Extended Data Fig. 2: Apoptosis induction upon EIF2B5 knockdown in APC^def cells is an on-target effect.

(a) PI cell cycle FACS analysis of shCTR-transduced or eIF2B5-depleted APC^def and APC^res cells (six days ethanol or doxycycline, respectively). Data show mean ± s.d. (n = 3 technical replicates), representative of two independent experiments with similar results. (b) Immunoblot of shCTR-transduced (-) or eIF2B5-depleted APC^def and APC^res cells (96 h ethanol or doxycycline, respectively), representative of two independent experiments with similar results. (c) Annexin V/PI FACS analysis of shCTR-transduced (-) or eIF2B5-depleted APC^def and APC^res cells treated as described in (b). Data show mean ± s.d. (n = 3 technical replicates), representative of two independent experiments with similar results. (d) Immunoblot of control (‘empty’) and eIF2B5mut-HA overexpressing shCTR-transduced or eIF2B5-depleted APC^def and APC^res cells (96 h ethanol or doxycycline, respectively), representative of two independent experiments with similar results. (e) mRNA expression of endogenous EIF2B5 of cells described in (d). Primers targeting the EIF2B5 3′UTR were used. Data show mean ± s.d. (n = 3 technical replicates), representative of two independent experiments with similar results. (f) Annexin V/PI FACS analysis of cells described in (d). Data show mean ± s.d. (n = 3 technical replicates), representative of two independent experiments with similar results. (g) Annexin V/PI FACS analysis of shCTR-transduced or eIF2B5-depleted HT29 and HCT116 cells. Data show mean ± s.d. (n = 3 biologically independent experiments); unpaired, two-tailed t-test. (h) Box plots of EIF2B5 mRNA expression in normal colorectal tissue and colorectal carcinoma of two independent datasets from Oncomine (n = number of biologically independent samples). Data sets are taken from ‘Kaiser’;⁵⁶ ‘Hong’⁵⁷. Points: minimum and maximum, whiskers: 10^th and 90^th percentile, box: 25^th and 75^th percentile, line: median; unpaired t-test. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 2.

Source data