Characterization of venetoclax sensitivity and the BCL2 family in myeloma cell lines. (A) Cells were treated with indicated doses of venetoclax for 24 hours followed by staining with Annexin V–fluorescein isothiocyanate to measure apoptosis. The table lists calculated IC₅₀ and cytogenetics (CTG) of each cell line. (B) RNA expression of BCL2, BCL2L1, and MCL1 in sensitive (green) and resistant (orange) cell lines as measured by RNA-sequencing, as well as calculated BCL2/BCL2L1 and BCL2/MCL1 ratios. (C) Protein lysates from 4 sensitive (Karpas 620, OCI-My5, PCM6, and H1112) and 5 resistant (KMS20, KMS28, KMS34, KMS21BM, and PE1) cell lines were prepared and then subjected to coimmunoprecipitation with anti-MCL1, anti-BCLXL, and anti-BCL2 antibodies. The resulting protein complexes were examined by western blot analysis using anti-BIM, anti-MCL1, anti-BCLXL, and anti-BCL2. (D) Protein expression of BIM, BAK, BAX, MCL1, BCLXL, BCL2, and actin from Cas9 parental cells or CRISPR knockout of BIM, BAK, BAX, or BAK and BAX in KMS12PE or OCI-My5. (E) Cells from panel D were treated with indicated doses of venetoclax for 24 hours followed by staining with Annexin V–fluorescein isothiocyanate to measure apoptosis. Data are presented in panels A and E as the mean ± standard error of 3 independent experiments. DKO, double knockout; FPKM, fragments per kilobase of transcript per million mapped reads; KO, knockout; WT, wild type.