Molecular mechanism underlying the synergism between Duv and Ven in RS cells. (A) Expression levels and activation status of a panel of proteins involved in survival signaling (Akt, GSK3β, Mcl-1, c-Myc) in RS1316 cells after ex vivo treatments (6 hours) with Duv, Ven, their combination, or dimethyl sulfoxide (DMSO). Bar plots represent protein bands intensities (5 independent experiments), measured using Image Lab, normalized on β-actin, and plotted as fold change (FC) over the band of untreated cells. Data are reported as mean ± SEM. (B) Anti-Mcl-1 and anti-c-Myc immunoprecipitation followed by immunoblotting for ubiquitin in RS1316 cells after ex vivo treatments (3 and 6 hours) with Duv, Ven, their combination, or DMSO. Bar plots represent the ratio of ubiquitinated over total protein. (C) Expression levels of Mcl-1 and c-Myc in RS1316 cells after ex vivo treatments (6 and 3 hours, respectively) with Duv, Ven, or their combination in the absence or presence of the proteasome inhibitor MG-132. Band intensities were normalized on β-actin. (D) Expression levels of p-GSK3β, GSK3β, Mcl-1, and c-Myc in RS1316 cells after ex vivo treatments with Duv, Ven, or Duv/Ven in the absence or presence of the GSK3β inhibitor tideglusib. Bar plots represent protein bands intensities (3 independent experiments), measured using Image Lab, normalized on β-actin, and plotted as ratio between tideglusib-treated and untreated samples. Data are reported as mean ± SEM. Statistical analysis was performed using 1-way ANOVA; *P < .05, **P < .01, ***P < .001, ****P < .0001.