Lysosomal damage has a global impact on the transcriptome and proteome

(A, B) The mean Log2 fold change (FC, LLOMe 2 h/control) and −Log10 P value of the transcriptome (A) and proteome (B) in RPE-1 cells are indicated on the x and y axes, respectively. Genes significantly upregulated or downregulated are labeled in red and blue, respectively.

(C) For genes showing significant upregulation in the transcriptome (Log2 FC >1 and P <0.05), the correlation between the mean Log2 FC of the transcriptome (from A, x axis) and the proteome (from B, y axis) is shown with a coefficient of correlation (R = 0.5899, n = 302).

(D, E) The bubble plots show the outcomes of gene enrichment analyses based on gene ontology molecular function (GOMF) (D) and MSigDB hallmark gene sets (E) for genes upregulated at the protein and RNA levels (top ten and five categories for GOMF and MSigDB hallmark gene sets, respectively). The color and size of the bubbles indicate the q value and gene ratio, respectively. The categories related to transcription, cytokine/growth factor, and apoptosis are labeled in red, blue, and green, respectively.

(F) The bubble plot illustrates the DoRothEA regulon-based prediction of transcription factors responsible for the induction of genes upregulated in cells treated with LLOMe (top five transcription factors). The color and size of the bubbles indicate the q value and gene ratio, respectively. NF-κB components are labeled in blue.

(G) Total cell lysates from RPE-1 cells treated with LLOMe for the indicated times were subjected to immunoblotting with the indicated antibodies.

(H) Schematic model of the cellular response to lysosomal damage.

Lysosomal damage activates TAK1 in a ubiquitin- and TAB-dependent manner

(A) RPE-1 cells treated with LLOMe for 5 min were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm.

(B) Total cell lysates from RPE-1 cells treated with LLOMe for the indicated times were subjected to immunoblotting with the indicated antibodies.

(C) Total cell lysates from RPE-1 cells pre-treated with TAK243 for 15 min and treated with LLOMe for 15 min were subjected to immunoblotting with the indicated antibodies.

(D) Total cell lysates from RPE-1 cells transfected with the indicated siRNAs and treated with LLOMe for 15 min were subjected to immunoblotting with the indicated antibodies.

The TAB–TAK1 pathway activates cytokines and transcription factors in response to lysosomal damage

(A) Heatmap showing the changes in the transcriptome of each sample. Clusters 4 and 5 include the genes upregulated by LLOMe in a TAB- and TAK1-dependent manner.

(B) The bubble plot shows the DoRothEA regulon-based prediction of transcription factors responsible for the induction of genes assigned to clusters 4 and 5 in (A) (top five transcription factors). The color and size of bubbles indicate q value and gene ratio, respectively. NF-κB components are labeled in blue.

(C, D) The bubble plots show the outcomes of gene enrichment analyses based on GOMF (C) and MSigDB hallmark gene sets (D) for genes assigned to clusters 4 and 5 in (A) (top five categories). The color and size of the bubbles indicate the q value and gene ratio, respectively. The categories related to transcription, cytokine/growth factor, and apoptosis, are labeled in red, blue, and green, respectively.

(E) Heatmaps showing the genes upregulated in a TAB- and TAK1-dependent manner within the categories of inflammatory response (left) and transcription-related factors (right). The color intensity indicates the Log2 FC (LLOMe 2 h/control).

(F) Total cell lysates from RPE-1 cells transfected with the indicated siRNAs, treated with LLOMe for 2 h, and washed for 2 h were subjected to immunoblotting with the indicated antibodies.

The K63Ub–TAB–TAK1–IKK–NF-κB pathway induces the expression of cytokines and transcription factors

(A) The mean Log2 FC (LLOMe 30 min/control) and −Log10 P value of the proteome are indicated on the x and y axes, respectively. Proteins significantly upregulated or downregulated are labeled in red and blue, respectively.

(B) IκBα protein abundance determined by mass spectrometry (MS) analysis. The individual values, mean, and standard deviation (SD) of the mean of protein abundance are presented. The mean ± SD values were calculated from two biological replicates. *P <0.05 (one-way ANOVA with Dunnett’s test).

(C) Total cell lysates from RPE-1 cells treated with LLOMe for the indicated times were subjected to immunoblotting with the indicated antibodies.

(D) Total cell lysates from RPE-1 cells pre-treated with TAK243 for 15 min and treated with LLOMe for 10 min were subjected to immunoblotting with the indicated antibodies.

(E, F) Total cell lysates from RPE-1 cells transfected with the indicated siRNAs and treated with LLOMe for 10 min were subjected to immunoblotting with the indicated antibodies.

