells for 24 hours. Whole-cell lysates were subjected to Western blot analyses using an anti-RARα (for detection of PMLRARα), anti-Fas and anti–β-actin antibodies. NB4 cells and U937 cells treated with ZnSO4 were used as a positive and a negative control for PMLRARα expression, respectively. (B) U937/PR9 cells were treated with 100 and 200μM ZnSO4 for 24 hours followed by incubation with 50 ng/mL CH-11 antibody for 8 and 24 hours (left panel). U937 and U937/PR9 cells were treated with ZnSO4 for 24 hours followed by incubation with 50 ng/mL CH-11 antibody for 24 hours (right panel). The cells were stained with propidium iodide (PI) and analyzed by flow cytometry for the rates of apoptosis. The values shown are the means ± SD of 3 independent experiments. (C) U937/PR9 cells were treated with ZnSO4 for 24 hours, washed and incubated with or without 50 ng/mL CH-11 antibody for 4 hours. The cells were stained with annexin V–FITC and analyzed by flow cytometry for the rates of apoptosis. Similar results were obtained in 2 independent experiments. (D) U937/PR9 cells were treated with or without ZnSO4 for 24 hours, then washed and incubated with or without FasL for 24 hours. The cells were stained with PI and analyzed for the apoptosis rates by flow cytometry. The data represent the means ± SD of 3 independent experiments. (E) U937/PR9 cells were treated with 0, 100, and 200μM ZnSO4 for 24 hours. Cells were then washed and incubated with CH-11 antibody (50 ng/mL) for 15 minutes. Whole cell lysates were subjected to Western blot analyses using anti–caspase-8 and anti–α-tubulin antibodies for detection of cleaved/active caspase-8 fragments and α-tubulin, respectively.
