Figure 4D-G ROS accumulation in WT and Perk KO neurons (N=3) and astrocytes (N=3) ± 20-24 hours of TM-induced ER stress. (D, F) Representative images obtained by high-content microscopy. Dendrites (MAP2, white), astrocytic soma (Phalloidin, white), nuclei ((Δ)Cre, red), ROS (CellROX green, green). Scale bar: 25 μm. (E, G) Quantification of CellROX green intensity. Data information: (E, G, Data are normalised to untreated WT. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test (E, G,H mRNA expression of Eif2ak1 (HRI) in HRI KD neurons determined by RT-qPCR, normalised to WT transduced with shRNA control (represented by a dashed line) (N=3). Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis One sample t-test with a hypothetical value of 100 (HI Quantification of the increase in nuclear ATF4 intensity in WT neurons ± HRI KD ± 20 hours of sodium arsenite (Ars) treatment (N=3). J The remaining ATF4 response (%) upon HRI KD, related to ATF4 response of WT transduced with shRNA control (100%, represented by a dashed line) (N=3). Calculated from data in (I): [[(KD_Ars+-KD_Ars-)/KD_Ars-]/[(WT_Ars+-WT_Ars-)/WT_Ars-]]*100. Data information: Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test I, One sample t-test with a hypothetical value of 100 J)K-M eIF2α phosphorylation in HRI KD WT and Perk KO neurons (N=3) ± 20-24 hours of TM-induced ER stress. Representative WB (K), quantification of the ER stress-induced p-eIf2α response (L) and absolute phosphorylated eIF2α level (p-eIF2α/eIF2α) (M). Data information: M, Data are normalised to untreated WT. (L, Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test L, Students t-test (M)N-S Protein synthesis (N=3) and ATF4 expression (N=4) in HRI KD WT and Perk KO neurons ± 20-24 hours of TM-induced ER stress. Representative images obtained by (N) confocal and (Q) high-content microscopy. Dendrites (MAP2, white/cyan), nuclei ((Δ)Cre, green/red), de novo synthesized proteins (Puromycin, green), ATF4 (magenta). Scale bar: 25 μm. Quantification of the ER stress-induced protein synthesis response (O) and puromycin intensity as measure for de novo protein synthesis (P). Quantification of the ER stress-induced ATF4 response (R) and absolute nuclear ATF4 intensity (S). Data information: P, S) Data are normalised to untreated WT. O, R) Data are related to untreated cells of the same genotype and normalised to WT without HRI KD. Baseline level (without ER stress) is depicted by a dashed line. Data are presented as mean ± SEM. N: Biological replicate. Relevant P-values are indicated: *p<0.05, ** p<0.01, **** p<0.0001 and ns: not significant. Statistical analysis: Two-way ANOVA with Tukey's post hoc test O, R); Nested t-test (P, S).