Ubiquitination-activated TAB–TAK1–IKK–NF-κB axis modulates gene expression for cell survival in the lysosomal damage response
- Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Japan
- Technology Research Division, Tokyo Metropolitan Institute of Medical Science, Japan
- Intracellular Quality Control Project, Tokyo Metropolitan Institute of Medical Science, Japan
Figures
Figure 1 with 1 supplement
Lysosomal damage has a global impact on the transcriptome and proteome.
(A, B) The mean Log2 fold change (FC, LLOMe 2 hr/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 Molecular Signatures Database (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) RPE-1 cells were treated with LLOMe for 2 hr and then washed. The total incubation times are indicated. Total cell lysates were subjected to immunoblotting with the indicated antibodies. (H) Schematic model of the cellular response to lysosomal damage.
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Figure 1—source data 1
Original files for western blot analysis for Figure 1G.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig1-data1-v1.zip
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Figure 1—source data 2
Uncropped full images displayed in Figure 1G.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig1-data2-v1.pdf
Figure 1—figure supplement 1
Representative targets induced by lysosomal damage.
(A) Heatmap showing genes upregulated at both the protein and mRNA levels, as derived from Figure 1C (top 30 in proteomic upregulation). The color scale represents Log2 fold change (FC) (LLOMe 2 hr/control). (B) RNA counts (top) and protein abundance (bottom) of representative genes, as measured by RNA sequencing and mass spectrometry (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).
Figure 2 with 1 supplement
Lysosomal damage activates TAK1 in a ubiquitin- and TAB-dependent manner.
(A) RPE-1 cells treated with L-leucyl–L-leucine methyl ester (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 TAK-243 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. (E) HeLa cells stably expressing FLAG-TAB2 WT, dNZF, and E685A were transfected with the indicated siRNAs and treated with LLOMe for 15 min. Total cell lysates were subjected to immunoblotting with the indicated antibodies.
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Figure 2—source data 1
Original files for western blot analysis for Figure 2B and C.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig2-data1-v1.zip
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Figure 2—source data 2
Uncropped full images displayed in Figure 2B and C.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig2-data2-v1.pdf
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Figure 2—source data 3
Original files for western blot analysis for Figure 2D and E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig2-data3-v1.zip
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Figure 2—source data 4
Uncropped full images displayed in Figure 2D and E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig2-data4-v1.pdf
Figure 2—figure supplement 1
The analysis in various lysosome-damaging conditions.
(A) RPE-1 cells treated with L-leucyl–L-leucine methyl ester (LLOMe) for 5 min were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm. (B) RPE-1 cells pre-treated with TAK-243 for 15 min and treated with LLOMe for 5 min were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm. (C, D) RPE-1 cells treated with glycyl-L-phenylalanine 2-naphthylamide (GPN) (C) and DC661 (D) for 4 hr were immunostained with the indicated antibodies and DAPI. Scale bar, 20 μm. (E) Total cell lysates from RPE-1 cells treated with GPN, DC661, and LLOMe for the indicated times were subjected to immunoblotting with the indicated antibodies.
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis for Figure 2—figure supplement 1E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Uncropped full images displayed in Figure 2—figure supplement 1E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig2-figsupp1-data2-v1.pdf
Figure 3 with 1 supplement
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 L-leucyl–L-leucine methyl ester (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 Gene Ontology Molecular Function (GOMF) (C) and Molecular Signatures Database (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 hr/control). (F) Total cell lysates from RPE-1 cells transfected with the indicated siRNAs, treated with LLOMe for 2 hr, and washed for 2 hr were subjected to immunoblotting with the indicated antibodies.
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Figure 3—source data 1
Original files for western blot analysis for Figure 3F.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig3-data1-v1.zip
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Figure 3—source data 2
Uncropped full images displayed in Figure 3F.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig3-data2-v1.pdf
Figure 3—figure supplement 1
TAB- and TAK1-dependent gene expression in the lysosomal damage response.
(A) Scatter plots showing the correlation between the mean Log2 fold change (FC) (L-leucyl–L-leucine methyl ester [LLOMe] 2 hr/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 Gene Ontology Molecular Function (GOMF). (C) Molecular Signatures Database (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).
