Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 5

Figure 5. IRGM depletion results in impaired mitophagy and accumulation of dysfunctional/deleterious mitochondria

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 antibody. Scale bar, 5 µm. Zoom panels are digital magnifications.

(B) The graph depicts the percentage of cells with swollen/rounded mitochondria in Irgm1+/+ and Irgm1-/- mouse BMDMs. Mean ± SD, n = 3 (biological repeats), **P < 0.005, Student's unpaired t-test.

(C) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Parkin antibodies. Line profile: Co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications.

(D) Representative confocal (STED) images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 and Pink1 antibodies. Line profile: Co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications.

(E) Representative confocal images of control siRNA and IRGM siRNA transfected THP-1 cells processed for IF analysis with Ubiquitin (green) and TOM20 (red) antibodies. Scale bar, 10 µm. Zoom panels are digital magnifications. (F) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells, probed with indicated antibodies. 2 biological replicates are shown.

(G) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells, probed with indicated antibodies (n=2 mice).

(H) Western blot analysis of mitochondrial fraction of Irgm1+/+ and Irgm1-/- mouse BMDM cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.

(I) Western blot analysis of mitochondrial fraction of control siRNA and IRGM siRNA transfected THP-1 cells untreated or treated with Bafilomycin A1 (300 nM, 3 h), probed with indicated antibodies.

(J) Seahorse XF Cell Mito stress test for analysis of mitochondrial function in Irgm1+/+ and Irgm1-/- mouse BMDM cells. The experiments were performed using the XF24 extracellular flux analyzer, and the flow chart showed the measurement of oxygen consumption rate (OCR) as described in material and methods. Results shown represent mean ± standard error (n=3 mice). *P<0.05.

(K) Representative confocal images of control and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye. Scale bar, 7.5 µm.

(L) Flow cytometry analysis of control siRNA and IRGM siRNA transfected THP-1 cells stained with CMXRos red dye (10 nM, 30 mins).

(M) Graph depicts the normalized mean fluorescent intensity of control and IRGM knockdown THP-1 cells stained with CMXRos. Mean± SD, n = 3 (biological repeats), *P < 0.05, Student's unpaired t-test.

(N) Representative dot plot showing flow cytometry analysis of control and IRGM siRNA knockdown THP-1 cells stained with JC-1 dye (2 µM, 30 mins). At low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence, whereas at mitochondrial membrane potential the dye aggregates producing a red to orange colored emission.

(O) The graph depicts the ratio of red vs. green fluorescent intensity in control and IRGM siRNA knockdown THP-1 cells stained with JC-1 dye. Mean ± SD, n = 3 (biological replicates), *P < 0.05, Student's unpaired t-test.

(P) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with MitoSox red dye (1µM, 20 min). The percentage of control and IRGM knockdown cells with increased red fluorescence (mitochondrial ROS generation) is depicted.

(Q) Representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells stained with CellRox-green and CellRox-orange dye (1µM, 30 min).