Figure 3

GSK-3 inhibition causes MM cell apoptosis and disruption of the mitochondrial membrane potential. (A) Histograms showing the percentage of surviving (annexin V negative) U-266, RPMI-8226 and INA-6 cells (left graph) and of normal PBMC control cells (right graph) after treatment without (black bars) or with (white bars) 4 μM SB415286 for 48 hours. Data represent mean ± SD, n = 5 (cell lines) or 4 (PBMC). * indicates p < 0.05 by Student's t-test. (B) Histograms showing FACS analysis of the amount of JC-1 monomer-containing, green fluorescent-INA-6 cells (left graph) or U-266 cells (right graph), treated with the same conditions as in A. Data are expressed as ratio over untreated cells. Data represent mean ± SEM, n = 5. * indicates p < 0.01 by Student's t-test. (C) Representative WB analysis of phospho Tyr 279 GSK-3α and phospho Tyr 216 GSK-3β, total GSK-3, PARP cleavage and Smac/DIABLO protein expression on cell lysates of RPMI-8226 cells grown with 5 μM or 10 μM SB216763 for 24 hours (top panels) and WB analysis of PARP protein in INA-6 cells grown with 4 μM SB415286 for 8 and 24 hours (bottom). β-Actin is used to check protein loading. (D) Representative WB analysis of nuclear (n) and cytosolic (c) proteins from U-266 cells untreated or treated with 5 μM SB216763, showing the levels of total GSK-3, Tyr phosphorylated GSK-3, β-catenin, total ERK 1/2, Thr/Tyr phosphorylated ERK 1/2, PARP and nucleophosmin and GAPDH as loading controls.