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REV1 downregulates CTH expression by promoting Rad18 binding and ubiquitinating CTH for degradation. A Heatmap generated from the results of enrichment of Gly/Ser/Thr signaling pathways in RNA sequencing. B REV1 was knocked down in A549 and H1299 cells, and the relative mRNA levels of CTH were detected by RT-PCR. *** P < 0.001, ( n = 3). C A549, H1299, and H1975 cells were transfected with siREV1, and the expression of the corresponding antibody was detected by Western blotting after 48 h ( n = 3). D DMSO and JH-RE-06-treated A549, H1299, and H1975 cells were harvested and CTH expression was detected by Western blotting ( n = 3). E-F Endogenous interaction of CTH with REV1 and Rad18 in A549 cells and H1299 cells. G Proteins of HEK293T cells transfected with Flag-Rad18 were collected and added to GST-only and GST-CTH fusion proteins, respectively. The expression of the corresponding molecules was detected by Western blotting after overnight enrichment and elution ( n = 3). H A549, H1299, and H1975 cells were transfected with siRad18, and the expression of the corresponding proteins was detected by Western blotting after 48 h ( n = 3). I Scrambled and Rad18-deleted A549 cells were treated with DMSO or MG132 for 4 h before protein collection, and the expression of the indicating molecules was examined by Western blotting ( n = 3). J Cells were transfected with the corresponding plasmids and siRNA, MG132 was added 4 h before protein collection treatment, and the in vitro CTH <t>ubiquitination</t> level was detected using the immunoprecipitation technique ( n = 3). K Left panel: Scrambled and Rad18-deleted A549 cells were treated with CHX (20 µg/mL). Cellular proteins were collected at the corresponding times, and the protein levels of CTH were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of CTH protein ( n = 3). L Proteins of HEK-293T cells transfected with the corresponding plasmids were harvested and incubated with GST-only or GST-CTH fusion proteins overnight and analyzed by Western blotting ( n = 3). M-N A549 and H1975 cells were transfected with the corresponding siRNA or plasmid and analyzed by Western blotting using the corresponding antibodies ( n = 3)
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REV1 downregulates CTH expression by promoting Rad18 binding and ubiquitinating CTH for degradation. A Heatmap generated from the results of enrichment of Gly/Ser/Thr signaling pathways in RNA sequencing. B REV1 was knocked down in A549 and H1299 cells, and the relative mRNA levels of CTH were detected by RT-PCR. *** P < 0.001, ( n = 3). C A549, H1299, and H1975 cells were transfected with siREV1, and the expression of the corresponding antibody was detected by Western blotting after 48 h ( n = 3). D DMSO and JH-RE-06-treated A549, H1299, and H1975 cells were harvested and CTH expression was detected by Western blotting ( n = 3). E-F Endogenous interaction of CTH with REV1 and Rad18 in A549 cells and H1299 cells. G Proteins of HEK293T cells transfected with Flag-Rad18 were collected and added to GST-only and GST-CTH fusion proteins, respectively. The expression of the corresponding molecules was detected by Western blotting after overnight enrichment and elution ( n = 3). H A549, H1299, and H1975 cells were transfected with siRad18, and the expression of the corresponding proteins was detected by Western blotting after 48 h ( n = 3). I Scrambled and Rad18-deleted A549 cells were treated with DMSO or MG132 for 4 h before protein collection, and the expression of the indicating molecules was examined by Western blotting ( n = 3). J Cells were transfected with the corresponding plasmids and siRNA, MG132 was added 4 h before protein collection treatment, and the in vitro CTH ubiquitination level was detected using the immunoprecipitation technique ( n = 3). K Left panel: Scrambled and Rad18-deleted A549 cells were treated with CHX (20 µg/mL). Cellular proteins were collected at the corresponding times, and the protein levels of CTH were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of CTH protein ( n = 3). L Proteins of HEK-293T cells transfected with the corresponding plasmids were harvested and incubated with GST-only or GST-CTH fusion proteins overnight and analyzed by Western blotting ( n = 3). M-N A549 and H1975 cells were transfected with the corresponding siRNA or plasmid and analyzed by Western blotting using the corresponding antibodies ( n = 3)

