Fig. 3: Altered metabolic profile, a hallmark of VEN resistance development.
A Paired RNA-Seq analysis of five RS4;11 VENsens compared to five VENins lines. Gene set enrichment analysis (GSEA, v4.0.3; http://www.broadinstitute.org/gsea) was performed to analyze the enrichment of gene sets annotated in the Molecular Signature Database as described. The ‘gene_set’ permutation configuration was used, and gene sets with a NOM p-value ≤ 0.05 and a FDR q-value ≤ 0.05 were considered significant. The enrichment plot of the Respiratory Electron Transport gene set is shown. B Basal oxygen consumption was measured in an XFe96 flux analyzer (Agilent) after treatment with DMSO or 10 nM and 100 nM VEN for 3 h. Mean values of five RS4;11 VENsens and VENins lines are shown (N = 1, in six technical replicates, ± standard error of mean). Uncoupled respiration was profiled by injection of 1.25 μM Oligomycin and full respiration capacity was determined by injecting 0.75 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Non-mitochondrial respiration was determined by injecting 0.5 μM antimycin A and rotenone, each. Oxygen consumption rates (OCRs) were determined by plotting the partial oxygen pressure against time. Basal respiration was analyzed by subtracting the minimal OCR after antimycin A/rotenone injection from the basal OCR. Data were normalized to cell number by Janus Green staining. Unpaired two-tailed Student’s T-test was used to calculate p-values. C To determine changes in mitochondrial membrane potential or mitochondrial mass, cells were stained with 2 μg/ml Tetramethylrhodaminemethylesterperchlorate (TMRM, Sigma-Aldrich) or 50 nM MitoTracker Green (ThermoFisher Scientific). Means of five RS4;11 VENsens and VENins lines are shown and median fluorescence intensities (MFI) were determined and normalized to MFIs of unstained controls (N = 3 in triplicates, ± standard deviation). Unpaired two-tailed Student’s T-test was used to calculate p-values. D Mitochondrial morphology of RS4;11 VENsens line 1 and VENins line 1 were analyzed by electron microscopy imaged in a JEM-1400 TEM (Jeol).
