Figure 3(A) The co-localization between mtDNA (SG-ALK, green) and mitochondria (MitoTracker, red) was examined in J774A.1 cells with or without knockdown of Phb1 or Phb2. White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (B) Quantitative analysis of data from (A). Data are from ten, eight, and seven images from three independent experiments (left to right, respectively) (mean ± SEM). n.s., no significance, *** p < 0.001 (Student's t-test).(C) J774A.1 cells with or without Phb1 knockdown were lysed, and DNA was extracted. qPCR assays were used to detect the mtDNA levels (D-loop) in the cytosol relative to nuclear DNA levels (Tert) in the whole cell lysates. Data are from six independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(D) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were fixed and subjected to IF analysis to detect the co-localization between mitochondria (MitoTracker, red) and mtDNA (SG-ALK, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. Scale bar: 10 μm. (E) Quantitative analysis of data from (D). Data are from ten and eight images from three independent experiments (mean ± SEM). ** p < 0.01 (Student's t-test).(F) PBMCs were extracted from Phb1F/F mice and Phb1MyeKO mice. Cells were lysed, and DNA was extracted. qPCR assays were used to detect the levels of mtDNA (D-loop) in the cytosol relative to the levels of nuclear DNA levels (Tert) in the whole cell lysates. Data of PBMC are from seven Phb1F/F mice and Phb1MyeKO mice, respectively (mean ± SEM). ** p < 0.01 (Student's t-test).(G) HeLa cells expressing TFAM-FLAG with or without Phb1 knockdown were incubated with CsA (2 μM) for 30 minutes. Cells were fixed and subjected to IF analysis to detect the co-localization between mitochondria (anti-TOMM20, red) and TFAM-FLAG (anti-FLAG, green). White boxed regions in the panels are enlarged. The white arrows indicate mtDNA outside mitochondria. The yellow arrows indicate mtDNA inside mitochondria. Scale bar: 10 μm.(H) Quantitative analysis of data from (G). Data from 30, 27, 25, and 24 images from three independent experiments (mean ± SEM). **** p < 0.0001 (Student's t-test).(I) HeLa cells with or without Phb1 knockdown were incubated with H2O2 (5 mM) for 20 minutes and CsA (4 μM) for 12 hours. Cells were lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels (ND1) in the cytosol and nuclear DNA levels (18S) in the whole cell lysates. Data are from four independent experiments (mean ± SEM). ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student's t-test).HeLa cells with or without Phb1 knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels (ND1 in HeLa cells in the cytosol relative to nuclear DNA levels (TERT in HeLa cells in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).J774A.1 cells with or without Phb1 knockdown were incubated with VBIT-4 (10 μM) for 1 hour and subsequently lysed, and DNA was extracted. qPCR assays were used to detect mtDNA levels D-loop in J774A.1 cells) in the cytosol relative to nuclear DNA levels Tert in J774A.1 cells) in the whole cell lysates. Data are from three independent experiments (mean ± SEM). n.s., no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t-test).(L) J774A.1 cells with or without Phb1 knockdown were lysed. The whole cell lysates were immunoprecipitated by IgG or anti-ASC antibody and the resulting proteins were detected by WB assay.(M) BMDMs isolated from Phb1F/F mice and Phb1MyeKO mice were incubated with MCC950 (50 nM) for 30 mins and were subsequently stimulated by LPS (200 ng/ml) for 6 hours and ATP (4 mM) for 45 minutes. Protein levels in whole cell lysates and culture medium were detected by WB assay.