Journal: Journal of Biomedical Science

Article Title: USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase

doi: 10.1186/s12929-024-01044-3

Figure Lengend Snippet: REV1 downregulates CTH expression by promoting Rad18 binding and ubiquitinating CTH for degradation. A Heatmap generated from the results of enrichment of Gly/Ser/Thr signaling pathways in RNA sequencing. B REV1 was knocked down in A549 and H1299 cells, and the relative mRNA levels of CTH were detected by RT-PCR. *** P < 0.001, ( n = 3). C A549, H1299, and H1975 cells were transfected with siREV1, and the expression of the corresponding antibody was detected by Western blotting after 48 h ( n = 3). D DMSO and JH-RE-06-treated A549, H1299, and H1975 cells were harvested and CTH expression was detected by Western blotting ( n = 3). E-F Endogenous interaction of CTH with REV1 and Rad18 in A549 cells and H1299 cells. G Proteins of HEK293T cells transfected with Flag-Rad18 were collected and added to GST-only and GST-CTH fusion proteins, respectively. The expression of the corresponding molecules was detected by Western blotting after overnight enrichment and elution ( n = 3). H A549, H1299, and H1975 cells were transfected with siRad18, and the expression of the corresponding proteins was detected by Western blotting after 48 h ( n = 3). I Scrambled and Rad18-deleted A549 cells were treated with DMSO or MG132 for 4 h before protein collection, and the expression of the indicating molecules was examined by Western blotting ( n = 3). J Cells were transfected with the corresponding plasmids and siRNA, MG132 was added 4 h before protein collection treatment, and the in vitro CTH ubiquitination level was detected using the immunoprecipitation technique ( n = 3). K Left panel: Scrambled and Rad18-deleted A549 cells were treated with CHX (20 µg/mL). Cellular proteins were collected at the corresponding times, and the protein levels of CTH were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of CTH protein ( n = 3). L Proteins of HEK-293T cells transfected with the corresponding plasmids were harvested and incubated with GST-only or GST-CTH fusion proteins overnight and analyzed by Western blotting ( n = 3). M-N A549 and H1975 cells were transfected with the corresponding siRNA or plasmid and analyzed by Western blotting using the corresponding antibodies ( n = 3)

Article Snippet: The main antibodies and reagents used in this study are as follows: mouse anti-REV1 (Santa Cruz, sc-393022, 1:100), rabbit anti-USP9X (Proteintech, 55054-1-AP, 1:1000), rabbit anti-CTH (Abcam. ab151769, 1:1000), mouse anti-GAPDH (ABclonal, AC033, 1:3000), rabbit anti-Rad51 (Abcam, ab133534, 1:1000), rabbit anti-USP24 (Proteintech, 13126 -1-AP, 1:1000), rabbit anti-USP5 (Proteintech, 10473-1-AP, 1:1000), rabbit anti-USP1 (Proteintech, 14346-1-AP, 1:1000), rabbit anti-USP14 ( Proteintech, 14517-1-AP, 1:1000), rabbit anti-UCHL5 (Proteintech, 11527-1-AP, 1:1000), mouse anti-Flag (Sigma-Aldrich, F1804, 1:3000), rabbit anti-GST (Abcolonal, AE006, 1:1000), rabbit anti-HA (Cell Signaling Technology, #3724, 1:3000), Ubiquitination Compound Library (Selleck, L6000), JH-RE-06 (Topscience, T15611), MG132 (Calbiochem, 474,790) and cycloheximide (Sigma-Aldrich, A8244).

