Fig. 2
AMPK activation potentiates MYC-dependent apoptosis by ABT-737. a Drug-targeted pathways. b Protocol for drug combination testing. MCF10A MycER cells were allowed to form mammospheres for 24 h. MYC was activated with 100 nM 4OHT for 24 h followed by 24 h incubation with drug combinations. c Combination drug testing to identify pharmacological triggers of MYC-dependent apoptosis. The drugs were administered as single agents or as ABT-737 with and without MYC activation. Each drug was tested in two concentrations. N = 3 biological repeats. Student’s t-test (unpaired), SD. d The relative level of apoptosis with and without MYC activity (+MYC: −MYC ratio). The ratio was calculated from fold-change in c. e Representative images of drug-treated mammospheres. f, g Sensitization to MYC-dependent apoptosis by 100 nM ABT-737 with either 1 μM A-769662 or 10 mM metformin. Mammospheres were treated as in b. Student’s t-test (unpaired), N = 3 biological replicates, SD. h Activation of AMPK alone does not sensitize to MYC-dependent apoptosis. N = 3 biological repeats, SD. i CRISPR/dead-Cas9-mediated transcriptional activation of MYC. HEK293 and MCF10A cells were transduced with vectors encoding dCas9-VP192 transcription-activating construct and MYC-promoter-targeted guide-RNAs. Western blot analysis shows MYC expression levels after 72 h treatment with doxycycline (DOX) and trimethoprim (TMP). Lamin B: Loading control. j CRISPR-mediated induction of endogenous MYC sensitizes cells to apoptosis by ABT-737+A-769662. Mammospheres with and without dCas9-VP192+MYC-gRNA were treated and analyzed as in b. Student’s t-test (unpaired), N = 3 biological replicates, SD
