Supplementary Figure 5: Hypoxia reduces immune response to cytosolic mtDNA.

(a) Hypoxia increased the level of cytosolic mtDNA. Upper: Western blot analysis for specific markers in cytosolic (β-actin), nucleus (Histone H3), and mitochondria (cytochrome c) fractions. Nucleus is referred to the fraction containing both nucleus and mitochondria fraction. Lower: qPCR analysis of cytosolic mtDNA in either hypoxic MCF7 or MDA-MB-231 cells using three sets of primers targeting different mitochondria DNA regions, as indicated. (b) Hypoxia induced mitophagy, as shown by accumulated level of LC-II and decreased level of p62. (c) Hypoxia suppressed mtDNA-induced immune response in either MCF7 (upper) or MDA-MB-231 cells (lower). The IFN-β expression was used as an indicator for mtDNA-induced immune response. (d,e) Loss of function of miR25/93 restored immune response in the presence of cytosolic mtDNA upon hypoxia. (f,g) The analysis of IFN-β expression in cGAS deficient cells indicated cGAS as a major mediator in mtDNA-induced immune response. The cells expressing shRNA targeting EGFP were used as control. N: normoxia H: hypoxia. All the data in graphs are presented as means ± SD (n = 3 independent experiments). Samples were compared using two-tailed Student’s t test. Asterisk indicates P < 0.05. The image shown in panel a,b is representative of three independent experiments. Unprocessed scans of western blot analysis is available in Supplementary Figure 9. Statistics source data is available in Supplementary Table 4.