Figure EV2. Induction of the ISR contributes to apoptosis in different cancer cell lines.

1. Relative Gadd34 mRNA expression levels in HCT116 cells treated as indicated for 16 h (NEN 1.2 µM, imipramine (Imi), desipramine (Desi), amitriptyline (Ami), each 30 µM, clomipramine (Clomi) 20 µM) ).
2. Immunoblot analysis of CHOP, ATF4, phosphorylated (p‐) and t‐) eIF2alpha (eIF2a) of U87 cells treated as indicated for 24 h (NEN 1.2 µM; amitriptyline (Ami), each 30 µM).
3. Densitometric quantifications of immunoblot analysis shown in Figs 2B and EV2B including CHOP, ATF4, phosphorylated (p‐) and t‐) eIF2alpha (eIF2a). HCT116 or U87 cells were treated as indicated for 16 or 24 h, respectively (NEN 1.2 µM; domperidone (Domp) amitriptyline (Ami), each 30 µM).
4. Test of knockdown efficiency for siATF4 and siCHOP upon transfection of HCT116 cells, determined by qPCR.
5. Caspase activity in BxPC3 cells grown as monolayer (2D) treated as indicated for 48 h (NEN 1.2 µM; domperidone (Domp), imipramine (Imi), desipramine (Desi), and amitriptyline (Ami), each 30 µM; clomipramine (Clomi) 20 µM, integrated stress response inhibitor (ISRIB) 1 µM).
6. Ratio of toxicity and viability in U87 spheroids (3D) treated as indicated for 48 h (NEN 1.2 µM; domperidone (Domp), imipramine (Imi), desipramine (Desi), and amitriptyline (Ami), each 30 µM; clomipramine (Clomi) 20 µM, integrated stress response inhibitor (ISRIB) 1 µM).

Data information: Data are presented as mean (SD) and were analyzed by a one‐way ANOVA with Dunnett’s post hoc test (A (N = 5, besides NEN + Clomi (N = 4) and NEN + Desi (N = 4)), two‐way ANOVA with Tukey post hoc test (C (N = 3), E (N = 4), F (N = 4) or Welsh’s t‐test (D (N = 3)). In (A), significance is indicated for the comparison of combinatorial treatments (NEN + TCAs) to controls. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Exact P‐values for all comparisons are listed in Appendix Table S1.

Source data are available online for this figure.