ER stress-induced inhibition of cap-dependent translation and loss of MCL1 is PERK-dependent.

<p><b>(A)</b> HCT116 cells were pre-treated for 1 h with the indicated concentration of GSK2606414 before addition of 100 nM Tg for 6 h. Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. <b>(B)</b> HCT116 cells were transfected with a dual luciferase reporter construct for assay of CAP/IRES-dependent translation. 24 h post-transfection, cells were pre-treated for 1 h with 100 nM GSK2606414 (GSK) before addition of 2 μg ml^-1 Tm or 1 μM AZD8055 for 24 h. Results shown are the mean ± S.D. luciferase activity within the whole cell lysates of one experiment performed in technical triplicate and are representative of three independent experiments. Statistics shown are the results of Student’s unpaired <i>t</i>-tests; N.S., not significant; *, p < 0.05. <b>(C)</b> HCT116 cells were pre-treated for 1 h with 100 nM GSK2606414 prior to the addition of the indicated concentration of Tm for 24 h. Whole cell lysates were fractionated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. Results in (A) and (C) are representative of 3 independent experiments.</p

Activation of ERK1/2 protects against cell death arising from PERK inhibition.

<p><b>(A)</b> NIH3T3 ΔCRAF:ER cells were pre-treated with 100 nM 4-HT for 1 h before addition of 100 nM GSK2606414 (GSK) and 0.1 μg ml^-1 Tm alone or in combination as indicated for 24 h. Whole cell lysates were fractionated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. Results are representative of two independent experiments. <b>(B)</b> NIH3T3 ΔCRAF:ER cells were pre-treated with 100 nM 4-HT for 1 h before addition of 100 nM GSK2606414 (GSK) and 0.1 μg ml^-1 Tm alone or in combination as indicated for 48 h. Cells were fixed and analysed by flow cytometry following propidium iodide staining. Results are means ± S.D. of three technical replicates from a single experiment and are representative of two independent experiments.</p

The PERK inhibitor GSK2606414 exacerbates ER stress-induced apoptosis.

<p><b>(A)</b> HCT116 cells were pre-treated for 1 h with 100 nM GSK2606414 prior to addition of the indicated concentration of Tm for 48 h. Cells were fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results shown are the means ± S.D. of 3 independent experiments each performed in technical triplicate. Statistics represent the results of two-way ANOVA and Bonferroni post-tests; ***, p < 0.001. <b>(B)</b> HCT116 cells were treated as in (A) for 6 h (top panel) or 24 h (bottom panel). Whole cell lysates were analysed by immunoblotting using the indicated antibodies following separation by SDS-PAGE. Results shown are representative of 3 independent experiments. <b>(C)</b> HCT116 and HCT116 BAK^-/-, BAX^-/- (DKO) cells were pre-treated for 1 h with 100 nM GSK2606414 (GSK) before 48 h treatment with 0.1 μg ml^-1 Tm. Cells were fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. <b>(D)</b> HCT116 cells were pre-treated for 1 h with 100nM GSK2606414 prior to addition of 0.1 μg ml^-1 Tm and 10 μM QVD-oPh (QVD), as indicated, for 48 h. Cells were fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results shown in (C) and (D) are the means ± S.D. of 3 independent experiments performed in technical triplicate. Statistics represent the results of Student’s unpaired <i>t</i>-tests; **, p < 0.01.</p

ER stress induces BAK/BAX-dependent, apoptotic cell death.

<p><b>(A)</b> HCT116 cells were treated with 100 nM Tg (top panel) or 2 μg ml^-1 Tm (bottom panel) for the indicated time, whole cell lysates were fractionated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Results are representative of at least 3 independent experiments. <b>(B)</b> HCT116 cells were treated with 100 nM Tg in the presence or absence of 10 μM QVD-oPh for 48 h, fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results are the means ± S.D. of 3 experiments each performed in technical triplicate. Student’s unpaired <i>t</i>-test results are indicated as follows; ***, p < 0.001. <b>(C)</b> Whole cell lysates of HCT116, HCT116 BAK^-/-, HCT116 BAX^-/- or HCT116 BAK^-/-, BAX^-/- (DKO) cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies to confirm their genotype. <b>(D)</b> HCT116, HCT116 BAK^-/-, HCT116 BAX^-/- or HCT116 BAK^-/-, BAX^-/- (DKO) cells were treated with 2 μg ml^-1 Tm for 48 h, fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results are the means ± S.D. of 3 independent experiments each performed in technical triplicate. Statistics represent the results of two-way ANOVA and Bonferroni post-tests comparing each genotype to WT; ***, p < 0.001. <b>(E)</b> HCT116, HCT116 BAK^-/-, HCT116 BAX^-/- or HCT116 BAK^-/-, BAX^-/- (DKO) cells were treated with 2 μg ml^-1 Tm for 4 h (top panel) or 24 h (bottom panel). Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Results are representative of three independent experiments.</p

Activation of ERK1/2 protects against ER stress-induced cell death.

<p><b>(A)</b> NIH3T3 cells were treated with either 100 nM Tg (left panel) or 2 μg ml^-1 Tm (right panel) for the indicated time. Whole cell lysates were separated by SDS-PAGE and analysed by immunoblotting using the indicated antibodies. <b>(B)</b> NIH3T3 ΔCRAF:ER cells were pre-treated for 1 h with 100 nM 4-HT prior to treatment with 2 μg ml^-1 Tm for the indicated time. Lysates were fractionated by SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Results in (A) and (B) are representative of 3 independent experiments. <b>(C)</b> NIH3T3 ΔCRAF:ER (left panel) or CCL39 ΔCRAF:ER (right panel) cells were pre-treated for 1 h with 100 nM 4-HT before addition of 2 μg ml^-1 Tm for 48 h. Cells were then fixed, stained with propidium iodide and the proportion of cells with sub-G1 DNA was measured by flow cytometry. Results are the means ± S.D. of 3 independent experiments each performed in technical triplicate. Statistics represent the results of Student’s unpaired <i>t</i>-tests; **, p < 0.01; ***, p < 0.001. <b>(D)</b> COLO205 cells were treated for 24 h with 1 μM Selumetinib (Sel) in addition to DMSO, 30 nM Tg or 0.5 μg ml^-1 Tm. Whole cell lysates were fractionated by SDS-PAGE and analysed by western blotting with the indicated antibodies. Results are representative of at least 3 independent experiments. <b>(E)</b> COLO205 cells were treated for 48 h as in (D). Cells were fixed, stained with propidium iodide and cell cycle distribution was determined by flow cytometry. Results are the means ± S.D. of an experiment performed in technical triplicate and representative of 3 independent experiments.</p