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. 2013 May 15;27(10):1115–1131. doi: 10.1101/gad.198630.112

Figure 5.

Figure 5.

Dysregulated mTORC1 activity promotes a magnified ER stress response under nutrient and O2 limitation. (A) To evaluate which sensors of the UPR are activated under ischemic stress, Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs were exposed for 24 h to 21% or 0.5% O2 in replete, S, and SG conditions. Whole-cell extracts were blotted for PERK, P-IRE1α (Ser 724), IRE1α, XBP1s, CHOP, and β-actin (see also Supplemental Fig. S5A–D). (B) Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs were exposed to 21% and 0.5% O2 in replete, S, or SG medium for 16 h, and levels of UPR target mRNAs Ho-1, Bip, Atf4, Xbp1s, Xbp1u, and Herp were determined by qRT–PCR. (C) The autophosphorylation of PERK and IRE1α and the induction of CHOP in Tsc2+/+, p53−/− and Tsc2−/−, p53−/− MEFs exposed to O, Ored, and SO conditions were assayed by Western blot analysis. (D) To determine whether oleic acid alters UPR signaling pathways under tumor-like stress, Tsc2−/−, p53−/− MEFs were cultured under SO conditions in the presence or absence of 50 μM oleic acid, and whole-cell extracts were blotted for PERK, P-IRE1α (Ser 724), and CHOP. (E) Representative electron micrographs are shown for Tsc2−/−, p53−/− MEFs cultured under SO conditions in the presence and absence of 50 μM oleic acid. Black arrows indicate the ER. (F) Tsc2−/−, p53−/− MEFs were depleted of PERK using siRNA pools and cultured under SO conditions. Viability was assessed by flow cytometry (see also Supplemental Fig. S5E,F). (G) Inhibition of IRE1α activity rescued the viability of Tsc2−/−, p53−/− MEFs under SO conditions (P < 0.005) (see also Supplemental Fig. S6A–D). (H) Inhibition of IRE1α activity rescued the viability of Tsc2−/−, p53−/− MEFs under SOG conditions. (*) P < 0.005.