Cellular transcriptional response during lysosomal damage. (A) RNAseq analysis of the change in gene expression (HEK293T cells) in response to treatment with 1 mM LLOMe for 30 min. Scatter (volcano) plot shows log2 fold change and −Log10 P value for the genes identified in three independent experiments (see Table S1, Tab 3). Red dots indicate the genes downregulated; green dots indicate the genes upregulated. Dashed line, significance cut-off (P < 0.05). (B) Immunoblot analysis of DUSP1 expression level and ERK2 (T185/187) phosphorylation in HEK293T cells treated with 1 mM LLOMe for 30 min. (C) Immunoblot analysis of TFEB (S142) phosphorylation in U2OS cells treated with 2 mM LLOMe for 30 min. (D) Immunoblot analysis of ERK2 (T185/187) and TFEB (S142) phosphorylation in Huh7 cells transfected with scrambled siRNA as control (SCR) or DUSP1 siRNA treated with 2 mM LLOMe for 30 min. (E) Quantification by HCM of TFEB nuclear translocation in Huh7 cells treated with or without 530 nM ERK2 inhibitor AZD6244 for 2 h followed by 2 mM LLOMe for 30 min. Blue: nuclei, Hoechst 33342. Red: anti-TFEB antibody, Alexa Fluor 568. White masks, computer-algorithm-defined cell boundaries. Pink masks, computer-identified nuclear TFEB based on the average intensity of Alexa Fluor 568 fluorescence. (F) Immunoblot analysis of ERK2 (T185/187) and TFEB (S142) phosphorylation in Huh7 cells treated with or without 530 nM ERK2 inhibitor AZD6244 for 2 h followed by 2 mM LLOMe for 30 min. Ctrl, control (untreated cells). Data, means ± SEM; HCM: n ≥ 3 (each experiment: 500 valid primary objects/cells per well, ≥5 wells/sample). **, P < 0.01, ANOVA. Source data are available for this figure: SourceData F2.
