Extended Data Fig. 4. The ISR induced by eIF2B5 depletion is MYC-dependent.
(a) Immunoblot of shCTR-transduced or eIF2B5-depleted APCdef and APCres cells (96 h ethanol or doxycycline, respectively), representative of two independent experiments with similar results. For ISR induction, cells were treated with 1 μg/ml tunicamycin (3 h), DMSO was used as solvent control.
(b) Immunoblot of shCTR-transduced or eIF2B5-depleted APCdef and APCres cells upon CHOP depletion (96 h ethanol or doxycycline, respectively), representative of three independent experiments with similar results. siRNA transfections were carried out using siCTR as non-targeting control or siCHOP for 72 h.
(c) Annexin V/PI FACS analysis of cells treated as described in (b). Data show mean ± s.d. (n = 3 biologically independent experiments); unpaired, two-tailed t-test.
(d) qPCR documenting MYC expression in shCTR-transduced or eIF2B5-depleted APCdef and APCres cells (96 h ethanol or doxycycline, respectively). Data show mean ± s.d. (n = 3 biologically independent experiments); unpaired, two-tailed t-test.
(e) Immunoblot of shCTR-transduced or eIF2B5-depleted APCdef and APCres cells (72 h ethanol or doxycycline, respectively) after cycloheximide (CHX) pulse chase. Experiment was performed once. Cells were treated with 100 μg/ml CHX for the indicated time points.
(f) Immunoblot of shCTR-transduced or eIF2B5-depleted APCdef and APCres cells (72 h ethanol or doxycycline, respectively), representative of three independent experiments with similar results.
(g) qPCR documenting MYC expression in shCTR-transduced or eIF2B5-depleted APCdef and APCres cells treated as described in (f). Data represent mean ± s.d. (n = 3 technical replicates), representative of two independent experiments with similar results.
(h) Immunoblot of shCTR-transduced or eIF2B5-depleted HT29 and HCT116 cells, representative of two independent experiments with similar results.
Unprocessed immunoblots are shown in Source Data Extended Data 4.