(G) Schematic model of the cellular signaling pathways activated in response to lysosomal damage.

The K63Ub–TAB–TAK1–IKK–NF-κB pathway promotes cell survival and intercellular signaling

(A, B) Total RNA from RPE-1 cells pre-treated with TAK243 (A) and HS-276 (B) for 15 min and treated with LLOMe for 2 h was analyzed by RT-qPCR. Target mRNA levels were normalized to GAPDH mRNA levels; the expression levels in control cells were set to 1. The individual values, mean, and standard error of the mean (SEM) of relative mRNA levels are presented. The mean ± SEM values were calculated from three biological replicates. ****P <0.0001 (one-way ANOVA with Dunnett’s test).

(C) Total RNA from RPE-1 cells transfected with the indicated siRNAs and treated with LLOMe for 2 h was analyzed by RT-qPCR. Target mRNA levels were normalized to GAPDH mRNA levels; the expression levels in cells treated with control siRNA were set to 1. The individual values, mean, and SEM of relative mRNA levels are presented. The mean ± SEM values were calculated from three biological replicates. ****P <0.0001 (one-way ANOVA with Dunnett’s test).

(D) Total cell lysates from HeLa cells pre-treated with TAK243 or HS-276 for 15 min, treated with LLOMe for 2 h, and washed for 6 h were subjected to immunoblotting with the indicated antibodies.

(E, F) Total cell lysates from HeLa cells transfected with the indicated siRNAs and treated with LLOMe for 2 h, and washed for 22 h were subjected to immunoblotting with the indicated antibodies.

(G) RPE-1 cells were stimulated for the indicated times with conditioned media (CM) from RPE-1 cells treated with LLOMe for 30 min and washed for 7.5 h. Total cell lysates were subjected to immunoblotting with the indicated antibodies.

(H, I) RPE-1 cells were stimulated for 15 min with CM from RPE-1 cells transfected with the indicated siRNAs, treated with LLOMe for 30 min, and washed for 7.5 h. Total cell lysates were subjected to immunoblotting with the indicated antibodies.

Model of the ubiquitin-mediated cellular response to lysosomal damage

(A) Heatmap showing genes upregulated at both the protein and mRNA levels, as derived from Fig. 1C (top 30 in proteomic upregulation). The color scale represents Log2 FC (LLOMe 2 h/control). (B) RNA counts (top) and protein abundance (bottom) of representative genes, as measured by RNA sequencing and MS analysis, respectively. The individual values, mean, and SEM are shown. The mean ± SEM values were calculated from three biological replicates. *P <0.05, ***P <0.001, and ****P <0.0001 (two-tailed Student’s t-test).

(A, B) RPE-1 cells treated with LLOMe for 5 min were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm.

(A) Scatter plots showing the correlation between the mean Log2 FC (LLOMe 2 h/control) in cells transfected with siControl (x axis) and siTAB2/3 (y axis) (left), and siControl (x axis) and siTAK1 (y axis) (right). (B) Venn diagram showing the overlap of genes upregulated by LLOMe treatment in a TAB- and TAK1-dependent manner. (C, D) The bubble plots show the outcomes of gene enrichment analyses based on GOMF (C) and MSigDB hallmark gene sets (D) for genes upregulated by LLOMe treatment in a TAB- and TAK1-dependent manner (top five categories). The color and size of the bubbles indicate the q value and gene ratio, respectively. The categories related to cytokine/growth factor and apoptosis are labeled in blue and green, respectively. (E) RNA counts of representative genes, as measured by RNA sequencing. The individual values, mean, and SEM are presented. The mean ± SD values were calculated from three biological replicates. ****P <0.0001 (one-way ANOVA with Dunnett’s test).

(A) The mean Log2 FC (LLOMe 2 h/control) and −Log10 P value of the phosphopeptides are indicated on the x and y axes, respectively. Phosphopeptides significantly upregulated or downregulated are labeled in red and blue, respectively. (B) Correlation between the mean Log2 FC (LLOMe 30 min/control) in cells treated with (y axis) or without HS-276 (x axis) is shown for phosphopeptides that were significantly upregulated 30 min after LLOMe treatment (Log2 FC >1 and P <0.05). (C) Phosphopeptides abundance measured by MS analysis. The individual values, mean, and SD of the mean of phosphopeptide abundance are shown. The mean ± SD values were calculated from three biological replicates. **P <0.01 and ****P <0.0001 (one-way ANOVA with Dunnett’s test).

(A) Total cell lysates from RPE-1 cells pre-treated with TAK243 or HS-276 for 15 min, treated with LLOMe for 2 h, and washed for 6 h were subjected to immunoblotting with the indicated antibodies.