Journal: Advanced Science

Article Title: Mitochondrial‐Derived Peptide MOTS‐c Suppresses Ovarian Cancer Progression by Attenuating USP7‐Mediated LARS1 Deubiquitination

doi: 10.1002/advs.202405620

Figure Lengend Snippet: MOTS‐c increases the ubiquitination level of LARS1 by impairing USP7‐mediated deubiquitination of LARS1. A) A2780 cell lysate was immunoprecipitated by anti‐LARS1 antibody, the lysate was separated by SDS‐PAGE and stained with Coomassie blue. LARS1‐bound proteins were identified by MS and overlapped with proteins that bind to MOTS‐c. B,C) Co‐IP assays were conducted to observe the interaction between endogenous LARS1 and USP7, and USP7 binding to MOTS‐c in OC cells. D) Western blot assay was performed to observe exogenous MOTS‐c binding to endogenous USP7 in OC cells. E) IF assay was conducted for intracellular co‐localization of USP7 and LARS1 proteins in OC cells. F) Western blot assay was used to analyze LARS1 protein abundance in OC cells transfected with His‐USP7 plasmid (0.5, 1, 2 µg). G) RT‐qPCR assay was performed to analyze the mRNA level of LARS1 in OC cells transfected with USP7 overexpressing or empty plasmids. H) Western blot and I) RT‐qPCR assays were conducted for analyzing the expression of LARS1 in OC cells transfected with USP7 siRNA. J) The stability of LARS1 protein was analyzed by Western blot, OC cells were transfected with USP7 siRNA and treated with CHX before harvesting. K,L) OC cells were transfected with USP7 siRNA, (K) MG132, and (L) CQ were then added 8 h before harvesting, LARS1 levels were detected by Western blot. M) IP and IB assays were conducted to analyze the ubiquitination levels of endogenous LARS1 in OC cells transfected with USP7 siRNA. N) IP and IB assays were conducted to analyze the ubiquitination levels of exogenous LARS1 in 293T cells transfected with HA‐Ub, Flag‐LARS1, His‐USP7 WT, and His‐USP7 C223S plasmids. O) IP and IB assays were conducted to analyze the ubiquitination levels of endogenous LARS1 in OC cells transfected with His‐USP7 plasmid treated with 30 µM MOTS‐c or scramble peptide. P) IP and IB assays for testing the LARS1 domains critical for USP7 binding in 293T cells transfected with different structural domains of LARS1 plasmids and His‐USP7 plasmids. Data are presented as the mean ± SEM, *** p < 0.001.

Article Snippet: [ ] The incubated antibodies include MOTS‐c (#MOTSC‐101AP, 1:500, FabGennix, USA), β‐actin (#81115‐1‐RR, 1:10 000, Proteintech, China), Bcl‐2 (#381 702, 1:1000, ZEN‐BIOSCIENCE, China), Bax (#380 709, 1:1000, ZEN‐BIOSCIENCE, China), Cleaved‐PARP (#380 374, 1:1000, ZEN‐BIOSCIENCE, China), Cleaved‐Caspase3 (#341 034, 1:1000, ZEN‐BIOSCIENCE, China), LARS1 (#21146‐1‐AP, 1:1000, Proteintech, China), Ubiquitin (#10201‐1‐AP, 1:1000, Proteintech, China), Flag (#66008‐4‐lg, 1:5000, Proteintech, China), GFP (#50430‐2‐AP, 1:1000, Proteintech, China), p‐mTOR (#5536, 1:1000, CST, USA), mTOR (#2983, 1:1000, CST, USA), p‐S6K (#9205, 1:1000, CST, USA), S6K (#2708, 1:1000, CST, USA), p‐4EBP1 (#2855, 1:1000, CST, USA), 4EBP1 (#9644, 1:1000, CST, USA), USP7 (#66514‐1‐lg, 1:5000, Proteintech, China) and His (#66005‐1‐lg, 1:5000, Proteintech, China).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, SDS Page, Staining, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Quantitative Proteomics, Transfection, Plasmid Preparation, Quantitative RT-PCR, Expressing