Extended Data Fig. 5: The formation of MCAM+ mCAFs was induced via TGF-β signaling, related to Fig. 4.
(a-b) Western blotting for key proteins of the FAK-AKT pathway in the indicated NPC cell lines, with or without collagen IV pretreatment (a), and with or without ITGA2 knockdown following collagen IV pretreatment (b). (c) TW03 and C666 cells were treated with or without the AKT inhibitor MK2206 in the presence of type IV collagen. After irradiation at a dose of 10 Gy, the distribution of γ-H2AX foci was visualized by immunofluorescence staining (n = 3 per group). The cell nuclei were counter-stained with DAPI (blue). Scale bars, 20 µm. (d) Volcano plot shows DEGs between C666 bulk samples (left) and RT sh-ITGA2 C666 bulk sample (right). The names of the reported irradiation-resistance related TFs are indicated in the plot1-6. (e) Correlation between the expression of TFs which highly expressed in C666 bulk samples and the cell score for FAK-PI3K_AKT in single malignant cells. Two-sided Spearman’s correlation test. (f) Enrichment of the TFs that are highly expressed in C666 bulk samples (versus sh-ITGA2 C666). (g) C666 cells combined with MCAM+ mCAFs (3:1) were injected subcutaneously into NCG mice (n = 6 per group). The mice were randomly subjected to gavage with MK2206 or DMSO, and treated with radiotherapy at a dose of 2 Gy for 5 consecutive days. (h) The deconvolution score of malignant and the score of the TGF-β pathway in malignant spots based on the ST data. Scale bars, 500 µm. (i) Violin plots showing the TGF-β score in malignant cells from PT (blue, n = 3,241) and RT (red, n = 11,105) samples based on the scRNA-seq data. Two-sided Wilcoxon rank-sum test. (j) Boxplot showing the TGF-β scores of NPC cells from wild type (W) and radioresistant tumor cells (R) based on the bulk RNA seq data (n = 3 per group). One-sided paired t-test. (k) Western blotting for key proteins in MCAM− CAFs after being treated with recombinant TGF-β in different concentration gradients. (l) MTT assay was performed after MCAM− mCAFs cells were treated with increasing doses of TGF-β. Two-sided ANOVA test.
