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. Author manuscript; available in PMC: 2021 Sep 22.
Published in final edited form as: Nat Genet. 2021 Mar 22;53(4):529–538. doi: 10.1038/s41588-021-00819-w

Extended Data Fig. 8. Validation of MCL1 dependency in pediatric cell lines.

Extended Data Fig. 8.

a, MCL1 gene effect scores for overlapping cell lines in DepMap 20Q1 (x-axis) and DRIVE RNAi (y-axis) for adult (gray) and pediatric cancer cell lines (red). b, MCL1 gene effect scores (x-axis) versus gene expression of BCL2L1 (y-axis) for adult (gray) and pediatric cancer cell lines (red). Gray and red lines in panels (a-b) represent linear model fits to adult or pediatric data, respectively. c, CRISPR-Cas9 mediated disruption of MCL1 by two independent sgRNAs reveals decreased cell growth in vitro as demonstrated by CellTiter-Glo luminescence (y-axis) versus time (x-axis), correlated with the larger screen. One representative experiment shown for each cell line; each time-point measured in replicate (n=8). Data presented as mean values +/− SEM. d, Western blotting after MCL1 disruption by CRISPR-Cas9 2 days post-selection (SKNBE2, SKNMC) or 3 days post-selection (Kelly). e, Western blotting after MCL1 inhibition with S63845 at 48 hours demonstrates increased protein expression of MCL1 after inhibition with S63845 at 48 hours with less induction of cleaved PARP or Caspase 3 at lower concentrations in SKNBE2 or EWS503 compared to the more sensitive neuroblastoma or Ewing cell lines, Kelly and SKNMC, respectively. f, Treatment with increasing concentrations of ZVAD, a pan-caspase inhibitor, reveals a concentration-dependent rescue of 2 μM S63845 treatment in Kelly and SKNMC at day 3 as demonstrated by the fraction of CellTiter-Glo luminescence compared to DMSO control (y-axis). One representative experiment shown for each cell line; each time-point measured in replicate (n=4). Data presented as mean values +/− SEM. g, Western blotting after one hour of pre-treatment with either DMSO or 20 μM ZVAD followed by either DMSO or 1 μM S63845 treatment at 48 hours show increased protein expression of MCL1 after inhibition with S63845 at 48 hours with decreased induction of cleaved PARP or Caspase 3 following pre-treatment with ZVAD in SKNMC. Experiments shown in panels (c-g) were performed independently at least in duplicate, with one representative experiment shown.