Figure 4 with 1 supplement
The K63 Ub–TAB–TAK1–IKK–NF-κB pathway induces the expression of cytokines and transcription factors.
(A) The mean Log2 fold change (FC) (L-leucyl–L-leucine methyl ester [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 TAK-243 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.
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Figure 4—source data 1
Original files for western blot analysis for Figure 4C.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data1-v1.zip
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Figure 4—source data 2
Uncropped full images displayed in Figure 4C.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data2-v1.pdf
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Figure 4—source data 3
Original files for western blot analysis for Figure 4D.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data3-v1.zip
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Figure 4—source data 4
Uncropped full images displayed in Figure 4D.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data4-v1.pdf
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Figure 4—source data 5
Original files for western blot analysis for Figure 4E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data5-v1.zip
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Figure 4—source data 6
Uncropped full images displayed in Figure 4E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data6-v1.pdf
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Figure 4—source data 7
Original files for western blot analysis for Figure 4F.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data7-v1.zip
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Figure 4—source data 8
Uncropped full images displayed in Figure 4F.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-data8-v1.pdf
Figure 4—figure supplement 1
Signaling pathways in the lysosomal damage response.
(A) The mean Log2 fold change (FC) (L-leucyl–L-leucine methyl ester [LLOMe] 30 min/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) Phosphopeptide abundance measured by mass spectrometry (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). (D) Total cell lysates from RPE-1 cells treated with LLOMe for the indicated times were subjected to immunoblotting with the indicated antibodies. (E) 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. (F) Total cell lysates from RPE-1 cells pre-treated with BX-795 for 15 min and treated with LLOMe for 10 min were subjected to immunoblotting with the indicated antibodies. (G) 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.
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Figure 4—figure supplement 1—source data 1
Original files for western blot analysis for Figure 4—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Uncropped full images displayed in Figure 4—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-figsupp1-data2-v1.pdf
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Figure 4—figure supplement 1—source data 3
Original files for western blot analysis for Figure 4—figure supplement 1F.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-figsupp1-data3-v1.zip
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Figure 4—figure supplement 1—source data 4
Uncropped full images displayed in Figure 4—figure supplement 1F.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-figsupp1-data4-v1.pdf
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Figure 4—figure supplement 1—source data 5
Original files for western blot analysis for Figure 4—figure supplement 1G.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-figsupp1-data5-v1.zip
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Figure 4—figure supplement 1—source data 6
Uncropped full images displayed in Figure 4—figure supplement 1G.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig4-figsupp1-data6-v1.pdf
Figure 5 with 1 supplement
The K63 Ub–TAB–TAK1–IKK–NF-κB pathway promotes cell survival and intercellular signaling.
(A, B) Total RNA from RPE-1 cells pre-treated with TAK-243 (A) and HS-276 (B) for 15 min and treated with L-leucyl–L-leucine methyl ester (LLOMe) for 2 hr 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 hr 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, E) HeLa cells were pre-treated with TAK-243 or HS-276 for 15 min, treated with LLOMe for 2 hr, and washed for 6 hr. Total cell lysates were subjected to immunoblotting with the indicated antibodies (D). Cells were stained with propidium iodide (PI), and the fractions of PI-positive cells were assessed using the flow cytometer. Error bars indicate SD (n=3). ****p<0.0001 (one-way ANOVA with Dunnett’s test) (E). (F, G) Total cell lysates from HeLa cells transfected with the indicated siRNAs, treated with LLOMe for 2 hr, and washed for 22 hr were subjected to immunoblotting with the indicated antibodies. (H) 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 hr. Total cell lysates were subjected to immunoblotting with the indicated antibodies. (I, J) 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 hr. Total cell lysates were subjected to immunoblotting with the indicated antibodies.
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Figure 5—source data 1
Original files for western blot analysis for Figure 5D, F, and G.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig5-data1-v1.zip
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Figure 5—source data 2
Uncropped full images displayed in Figure 5D, F, and G.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig5-data2-v1.pdf
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Figure 5—source data 3
Original files for western blot analysis for Figure 5H–J.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig5-data3-v1.zip
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Figure 5—source data 4
Uncropped full images displayed in Figure 5H–J.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig5-data4-v1.pdf
Figure 5—figure supplement 1
Anti-apoptotic regulation in the lysosomal damage response.
