Fig. 5: Stress-induced plasticity is mediated by the ISR.

a eIF2α-p, eIF2α, and 4E-BP1 immunoblots from 0, 3, or 6 h INK (20 nM) treated T47D cells (n = 3). b NODAL immunoblot of 0–24 h AZE (5 mM) treated T47D cells (n = 3). c Control MDA-MB-231(EV) and MDA-MB-231 cells depleted for endogenous eIF2α and stably expressing wild type eIF2α (WT) or HA-tagged eIF2α with the S51A mutation (knock-in, KI) were treated with vehicle or TG. Immunoblots for eIF2α-p and ATF4 with eIF2α and β-Actin loading controls. d Mean log₂ fold change of qRT-PCR quantification of TWIST, ZEB1, SNAIL, VIM transcripts in MDA-MB-231 WT (white) and KI (black) cultured for 24 h in normoxia or hypoxia (n = 3). e Mean sphere counts for MDA-MB-231 WT and KI pre-exposed to hypoxia or normoxia for 24 h relative to normoxia MDA-MB-231 WT spheres (n = 3) with representative images. f eIF2α-p, eIF2α and NODAL immunoblots from 24 h vehicle (DMSO), ISRIB (10 nM), INK128 (20 nM) or Salubrinal (20μM) treated T47D cells with β-actin and eIF2α loading controls (n = 3). g Mean log₂-fold change of NANOG and SNAIL mRNA levels by qRT-PCR in Salubrinal-treated T47D cells (0, 3, or 6 h with (20 μM)) (n = 4). h NANOG and SNAIL immunoblot analyses of 0, 3, or 6 h Salubrinal (20 μM)-treated T47D cells with β-actin and β-tubulin loading controls (n = 3). i Mouse lung colonies 8 weeks following tail vein injection of SUM149 cells pretreated for 24 h with DMSO or Salubrinal (20 μM) (n = 10) with representative lung colony images (brown). j Mean SUM149 tumor volumes in mice treated every second day for 2 weeks (DMSO, INK (30 mg/kg) or INK + ISRIB (2.5 mg/kg)). Arrow indicates treatment cessation (p < 0.01, n = 7). k Mean sphere counts from 96,000 SUM149 cells dissociated from endpoint tumors from j (n = 12) with representative spheres images. Micron bars = 500 μm. Data represents independent experiments. Error bars indicate mean ± SD. Box and whisker plot represents median, interquartile range (IQR), whiskers extend to maximum and minimum. Two-sided t-test for paired samples. The asterisks denote p-values < *0.05, **0.01. Multiple comparisons tested by ANOVA. The same letters indicate relationships with a p ≥ 0.05. Different letters indicate statistical differences (p < 0.05).