Downregulation of PKCα suppresses stress granule assembly.

<p>MDA-MB-231 cells were transfected with three different siRNA oligonucleotides against PKCα, and were thereafter subjected to heat shock at 44°C for 1 h. Stress granules were visualized with immunofluorescence using a G3BP2 antibody (A). Western blot of cell lysates demonstrating PKCα downregulation and quantification of the percentage of cells containing stress granules (B) (mean ± SEM, n = 3). (C) MDA-MB-231 cells were treated with siRNAs targeting PKCα or PKCε, followed by heat shock for indicated time periods. Cell lysates were analyzed with Western blot demonstrating downregulation of respective isoforms. Stress granules were visualized by G3BP2 immunofluorescence and the percentage of cells with stress granules was quantified (mean ± SEM, n = 3). (D) MDA-MB-231 cells with downregulated PKCα were treated with 300 µM As₂O₃ for 30 or 60 minutes. Cell lysates were analyzed with Western blot and the percentage of cells with stress granules, identified by PABPC1 immunofluorescence was quantified (mean ± SEM, n = 3). (E) MDA-MB-231 cells were treated with 16 nM TPA and/or 2 µM GF109203X (GFX) during heat shock. Stress granule-positive cells were thereafter quantified. * denotes statistically significant (p<0.05) difference compared to control using ANOVA followed by Duncan's multiple range test.