Techniques: Expressing, Binding Assay, Generated, Protein-Protein interactions, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, In Vitro, Ubiquitin Proteomics, Immunoprecipitation, Incubation, Plasmid Preparation

USP9X positively regulates REV1 expression through the ubiquitin-proteasome system pathway. A Protein extracted from A549 and H1299 lung cancer cells after 4 h of MG132 treatment and protein expression of REV1 detected by Western blotting. B Flow chart of the ubiquitination compound library screening system. C The four drugs obtained from the screening that inhibited REV1 protein expression most significantly were used to treat A549 cells for 24 h. The proteins were extracted, and the expression of REV1 protein was detected by Western blotting. D The targets of the four small-molecule drugs obtained from the screening. Interaction between USP9X and REV1 ( n = 3). E Endogenous interaction between REV1 and small molecule drug targets. F A549 cells were transfected with SFB-REV1 plasmid, and the interaction between USP9X and REV1 was detected by co-IP assay after 24 h ( n = 3). G siUSP9X was transfected into A549 and H1299 lung cancer cells, and REV1 expression was detected by Western blotting after 48 h ( n = 3). H A549 cells were transfected with the corresponding plasmids for 24 h, treated with MG132 for 4 h before protein extraction, and treated with S beads for immunoprecipitation. I A549 cells were transfected with Scramble or siUSP9X for 48 h, DMSO or MG132 was added 4 h before protein extraction, and the expression of the corresponding proteins was detected by Western blotting ( n = 3). J Cells were transfected with the corresponding siRNA or plasmid, MG132 was added 4 h before protein extraction, and immunoprecipitation experiments were performed with S beads for immunoprecipitation experiments ( n = 3). K Left panel: A549 lung cancer cells transfected with scramble or siUSP9X for 48 h before CHX (100 µg/mL) treatment. Cellular proteins were collected at the corresponding times, and the protein levels of REV1 were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of REV1 protein ( n = 3)

Journal: Journal of Biomedical Science

Article Title: USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase

doi: 10.1186/s12929-024-01044-3

Figure Lengend Snippet: USP9X positively regulates REV1 expression through the ubiquitin-proteasome system pathway. A Protein extracted from A549 and H1299 lung cancer cells after 4 h of MG132 treatment and protein expression of REV1 detected by Western blotting. B Flow chart of the ubiquitination compound library screening system. C The four drugs obtained from the screening that inhibited REV1 protein expression most significantly were used to treat A549 cells for 24 h. The proteins were extracted, and the expression of REV1 protein was detected by Western blotting. D The targets of the four small-molecule drugs obtained from the screening. Interaction between USP9X and REV1 ( n = 3). E Endogenous interaction between REV1 and small molecule drug targets. F A549 cells were transfected with SFB-REV1 plasmid, and the interaction between USP9X and REV1 was detected by co-IP assay after 24 h ( n = 3). G siUSP9X was transfected into A549 and H1299 lung cancer cells, and REV1 expression was detected by Western blotting after 48 h ( n = 3). H A549 cells were transfected with the corresponding plasmids for 24 h, treated with MG132 for 4 h before protein extraction, and treated with S beads for immunoprecipitation. I A549 cells were transfected with Scramble or siUSP9X for 48 h, DMSO or MG132 was added 4 h before protein extraction, and the expression of the corresponding proteins was detected by Western blotting ( n = 3). J Cells were transfected with the corresponding siRNA or plasmid, MG132 was added 4 h before protein extraction, and immunoprecipitation experiments were performed with S beads for immunoprecipitation experiments ( n = 3). K Left panel: A549 lung cancer cells transfected with scramble or siUSP9X for 48 h before CHX (100 µg/mL) treatment. Cellular proteins were collected at the corresponding times, and the protein levels of REV1 were detected by Western blotting ( n = 3). Right panel: quantitative statistical analysis of REV1 protein ( n = 3)