(A) Total cell lysates from RPE-1 cells pre-treated with TAK-243 or HS-276 for 15 min, treated with L-leucyl–L-leucine methyl ester (LLOMe) for 2 hr, and washed for 6 hr were subjected to immunoblotting with the indicated antibodies. (B) Total cell lysates from HeLa cells pre-treated with JNK-IN-8 for 15 min, treated with LLOMe for 2 hr, and washed for 10 hr were subjected to immunoblotting with the indicated antibodies.
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Figure 5—figure supplement 1—source data 1
Original files for western blot analysis for Figure 5—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Uncropped full images displayed in Figure 5—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/106901/elife-106901-fig5-figsupp1-data2-v1.pdf
Figure 6
Model of the ubiquitin-mediated cellular response to lysosomal damage.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | RPE-1 | ATCC | CRL-4000 RRID:CVCL_4388 | |
| Cell line (Homo sapiens) | HeLa | ATCC | CCL-2 RRID:CVCL_0030 | |
| Antibody | anti-IL6 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 12153 RRID:AB_2687897 | WB (1:1000) |
| Antibody | anti-IRF1 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 8478 RRID:AB_10949108 | WB (1:1000) |
| Antibody | anti-NKX3.1 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 92998; RRID:AB_2800197 | WB (1:1000) |
| Antibody | anti-c-Fos (rabbit monoclonal) | Cell Signaling Technology | Cat#: 2250 RRID:AB_2247211 | WB (1:1000) |
| Antibody | anti-c-Jun (rabbit monoclonal) | Cell Signaling Technology | Cat#: 9165 RRID:AB_2130165 | WB (1:1000) |
| Antibody | HRP-conjugated anti-α-tubulin (rabbit polyclonal) | MBL | Cat#: PM054-7 RRID:AB_10695326 | WB (1:1000) |
| Antibody | anti-K63 ubiquitin (rabbit monoclonal) | Millipore | Cat#: 05-1308 RRID:AB_1587580 | WB (1:500) IF (1:200) |
| Antibody | anti-ubiquitin (mouse monoclonal) | Santa Cruz Biotechnology | Cat# sc-8017 RRID:AB_628423 | WB (1:200) |
| Antibody | anti-phospho-TAK1 (T184/T187) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 4508 RRID:AB_561317 | WB (1:1000) |
| Antibody | anti-TAK1 (rabbit polyclonal) | Cell Signaling Technology | Cat#: 4505 RRID:AB_490858 | WB (1:1000) |
| Antibody | anti-TAB2 (rabbit polyclonal) | Abcam | Cat#: ab222214 | WB (1:500) |
| Antibody | anti-TAB3 (rabbit polyclonal) | Abcam | Cat#: ab85655 RRID:AB_2140510 | WB (1:500) |
| Antibody | HRP-conjugated anti-FLAG (mouse monoclonal) | Sigma-Aldrich | Cat#: A8592 RRID:AB_439702 | WB (1:1000) |
| Antibody | anti-TFEB (rabbit monoclonal) | Cell Signaling Technology | Cat#: 37785 RRID:AB_2799119 | WB (1:1000) |
| Antibody | anti-phospho-IKKα/β (S176/S180) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 2697 RRID:AB_2079382 | WB (1:1000) |
| Antibody | anti-IKKα (rabbit polyclonal) | Cell Signaling Technology | Cat#: 2682 RRID:AB_331626 | WB (1:1000) |
| Antibody | anti-IKKβ (rabbit monoclonal) | Cell Signaling Technology | Cat#: 8943 RRID:AB_11024092 | WB (1:1000) |
| Antibody | anti-phospho-IκBα (S32) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 2859 RRID:AB_561111 | WB (1:1000) |
| Antibody | anti-IκBα (rabbit monoclonal) | Cell Signaling Technology | Cat#: 4812 RRID:AB_10694416 | WB (1:1000) |
| Antibody | anti-phospho-JNK (T183/Y185) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 4668 RRID:AB_823588 | WB (1:1000) |
