Journal: Cell proliferation

Article Title: Deciphering the Immunomodulatory Function of GSN + Inflammatory Cancer-Associated Fibroblasts in Renal Cell Carcinoma Immunotherapy: Insights From Pan-Cancer Single-Cell Landscape and Spatial Transcriptomics Analysis.

doi: 10.1111/cpr.70062

Figure Lengend Snippet: FIGURE 6 | Silencing of GSN in CAFs inhibits intra-tumoral CD8+ T cells function and drives them towards a dysfunctional state. (A) tSNE plot of iCAF marker gene GSN, and expression of GSN in iCAFs from responders and non-responders. (B) Survival analysis of GSN (in terms of OS) in TCGA-KIRC cohort. (C) Immunohistochemistry (IHC) staining in tumour samples of three non-responders and three responders. (D) Workflow of the co-culture system with primary CAFs (NC, siGSN-1 and siGSN-2), primary ccRCC tumour cells and CD8+ T cells. (E) qRT-PCR analysis of GSN mRNA in three groups (NC, siGSN-1 and siGSN-2). (F) ELISA analysis of the IFN-γ and TNF-α levels in supernatant from the co-culture system. (G) Flow cytometry analysis of IFN-γ, TNF-α, GZMB and Perforin in CD8+ T cells isolated from the co-culture system. (H) GSEA analysis in TCGA-KIRC cohort (low GSN vs. high GSN), as well as in pan-cancer scRNA-seq landscape of ICI therapy (non-responders vs. responders). (I) Representative western blot of GSN, p65, p-p65, IKB, p-IKB and IKKα + β protein expression levels in the co-culture system. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: These primary antibodies were used in our western blot analysis: p65 (8242, Cell Signalling Technology, CST), p- p65 (3033, CST), IKB (4812, CST), p- IKB (2859, CST), IKKα + β (2697, CST), anti- human GSN (11644- 2- AP, Protein tech), β- tubulin (10094- 1- AP, Protein tech).

Techniques: Marker, Expressing, Immunohistochemistry, Co-Culture Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Western Blot