Article Snippet: The main antibodies and reagents used in this study are as follows: mouse anti-REV1 (Santa Cruz, sc-393022, 1:100), rabbit anti-USP9X (Proteintech, 55054-1-AP, 1:1000), rabbit anti-CTH (Abcam. ab151769, 1:1000), mouse anti-GAPDH (ABclonal, AC033, 1:3000), rabbit anti-Rad51 (Abcam, ab133534, 1:1000), rabbit anti-USP24 (Proteintech, 13126 -1-AP, 1:1000), rabbit anti-USP5 (Proteintech, 10473-1-AP, 1:1000), rabbit anti-USP1 (Proteintech, 14346-1-AP, 1:1000), rabbit anti-USP14 ( Proteintech, 14517-1-AP, 1:1000), rabbit anti-UCHL5 (Proteintech, 11527-1-AP, 1:1000), mouse anti-Flag (Sigma-Aldrich, F1804, 1:3000), rabbit anti-GST (Abcolonal, AE006, 1:1000), rabbit anti-HA (Cell Signaling Technology, #3724, 1:3000), Ubiquitination Compound Library (Selleck, L6000), JH-RE-06 (Topscience, T15611), MG132 (Calbiochem, 474,790) and cycloheximide (Sigma-Aldrich, A8244).

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Drug discovery, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Protein Extraction, Immunoprecipitation

Schematic. Left: REV1 induces metabolic abnormalities involved in the development of radioresistance in lung cancer. USP9X mediates REV1 deubiquitination to stabilize its expression in lung cancer. Aberrantly expressed REV1 acts as a scaffolding protein to assist the E3 ubiquitin ligase Rad18 in interacting with CTH, promoting CTH ubiquitination degradation and upregulating intra- and extracellular Gly/Ser/Thr levels, leading to lung cancer radiotherapy resistance. Right: Therapeutic potential of targeting REV1 in lung cancer. JH-RE-06 promotes the formation of loss-of-function dimers of REV1 and induces remodeling of intra- and extracellular amino acid metabolism, thereby enhancing the radiosensitivity of lung cancer cells

Journal: Journal of Biomedical Science

Article Title: USP9X-mediated REV1 deubiquitination promotes lung cancer radioresistance via the action of REV1 as a Rad18 molecular scaffold for cystathionine γ-lyase

doi: 10.1186/s12929-024-01044-3

Figure Lengend Snippet: Schematic. Left: REV1 induces metabolic abnormalities involved in the development of radioresistance in lung cancer. USP9X mediates REV1 deubiquitination to stabilize its expression in lung cancer. Aberrantly expressed REV1 acts as a scaffolding protein to assist the E3 ubiquitin ligase Rad18 in interacting with CTH, promoting CTH ubiquitination degradation and upregulating intra- and extracellular Gly/Ser/Thr levels, leading to lung cancer radiotherapy resistance. Right: Therapeutic potential of targeting REV1 in lung cancer. JH-RE-06 promotes the formation of loss-of-function dimers of REV1 and induces remodeling of intra- and extracellular amino acid metabolism, thereby enhancing the radiosensitivity of lung cancer cells

Article Snippet: The main antibodies and reagents used in this study are as follows: mouse anti-REV1 (Santa Cruz, sc-393022, 1:100), rabbit anti-USP9X (Proteintech, 55054-1-AP, 1:1000), rabbit anti-CTH (Abcam. ab151769, 1:1000), mouse anti-GAPDH (ABclonal, AC033, 1:3000), rabbit anti-Rad51 (Abcam, ab133534, 1:1000), rabbit anti-USP24 (Proteintech, 13126 -1-AP, 1:1000), rabbit anti-USP5 (Proteintech, 10473-1-AP, 1:1000), rabbit anti-USP1 (Proteintech, 14346-1-AP, 1:1000), rabbit anti-USP14 ( Proteintech, 14517-1-AP, 1:1000), rabbit anti-UCHL5 (Proteintech, 11527-1-AP, 1:1000), mouse anti-Flag (Sigma-Aldrich, F1804, 1:3000), rabbit anti-GST (Abcolonal, AE006, 1:1000), rabbit anti-HA (Cell Signaling Technology, #3724, 1:3000), Ubiquitination Compound Library (Selleck, L6000), JH-RE-06 (Topscience, T15611), MG132 (Calbiochem, 474,790) and cycloheximide (Sigma-Aldrich, A8244).

Techniques: Expressing, Scaffolding, Ubiquitin Proteomics