Figure 5
From: Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death
Effects of GSK-3α and β knockdown by RNA interference on MM cell survival. (A) WB analysis of the levels of GSK-3α (left panels) or GSK-3β (right panels) upon nucleofection with: un = untransfected; scr = scrambled siRNAs oligos; GSK-3α or GSK-3β=GSK-3α or GSK-3β-directed siRNAs oligos. (B, left panels) Light microscopy microphotographs of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos. Scale bars = 10 μm. (B, middle plots) Representative dot plot graphs and FACS analysis of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos. (B, right panels) Histograms summarizing the results of FACS analysis of U-266 cells transfected with scrambled (scr) siRNAs oligos, GSK-3α (top) or GSK-3β (bottom)-specific siRNAs oligos,. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 6. * indicates p < 0.05 (Student's t test). (C) Representative WB analysis of the levels of unprocessed PARP after nucleofection of U-266 cells with: scrambled, GSK-3α or GSK-3β siRNAs oligos. (D) Light microscopy microphotographs and histograms summarizing the results of FACS analysis of U-266 cells transfected with scrambled (scr), GSK-3α or GSK-3β-directed siRNAs oligos and treated with 5 nM BZ. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 3. * indicates p < 0.05 (one-way ANOVA test). (E) Histogram plot summarizing the results of the FACS analysis of U-266 cells transfected with scrambled (scr) or GSK-3α/GSK-3β-directed siRNA oligos and treated with 5 nM BZ. In y-axis dead cells are cells displaying a low FSC/high SSC cyto-morphological profile. Data represent mean ± SEM, n = 3. * indicates p < 0.05 (one-way ANOVA). (F) WB analysis of GSK-3α, GSK-3β, AKT, Ser 473-phosphorylated AKT and MCL-1 protein levels in U-266 cells untransfected (lane 1), treated with 5 nM BZ (lane2), transfected with scrambled (scr, lane 3) or GSK-3α/β (lane 4), GSK-3α (lane 5) or GSK-3β (lane 6)-directed siRNAs oligos without (-, lanes 3-6) or with (+, lanes 7-10) exposure to 5 nM BZ for 18 hours.