Extended Data Fig. 2: Senolytic screens and validation in additional models.
From: cFLIP suppression and DR5 activation sensitize senescent cancer cells to senolysis
a, Schematic of CRISPR-based senolytic screen workflow in SK-Hep1 and PC3. Genome-wide Brunello gRNA collection lentivirus was introduced to the SK-Hep1-iCas9 or PC3-iCas9 cells. After 7 days of 0.5 µM alisertib treatment, cells were switched to 1 µg/ml doxycycline (DOX) for 10 days. Illumina deep sequencing was used to determine changes in library representation. b, Top hits from SK-Hep1 and PC3 were selected based on the fold depletion of S2 divided by S1 (N = 3 independently viral infected cell cultures). c, Real-time PCR analysis of cFLIP expression on SK-Hep1 cFLIPKO clone. d, Senescence inducers treated SK-Hep1 parental and cFLIPKO cells were incubated with caspase-3/7 green apoptosis assay reagent. The black scale bars present 100 µm. e, CellTiter-Blue measurement in the SK-Hep1 parental and cFLIPKO cells treated with senescence inducers for one week. f, Quantification of colony-formation result in 0.5 µM alisertib and 10 µM Z-VAD-FMK treated A549 parental and cFLIP-KO cells. g, Senescence-associated beta-galactosidase staining on PC3 cells with one week of 0.5 µM Alisertib treatment. The black scale bars present 100 µm. h, Real-time PCR analysis of cFLIP expression on PC3 cells infected with shRNAs against cFLIP. i, Quantification of colony-formation result in 0.5 µM alisertib treated PC3 cells infected with shRNAs against cFLIP. Error bars in this figure panel c-i represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
