Extended Data Fig. 8: ADSL inhibits STING independently on the mitochondrial membrane potential alteration or mtDNA leakage.
From: ADSL-generated fumarate binds and inhibits STING to promote tumour immune evasion
(a, b) MCF-10A and HMEC cells were treated with or without hypoxia in the presence of 10 μM or 50 μM DMF for 6 h. Relative intercellular cGAMP level were measured (a) (n = 3 biological experiments). Total lysates were harvested for immunoprecipitation and immunoblotting analyses as indicated (b). (c) MDA-MB-231 cells were treated with or without hypoxia in the presence of 10 mM or 20 mM NAC for 24 h. Total lysates were harvested for immunoblotting analyses with the indicated antibodies (left). Intracellular fumarate level was measured (right) (n = 3 biological experiments). (d, e) Parental MDA-MB-231 cells and the indicated cells with knock-in expression of ADSL T350A, E481/L482A and A291V mutants were treated with increased amount of DMF. The cellular fumarate levels were detected (d) (n = 3 biological experiments). The mRNA expression levels of IRF3 target genes were measured using quantitative PCR (e) (n = 3 biological experiments). (f-h) Parental MDA-MB-231 cells and the indicated cells with knock-in expression of ADSL T350A, E481/L482A and A291V mutants were treated with increased amount of DMF. The mitochondrial membrane potential was measured with TRME staining. Mitotracker Green (MTG) was used as a control, which is not affected by transmembrane potential. Representative staining images are shown (f). 20 cells from each independent experiment were analysed and representative data are shown (g). Each data point corresponds to the mean fluorescence intensity value derived from the analysis of two individual cells. And the mitochondrial membrane potential was measured with flow cytometry (h) (n = 3 biological experiments). (i-k) Parental MDA-MB-231 cells as well as the indicated cells with knock-in expression of ADSL T350A, E481/L482A and A291V mutants were treated with increased amount of DMF, immunofluorescence analyses were performed with the indicated antibodies. Representative staining images are shown (i). White arrowheads indicate cytosolic DNA foci. Percentage of cells showing cytosolic DNA foci (j) and number of cytosolic DNA foci per cell (k) from (i). over 30 cells from three independent experiments were analysed. (l) Quantification of mtDNA copy number by QPCR, from isolated cytosolic fractions of indicated cells (n = 3 biological experiments). Data are representative of as mean ± SD; Statistical significance was determined by two-tailed Student’s t-test. Experiment was repeated three times (b) with similar results.
