Journal: Cell Death Discovery

Article Title: mTORC1 accelerates osteosarcoma progression via m 6 A-dependent stabilization of USP7 mRNA

doi: 10.1038/s41420-024-01893-9

Figure Lengend Snippet: A Eight patients with high levels of E2 were collected. The E2 content in 8 OS tissues or normal tissues. B The corresponding E2 content between each OS tissues and normal tissues. C Immunoblot analysis of p-S6, and S6 in 8 OS tissues or normal tissues. D Quauntification of p-S6 protein expression in OS tissues or normal tissues ( n = 8). E , F The formation and quantification of ASC speck in OS tissues or normal tissues ( n = 8). G IHC analysis of the expression of CYP19a1 in OS tissues or normal tissues, scale bar: 50 µm. H Quantification analysis of CYP19a1 positive cells in OS tissues or normal tissues. I The relative expression of USP7 in OS tissues and normal tissues ( n = 8). J The m 6 AqPCR assay was performed to analyze the m 6 A enrichment in 5′UTR, CDS, and 3’UTR of USP7 mRNA in vector or Raptor transfected U2OS cells. K Schematic illustrations of mutation in m6A motif in CDS of USP7. L F-Luc/R-Luc activity in vector or Raptor transfected U2OS cells following treatment with USP7-CDS-WT or USP7-CDS-MUT. M Lifetime USP7 mRNA levels in vector or Raptor transfected U2OS cells following treatment with 0.5 mg/mL of actinomycin D for 0–4 h. N Lifetime USP7 mRNA levels in shCtrl or shRaptor transfected U2OS cells following treatment with 0.5 mg/mL of actinomycin D for 0–4 h. O The USP7 and Raptor mRNA levels in in vector or Raptor transfected U2OS cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs control group. ns not significant.

Article Snippet: After blocking, the slides were incubated with Alexa Fluor 594 conjugated USP7 antibody (1:200, Novus Biologicals, CO, USA), Alexa Fluor 488 conjugated NLRP3 antibody (1:100, R&D Systems, USA) or ASC antibody (1:100, Cell Signaling Technology) at 4 °C for 60 min, then washed by PBS.

Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Mutagenesis, Activity Assay, Control

Journal: Cell Death Discovery

Article Title: mTORC1 accelerates osteosarcoma progression via m 6 A-dependent stabilization of USP7 mRNA

doi: 10.1038/s41420-024-01893-9

Figure Lengend Snippet: A The SDS-PAGE band pattern of immunoprecipitated proteins using anti-USP7 and IgG. The bands of protein were excised to mass spectrometry (MS) analysis for identification. The red arrow marks the distinct band (NLRP3) in USP7 immunoprecipitated samples. B The immunoprecipitated protein of NLRP3 were analyzed using anti-USP7 and IgG. C The protein expression levels of NLRP3, pro-caspase1, and caspase1 in MG63 and U2OS cells treated with 100 nM of E2 for 48 h with or without pretreatment of 1 μM of torin1. D The protein expression levels of NLRP3 and USP7 in U2OS cells treated with shCtrl or shUSP7 for 24 h. E U2OS cells were transfected with shCtrl or shUSP7 for 24 h. The protein extracts were immunoprecipitated with IgG beads of anti-NLRP3, and the protein expression levels of K48-ubiquitin, K63-ubiquitin and NLRP3 were detected. F The relative expression of NLRP3 in OS tissues and normal tissues ( n = 8). G The correlation between USP7 and NLRP3 in OS tissues or normal tissues ( n = 8). H The colabel staining of USP7 and NLRP3 in OS tissues or normal tissues, scale bar: 50 µm. Arrow indicates USP7 + NLRP3 + cells. I Quantification of the USP7 + NLRP3 + cells in OS tissues or normal tissues ( n = 8). ** P < 0.01, **** P < 0.0001.

Article Snippet: After blocking, the slides were incubated with Alexa Fluor 594 conjugated USP7 antibody (1:200, Novus Biologicals, CO, USA), Alexa Fluor 488 conjugated NLRP3 antibody (1:100, R&D Systems, USA) or ASC antibody (1:100, Cell Signaling Technology) at 4 °C for 60 min, then washed by PBS.

Techniques: SDS Page, Immunoprecipitation, Mass Spectrometry, Expressing, Transfection, Ubiquitin Proteomics, Staining

Journal: Cell Death Discovery

Article Title: mTORC1 accelerates osteosarcoma progression via m 6 A-dependent stabilization of USP7 mRNA

doi: 10.1038/s41420-024-01893-9

Figure Lengend Snippet: A The flank of the nude mice were subcutaneously injected with 1 × 10 6 of USP7 KO and control cells. Tumor volume ( B ) and tumor weight ( C ) were calculated and observed ( n = 5). D , E IHC analysis and quantification of the expression of Ki-67 in USP7 KO or control mice ( n = 5), scale bar: 100 µm. F The flank of the nude mice were subcutaneously injected with 1 × 10 6 of shCtrl or shRaptor treated U2OS cells. Tumor volume was calculated ( n = 5). G , H IHC analysis and quantification of the expression of Ki-67 in shCtrl or shRaptor treated mice ( n = 5), scale bar: 100 µm. I Tumor weight was collected in shCtrl or shRaptor treated mice ( n = 5). J , K The formation and quantification of ASC speck in shCtrl or shRaptor treated mice, scale bar: 50 µm. ** P < 0.01, **** P < 0.0001 vs control group.

Article Snippet: After blocking, the slides were incubated with Alexa Fluor 594 conjugated USP7 antibody (1:200, Novus Biologicals, CO, USA), Alexa Fluor 488 conjugated NLRP3 antibody (1:100, R&D Systems, USA) or ASC antibody (1:100, Cell Signaling Technology) at 4 °C for 60 min, then washed by PBS.

Techniques: Injection, Control, Expressing

Journal: Cell Death Discovery

Article Title: mTORC1 accelerates osteosarcoma progression via m 6 A-dependent stabilization of USP7 mRNA

doi: 10.1038/s41420-024-01893-9

Figure Lengend Snippet: E2-activated mTORC1 promotes stabilization of USP7 through m 6 A modification. USP7 interacts directly with NLRP3 and deubiquitinases NLRP3. The ASC speck is increased following the assembling and activating of NLRP3 inflammasome, which leads to activation of caspase1.

Article Snippet: After blocking, the slides were incubated with Alexa Fluor 594 conjugated USP7 antibody (1:200, Novus Biologicals, CO, USA), Alexa Fluor 488 conjugated NLRP3 antibody (1:100, R&D Systems, USA) or ASC antibody (1:100, Cell Signaling Technology) at 4 °C for 60 min, then washed by PBS.

Techniques: Modification, Activation Assay