Fig. 2: Succinyl-CoA enhanced CTL activity by decreasing PD-L1.
From: Alterations in PD-L1 succinylation shape anti-tumor immune responses in melanoma
a,b, Spearman’s rank correlation between the SucciCoA score and immune checkpoints in the cancer cell lines dataset from the CCLE database (a; n = 375 cell lines) or in the NSCLC protein cohort (b; n = 110 patients). c, Western blot analysis of PD-L1 and other immune checkpoints in A375 cells treated with DES or DMK (n = 3). d, PD-L1 expression in tumor cells quantified by flow cytometry in B16F10 tumor-bearing mice treated with DES or DMK (n = 5 per group). e, Tumor volume measured in mice injected with control or PD-L1 stable KO B16F10 cells, treated with DES or DMK (n = 5 per group). f, Tumor weight measured after euthanasia of mice (n = 5 per group). g, Quantification of GZMB+/CD8+ CTL cells in tumors (n = 5 per group). h, Crystal Violet staining of A375 WT and PD-L1 KO cells co-cultured with activated T cells with or without DES or DMK (n = 3). i,j, Western blot analysis of PD-L1 protein levels in A375 and SK-MEL-28 cells treated with SDH inhibitors (DMM, 3-NPA) (i) or OGDH inhibitor (CPI-613; j) (n = 3). k, Membrane PD-L1 levels after treatment with DES, DMK, DMM, 3-NPA and CPI-613 for 24 h (n = 3). l, The mRNA levels of PD-L1 in A375 cells after treatment with DES or DMK (n = 3). m, Western blot analysis of PD-L1 in A375 cells overexpressing PD-L1, treated with DES or DMK (n = 3). n, PD-L1 protein levels in A375 cells treated with DES or DMK and CHX for various times (in hours) (n = 3) o, PD-L1 overexpression cells treated with DES or DMK, followed by MG132 or pretreated with bafilomycin A1 (n = 3). p, Immunofluorescence showing PD-L1 co-localization with specific marker for recycling endosomes (Rab11), late endosomes (Rab7) and lysosomes (Lamp1) (n = 3). Scale bar, 10 or 5 μm. q, Western blot analysis of PD-L1 in endosomal fractions after treatment with DES or DMK (n = 3). For details on visualization, statistics and reproducibility, see Methods. Note, in d–g, n represents biologically independent samples and, in c, h and q, independent experiments.
