Fig. 2: MCL-1, a possible mediator of VEN-resistance development.

A Western Blot analysis of BCL-2, BCL-XL and MCL-1 protein expression comparing RS4;11 VEN^sens and VEN^ins lines under continuous VEN treatment. ImageJ analysis of protein expression normalized to Vinculin is shown. Unpaired two-tailed Student’s T-test was used to calculate p-values comparing five VEN^sens and five VEN^ins lines (± standard deviation is shown). B Immunoprecipitation (IP) analysis of BIM was performed in RS4;11 VEN^sens and VEN^ins line 1. Binding of BIM to BCL-2 or MCL-1 was analyzed upon exposure to VEN (1 nM in VEN^sens and 1 µM in VEN^ins cells), 2 µM S63845 or the combination of both inhibitors for 4 h. Cell death rates after drug treatment were analyzed by FSC/SSC criteria as stated. The IP:BIM lanes show the interaction of BIM with BCL-2 and MCL-1, while input lanes show the total proteins in the lysates (N = 1). C RS4;11 VEN^sens and VEN^ins lines were incubated with increasing concentrations of S63845, VEN (DMSO; 1 nM; 5 nM; 10 nM; 25 nM; 50 nM; 100 nM; 250 nM; 500 nM; 1 µM; 2.5 µM; 5 µM; 7.5 µM and 10 µM) or their combination for 72 h. Cell death was estimated using FSC/SSC criteria and normalized to the corresponding DMSO control. The mean of all five VEN^sens and VEN^ins lines per drug treatment is shown (N = 3, in triplicates). D A graphical schematic of baseline BH3 profiling analyzing RS4;11 VEN^sens and VEN^ins lines. E For baseline BH3 profiling, cells of all five RS4;11 VEN^sens and VEN^ins lines were permeabilized and incubated with the pro-apoptotic BH3-peptides BAD (indicating BCL-2 dependence), HRK (BCL-XL dependence) and MS1 (MCL-1 dependence). Cells were then fixed and stained with an anti-cytochrome c antibody binding exclusively to mitochondrial cytochrome c. Results show decreased BCL-2 priming in RS4;11 VEN^ins cells (BAD-HRK), while increased MS1 dependency upon VEN resistance was shown. (N = 5 biological replicates/N = 1 technical triplicates, ± standard deviation). Unpaired two-tailed Student’s T-test was used to calculate p-values comparing five VEN^sens to VEN^ins lines. F Western blot analysis of MCL-1 protein expression comparing RS4;11 VEN^sens and VEN^ins lines under continuous exposure to VEN (left panel) and RS4;11 VEN^sens and VEN^ins lines after 20 weeks drug removal (drug holiday, right panel) with respective loading controls (Vinculin) is shown. G Densitometric quantification of protein expression normalized to the corresponding Vinculin control and shown as a relative expression to the mean of the VEN^sens lines 1-5. Unpaired two-tailed Student’s T-test was used to calculate p-values comparing five VEN^sens and five VEN^ins lines (± standard deviation is shown). H Baseline BH3 profiling of 20 weeks drug holiday cells comparing all five RS4;11 VEN^sens and VEN^ins lines (± standard deviation is shown). I Immunoprecipitation (IP) analysis of BIM was performed in RS4;11 VEN^sens and VEN^ins line 1, also under 20 weeks drug holiday conditions. Basal binding of BIM to BCL-2 or MCL-1 was analyzed. The IP:BIM lanes show the interaction of BIM with BCL-2 and MCL-1, while input lanes show the total protein expression of whole cell lysates (N = 1).