Journal: Frontiers in Immunology

Article Title: A novel necroptosis-related gene index for predicting prognosis and a cold tumor immune microenvironment in stomach adenocarcinoma

doi: 10.3389/fimmu.2022.968165

Figure Lengend Snippet: Individual assessment of NRGPI genes and association with necroptosis. (A) Spearman’s correlation between NRGPI genes and infiltration of macrophages in the TCGA STAD dataset assessed with TIMER database. (B) Spearman’s correlation between NRGPI genes and KEGG TGF-β signaling pathway signature genes and (C) WNT signaling pathway signature genes. (D) mRNA expression level of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), and markers of WNT signaling pathway (WNT2B, WNT9A) and TGF- signaling pathway (TGFB1, TGFB3) in gastric cancer cells (AGS and MNK45) after being treated with a combination of human recombinant TNF-α, SMAC mimetic, and zVAD-FMK (TSZ) to induce necroptosis or added necroptosis inhibitor (necrostatin-1) to inhibit necroptosis. Graph shows mean ± SD. *P<0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not signficant.

Article Snippet: After washing with 10% phosphate buffered saline (PBS), the sections were blocked with 5% BSA and incubated with primary antibodies against RIPK1 (Proteintech, #17519-1-AP, Rabbit, 1:50), RIPK3 (Proteintech, #17563-1-AP, Rabbit, 1:100), MLKL (Proteintech, #21066-1-AP, Rabbit, 1:50), SERPINE1 (Proteintech, #13801-1-AP, Rabbit, 1:50), FCN1 (Proteintech, #11775-1-AP, Rabbit, 1:50), CNTN1 (Proteintech, #13843-1-AP, Rabbit, 1:50), CYTL1 (Proteintech, #15856-1-AP, Rabbit, 1:50), PLCL1 (Abcam, #EPR11213, Rabbit, 1:100), GRP (Proteintech, #28482-1-AP, Rabbit, 1:500), GPX3 (Affinity Biosciences, #DF6765, Rabbit, 1:50), APOD (Proteintech, #10520-1-AP, Rabbit, 1:50), TGFB1 (Affinity Biosciences, #AF1027, Rabbit, 1:100), TGFB3 (Proteintech, #18942-1-AP, Rabbit, 1:50), WNT2B (Affinity Biosciences, #DF12538, Rabbit, 1:100), WNT9A (Affinity Biosciences, #DF9044, Rabbit, 1:100), CD68 (Abcam,ab955,1:3000), CD206 (Cell Signaling,#24595,1:200), and CD163 (Cell Signaling,#93498,1:250) at 4°C overnight.

Techniques: Expressing, Recombinant

Journal: Frontiers in Immunology

Article Title: A novel necroptosis-related gene index for predicting prognosis and a cold tumor immune microenvironment in stomach adenocarcinoma

doi: 10.3389/fimmu.2022.968165

Figure Lengend Snippet: Representative images of expression (brown, cell cytoplasmic/nucleus stain) of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), markers of WNT signaling pathway (WNT2B, WNT9A), TGF-β signaling pathway (TGFB1, TGFB3), and macrophage (CD63, CD206, CD163) in the clinical samples of stomach adenocarcinoma.

Article Snippet: After washing with 10% phosphate buffered saline (PBS), the sections were blocked with 5% BSA and incubated with primary antibodies against RIPK1 (Proteintech, #17519-1-AP, Rabbit, 1:50), RIPK3 (Proteintech, #17563-1-AP, Rabbit, 1:100), MLKL (Proteintech, #21066-1-AP, Rabbit, 1:50), SERPINE1 (Proteintech, #13801-1-AP, Rabbit, 1:50), FCN1 (Proteintech, #11775-1-AP, Rabbit, 1:50), CNTN1 (Proteintech, #13843-1-AP, Rabbit, 1:50), CYTL1 (Proteintech, #15856-1-AP, Rabbit, 1:50), PLCL1 (Abcam, #EPR11213, Rabbit, 1:100), GRP (Proteintech, #28482-1-AP, Rabbit, 1:500), GPX3 (Affinity Biosciences, #DF6765, Rabbit, 1:50), APOD (Proteintech, #10520-1-AP, Rabbit, 1:50), TGFB1 (Affinity Biosciences, #AF1027, Rabbit, 1:100), TGFB3 (Proteintech, #18942-1-AP, Rabbit, 1:50), WNT2B (Affinity Biosciences, #DF12538, Rabbit, 1:100), WNT9A (Affinity Biosciences, #DF9044, Rabbit, 1:100), CD68 (Abcam,ab955,1:3000), CD206 (Cell Signaling,#24595,1:200), and CD163 (Cell Signaling,#93498,1:250) at 4°C overnight.

Techniques: Expressing, Staining

Journal: Frontiers in Immunology

Article Title: A novel necroptosis-related gene index for predicting prognosis and a cold tumor immune microenvironment in stomach adenocarcinoma

doi: 10.3389/fimmu.2022.968165

Figure Lengend Snippet: (A) Expression level (IHC quantification) of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), and markers of WNT signaling pathway (WNT2B, WNT9A), TGF-β signaling pathway (TGFB1, TGFB3), and macrophage (CD63, CD206, CD163) in the clinical samples (n = 4) of stomach adenocarcinoma. The scattered dots represent the IHC score of each individual sample. The thick middle lines represent the median value and error bars indicate the standard deviation. The bottom and top of the boxes are the 25th and 75th percentiles (interquartile range), respectively. (B) A correlation matrix illustrating Pearson’s correlation coefficients indicating the relationship among expression levels of RIPK1, RIPK3, MLKL (necroptosis core mediators), NRGPI oncogenes (SERPINE1, GPX3, GRP, FCN1, CYTL1, CNTN1, PLCL1, and APOD), and markers of WNT signaling pathway (WNT2B, WNT9A), TGF-β signaling pathway (TGFB1, TGFB3), and macrophage (CD63, CD206, CD163) in the clinical samples (n = 4) of stomach adenocarcinoma. P values are shown as: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After washing with 10% phosphate buffered saline (PBS), the sections were blocked with 5% BSA and incubated with primary antibodies against RIPK1 (Proteintech, #17519-1-AP, Rabbit, 1:50), RIPK3 (Proteintech, #17563-1-AP, Rabbit, 1:100), MLKL (Proteintech, #21066-1-AP, Rabbit, 1:50), SERPINE1 (Proteintech, #13801-1-AP, Rabbit, 1:50), FCN1 (Proteintech, #11775-1-AP, Rabbit, 1:50), CNTN1 (Proteintech, #13843-1-AP, Rabbit, 1:50), CYTL1 (Proteintech, #15856-1-AP, Rabbit, 1:50), PLCL1 (Abcam, #EPR11213, Rabbit, 1:100), GRP (Proteintech, #28482-1-AP, Rabbit, 1:500), GPX3 (Affinity Biosciences, #DF6765, Rabbit, 1:50), APOD (Proteintech, #10520-1-AP, Rabbit, 1:50), TGFB1 (Affinity Biosciences, #AF1027, Rabbit, 1:100), TGFB3 (Proteintech, #18942-1-AP, Rabbit, 1:50), WNT2B (Affinity Biosciences, #DF12538, Rabbit, 1:100), WNT9A (Affinity Biosciences, #DF9044, Rabbit, 1:100), CD68 (Abcam,ab955,1:3000), CD206 (Cell Signaling,#24595,1:200), and CD163 (Cell Signaling,#93498,1:250) at 4°C overnight.

Techniques: Expressing, Standard Deviation