| Antibody | anti-JNK (rabbit polyclonal) | Cell Signaling Technology | Cat#: 9252 RRID:AB_2250373 | WB (1:1000) |
| Antibody | anti-phospho-p38 (T180/Y182) (rabbit polyclonal) | Cell Signaling Technology | Cat#: 9211 RRID:AB_331641 | WB (1:1000) |
| Antibody | anti-p38 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 8690 RRID:AB_10999090 | WB (1:1000) |
| Antibody | anti-phospho-ERK (T202/Y204) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 4370 RRID:AB_2315112 | WB (1:1000) |
| Antibody | anti-ERK (rabbit monoclonal) | Cell Signaling Technology | Cat#: 4695 RRID:AB_390779 | WB (1:1000) |
| Antibody | anti-cleaved caspase-3 (rabbit polyclonal) | Cell Signaling Technology | Cat#: 9661 RRID:AB_2341188 | WB (1:1000) |
| Antibody | anti-phospho-STAT3 (Y705) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 9145 RRID:AB_2491009 | WB (1:1000) |
| Antibody | anti-STAT3 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 4904 RRID:AB_331269 | WB (1:1000) |
| Antibody | anti-phospho-TBK1 (S172) (rabbit monoclonal) | Cell Signaling Technology | Cat#: 5483 RRID:AB_10693472 | WB (1:1000) |
| Antibody | anti-TBK1 (rabbit monoclonal) | Abcam | Cat#: ab40676 RRID:AB_776632 | WB (1:2000) |
| Antibody | anti-RNF31 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 99633 RRID:AB_2891320 | WB (1:1000) |
| Antibody | HRP-conjugated goat anti-rabbit IgG | Promega | Cat#: W4011 RRID:AB_430833 | WB (1:20,000) |
| Antibody | HRP-conjugated goat anti-mouse IgG | Promega | Cat#: W4021 RRID:AB_430834 | WB (1:20,000) |
| Antibody | anti-TAB2 (mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-398188 RRID:AB_2885043 | IF (1:50) |
| Antibody | anti-LAMP1 (rabbit monoclonal) | Cell Signaling Technology | Cat#: 9091 RRID:AB_2687579 | IF (1:100) |
| Antibody | anti-TAK1 (mouse monoclonal) | Santa Cruz Biotechnology | Cat#: sc-7967 RRID:AB_627929 | IF (1:50) |
| Antibody | anti-Galectin-3 (rat monoclonal) | Santa Cruz Biotechnology | Cat#: sc-23938 RRID:AB_627658 | WB (1:50) |
| Antibody | Alexa Fluor 488-conjugated anti-mouse (goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11029 RRID:AB_2534088 | IF (1:1000) |
| Antibody | Alexa Fluor 488-conjugated anti-rat (goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11006 RRID:AB_2534074 | IF (1:1000) |
| Antibody | Alexa Fluor 594-conjugated anti-rabbit (goat polyclonal) | Thermo Fisher Scientific | Cat#: A-11012 RRID:AB_2534079 | IF (1:1000) |
| Sequence-based reagent | siRNA: non-targeting | Horizon Discovery | Cat#: D-001810-03 | On-TARGETplus |
| Sequence-based reagent | siRNA: TAB2 | Horizon Discovery | Cat#: L-004771 | On-TARGETplus SMARTpool |
| Sequence-based reagent | siRNA: TAB3 | Horizon Discovery | Cat#: L-015572 | On-TARGETplus SMARTpool |
| Sequence-based reagent | siRNA: TAK1 | Horizon Discovery | Cat#: L-003790 | On-TARGETplus SMARTpool |
| Sequence-based reagent | siRNA: IKKα | Horizon Discovery | Cat#: L-003473 | On-TARGETplus SMARTpool |
| Sequence-based reagent | siRNA: IKKβ | Horizon Discovery | Cat#: L-003503 | On-TARGETplus SMARTpool |
| Sequence-based reagent | siRNA: IKKγ | Horizon Discovery | Cat#: L-003763 | On-TARGETplus SMARTpool |
| Sequence-based reagent | siRNA: RNF31 | Horizon Discovery | Cat#: L-021419 | On-TARGETplus SMARTpool |
| Commercial assay or kit | RNeasy kit | QIAGEN | Cat#: 74104 | |
| Commercial assay or kit | DNase | QIAGEN | Cat#: 79254 | |
