Fig. 3. Isoform-specific translation of BCSC transcripts during mTOR inhibition.

a Diagram of a working model wherein hypoxia causes a reduction in mTOR activity and the activation of the integrated stress response (ISR), signified by eIF2α phosphorylation. These changes in the translational machinery lead to diminished global protein synthesis and selective translation of a subset of mRNA isoforms of NANOG, SNAIL, and NODAL that contain specific 5′UTR features, including uORFs. The experiments presented below are designed to test whether mTOR inhibition (treatment with INK128) can replicate the phenomena observed during hypoxia and whether ISRIB can mitigate these effects. b Polysome profiles and immunoblot analysis of lysates from MCF7 breast cancer cells exposed to vehicle (DMSO), MLN0128/INK128 (INK; 20 nM), ISRIB (10 nM), or INK + ISRIB for 24 h show that INK reduces global translation (INK and ISRIB + INK p < 0.01) concomitant with reduced 4E-BP1 and rpS6 phosphorylation and increased eIF2α phosphorylation. These readouts are unaffected by ISRIB. Total 4E-BP, rpS6, and eIF2α levels were unchanged. β-actin is used as a loading control. c ATF4 mRNA and d–f mRNA isoforms of f NANOG, e SNAIL, and f NODAL associated with mononsomes, low MW polysomes and high MW polysomes from MCF7 cell lysates fractionated in b. Bars represent the mean percent of transcript associated with each fraction in each condition ± SD (n = 3). Error bars indicate mean ± SD. Multiple comparisons tested by ANOVA. The asterisks denote p-values < *0.05, **0.01 compared to control treatment. Multiple comparisons tested by ANOVA. The same letters indicate relationships with a p ≥ 0.05. Different letters indicate statistical differences (p < 0.05).