Journal: Journal of translational internal medicine

Article Title: WWP2 protects against sepsis-induced cardiac injury through inhibiting cardiomyocyte ferroptosis.

doi: 10.2478/jtim-2024-0004

Figure Lengend Snippet: Figure 4: WWP2 is predominantly responsible for reducing ferroptosis in sepsis-induced myocardial damage. (A) Detection of LDH levels in NC-saline, NC-LPS, WWP2sh-LPS, WWP2sh+Fer-1-LPS and WWP2sh+z-VAD-FMK-LPS myocardium (N = 6). (B) Cell viability quantified among NC-saline, NC-LPS, WWP2sh-LPS, WWP2sh+Fer-1-LPS and WWP2sh+z-VAD-FMK-LPS H9C2 cells by CCK-8 assay (N = 6). (C) Western blotting and quantification of GPX4 and PTGS2 in mice treated with LPS or saline and injected with AAV9-CTNT-WWP2sh or AAV9-CTNT-CONi (N = 3). (D) Representative transmission electron microscopy (TEM) images of mitochondrial morphology and function. Scale bar: 800 nm. (E-F) Representative images and quantification of lipid peroxidation products in H9C2 cells. Scale bar: 20 μm (N = 6). (G) Determination of Malondialdehyde (MDA) levels in mice treated with LPS or saline and injected with AAV9-CTNT-WWP2, AAV9-CTNT-WWP2sh or AAV9-CTNT-CON (N = 6). (H) Determination of glutathione (GSH), Oxidized glutathione (GSSG) and GSH/GSSG ratio in H9C2 cells (N = 6). (I) Representative images of mitochondrial membrane potential stained by JC-1 kit. Scale bar: 50 μm. Data were presented as the mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The primary antibodies used in this study are as follows: α-TUBULIN (Proteintech, 11224–1- AP, diluted 1: 1000), WWP2 (Proteintech, 12197–1-AP, diluted 1: 1000), GPX4 (Proteintech, 67763–1-Ig, diluted 1: 1000), FACL4 (Proteintech, 22401–1-AP, diluted 1: 1000), PTGS2 (Proteintech, 66351–1-Ig, diluted 1: 1000), Flag (Proteintech, 80010-1-RR, diluted 1: 1000).

Techniques: Saline, CCK-8 Assay, Western Blot, Injection, Transmission Assay, Electron Microscopy, Membrane, Staining

Journal: Journal of translational internal medicine

Article Title: WWP2 protects against sepsis-induced cardiac injury through inhibiting cardiomyocyte ferroptosis.

doi: 10.2478/jtim-2024-0004

Figure Lengend Snippet: Figure 7: Knockdown of FACL4 rescues the adverse effects of WWP2 knockout on cardiac injury and cardiomyocytes ferroptosis. (A) Western blotting and quantification of GPX4 and PTGS2 in WT and WWP2 knockout (WWP2KO) mice injected with AAV9-CTNT-FACL4sh and treated with LPS (N = 3). (B) Quantification of EF and FS in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS (N = 6). (C) Representative H& E staining of myocardium in injected with AAV9-CTNT-FACL4sh and treated with LPS. Scale bar: 50 μm. (D) Detection of plasma levels of CK-MB, cTnI and TNF-α in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS (N = 6). (E) Detection of LDH levels in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS (N = 6). (F) Cell viability quantified by CCK-8 assays (N = 6). (G) Representative TEM images of mitochondrial morphology and function in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS. Scale bar: 800 nm. (H) Determination of MDA levels in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS (N = 6). (I) Determination of glutathione (GSH), Oxidized glutathione (GSSG) and GSH/GSSG ratio in H9C2 cells (N = 6). (J) Representative IF staining and quantification of DHE in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS. Scale bar: 75 μm (N = 6). (K) Representative IF staining and quantification of 4-HNE in WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh and treated with LPS. Scale bar: 75 μm. (L) Survival rate of WT and WWP2KO mice injected with AAV9-CTNT-FACL4sh after treated with saline or LPS. Data were presented as the mean±SEM. *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: The primary antibodies used in this study are as follows: α-TUBULIN (Proteintech, 11224–1- AP, diluted 1: 1000), WWP2 (Proteintech, 12197–1-AP, diluted 1: 1000), GPX4 (Proteintech, 67763–1-Ig, diluted 1: 1000), FACL4 (Proteintech, 22401–1-AP, diluted 1: 1000), PTGS2 (Proteintech, 66351–1-Ig, diluted 1: 1000), Flag (Proteintech, 80010-1-RR, diluted 1: 1000).

Techniques: Knockdown, Knock-Out, Western Blot, Injection, Staining, Clinical Proteomics, CCK-8 Assay, Saline

Journal: International immunopharmacology

Article Title: IL17A/F secreted by ASCT2-overexpression ovarian cancer cells contributes to immune escape through the suppression of natural killer (NK) cells cytotoxicity by the activation of c-JUN/ PTGS2 pathway.

doi: 10.1016/j.intimp.2025.114226

Figure Lengend Snippet: Fig. 5. Analysis of gene expression changes in NK cells co-cultured with ASCT2-overexpressing ovarian cancer cells. A: Volcano plot of differentially expressed genes, with significantly upregulated genes shown in red points, downregulated genes shown in blue points, and genes with no significant differential expression shown in gray points; the x-axis represents the fold change in gene expression between different groups, and the y-axis represents the statistical significance of the difference in gene expression. B: KEGG pathway enrichment plot of differentially expressed genes, with the x-axis representing the ratio of differentially expressed genes annotated to KEGG pathways to the total number of differentially expressed genes, and the y-axis representing KEGG pathways. C: Protein interaction plot of the top 100 differentially significant genes in NK cells. Genes shown in orange are upregulated, while genes shown in green are downregulated. D: Schematic diagram of the IL-17 signaling pathway. E, F, G: Detection of JUN and PTGS2 expression using RNA sequencing, qPCR, and Western blot analysis. OE vs NC, Comparison between NK cells co-cultured with ASCT2-overexpressing ovarian cancer cells and NK cells co-cultured with ASCT2-normal ovarian cancer cells. OE-NC, NK cells after co-culture with ovarian cancer cells with normal ASCT2 expression; OE-ASCT2, NK cells after co-culture with ASCT2-overexpressed ovarian cancer cells. The expression of JUN and PTGS2 in NK cells co-cultured with ASCT-2 overexpression was higher than that in NK cells co-cultured with normal ASCT-2 expression.

Article Snippet: The membrane was blocked in Tris-buffered saline (pH 7.4) containing 5 % skim milk and 0.1 % Tween-20, followed by overnight incubation at 4 ◦C with the primary antibody PTGS2 (66351-1-Ig, Proteintech, 1:4000)、 JUN (22114-1-AP, Proteintech Dilution: 1:1000) 、GADPH (GB15002, seivicebio Dilution: 1:2000) and subsequent incubation with the appropriate secondary antibody (AB 228338 Invitrogen, 1:3000) for 1 h. Protein bands were visualized using a Gel Imaging System (BIO-RAD, USA), and band intensities were quantified using ImageJ software.

Techniques: Gene Expression, Cell Culture, Quantitative Proteomics, Expressing, RNA Sequencing, Western Blot, Comparison, Co-Culture Assay, Over Expression