| Commercial assay or kit | S-Trap micro columns | ProtiFi | Cat#: C02-micro | |
| Commercial assay or kit | S-Trap mini columns | ProtiFi | Cat#: C02-mini | |
| Commercial assay or kit | S-Trap mini columns | ProtiFi | Cat#: C02-mini | |
| Commercial assay or kit | Trypsin and lysyl-endopeptidase (Lys-C) | Thermo Fisher Scientific | Cat#: A41009 | |
| Commercial assay or kit | High-Select Fe-NTA Phosphopeptide Enrichment Kit | Thermo Fisher Scientific | Cat#: A32992 | |
| Commercial assay or kit | C18 spin tips | Thermo Fisher Scientific | Cat#: 84850 | |
| Chemical compound, drug | L-Leucyl–L-Leucine methyl ester (LLOMe) | Cayman | Cat#: 16008 | |
| Chemical compound, drug | Glycyl-L-phenylalanine 2-naphthylamide (GPN) | Cayman | Cat#: 14634 | |
| Chemical compound, drug | TAK-243 | Active Biochem | Cat#: A-1384 | |
| Chemical compound, drug | HS-276 | Sigma-Aldrich | Cat#: SML-3629 | |
| Chemical compound, drug | BX-795 | Abcam | Cat#: ab142016 | |
| Chemical compound, drug | JNK-IN-8 | Cayman | Cat#: 18096 | |
| Software, algorithm | DRAGEN RNA Pipeline Application | Illumina | v.3.10.12 | |
| Software, algorithm | DIA-NN | Demichev et al., 2020 | RRID:SCR_022865 | v.1.9.1 |
| Software, algorithm | Perseus | Tyanova et al., 2016 | RRID:SCR_015753 | v.2.0.3 |
| Software, algorithm | RNAseqChef | Etoh and Nakao, 2023 | v.1.1.4 | |
| Software, algorithm | Morpheus | https://software.broadinstitute.org/morpheus/ | RRID:SCR_017386 | |
| Software, algorithm | GraphPad Prism | Demichev et al., 2020 | RRID:SCR_002798 | v.8.1.0 |
| Software, algorithm | Proteome Discoverer | Thermo Fisher Scientific | RRID:SCR_014477 | v.3.1 |
| Software, algorithm | ZEN | Carl Zeiss | RRID:SCR_013672 | v.3.8 |
Additional files
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Supplementary file 1
Transcriptome analysis L-leucyl–L-leucine methyl ester (LLOMe) 2 hr.
- https://cdn.elifesciences.org/articles/106901/elife-106901-supp1-v1.xlsx
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Supplementary file 2
Proteome analysis L-leucyl–L-leucine methyl ester (LLOMe) 2 hr.
- https://cdn.elifesciences.org/articles/106901/elife-106901-supp2-v1.xlsx
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Supplementary file 3
Correlation of transcriptome and proteome L-leucyl–L-leucine methyl ester (LLOMe) 2 hr.
- https://cdn.elifesciences.org/articles/106901/elife-106901-supp3-v1.xlsx
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Supplementary file 4
Transcriptome analysis RNAi L-leucyl–L-leucine methyl ester (LLOMe) 2 hr.
- https://cdn.elifesciences.org/articles/106901/elife-106901-supp4-v1.xlsx
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Supplementary file 5
Proteome analysis L-leucyl–L-leucine methyl ester (LLOMe) 30 min.
- https://cdn.elifesciences.org/articles/106901/elife-106901-supp5-v1.xlsx
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Supplementary file 6
Phosphoproteome analysis with HS-276.
- https://cdn.elifesciences.org/articles/106901/elife-106901-supp6-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/106901/elife-106901-mdarchecklist1-v1.docx
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Ubiquitination-activated TAB–TAK1–IKK–NF-κB axis modulates gene expression for cell survival in the lysosomal damage response
eLife 14:RP106901.
https://doi.org/10.7554/eLife.106901.3
