Autoimmunity Gene IRGM Suppresses cGAS-STING and RIG-I-MAVS Signaling to control Interferon Response (2020)
Figure 6

Figure 6. IRGM depleted cells are soiled with DAMPs that induces IFN response.

(A) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with Tom20 (red) and dsDNA (green) antibody. Scale bar, 5 µm.

(B) Representative STED microscopy images of control THP-1 cells processed for IF analysis with cGAS (green), dsDNA (red), and Lamin B1 (cyan) antibodies. Scale bar, 3 µm. Zoom panels are digital magnifications.

(C) The graph depicts the percentage of cells with micronuclei in control siRNA and IRGM siRNA knockdown THP-1 cells. Mean ± SD, n = 3 (biological replicates), **p < 0.005, Student's unpaired t-test.

(D-E) Representative STED microscopy images of Irgm1-/- mouse BMDMs (D) transfected with mCherry-cGAS and immunostained with dsDNA (green) antibody. The white line depicts the periphery of the cells. Scale bar, 3 µm. (E) The graph depicts the percentage of cells with micronuclei in Irgm1+/+ and Irgm1-/- mouse BMDMs. Mean ± SD, n = 3 (biological replicates), **p < 0.005, Student's unpaired t-test. Zoom panels are digital magnifications.

(F) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs. Mean ± SE, n = 3 mice, ***p < 0.0005, Student's unpaired t-test.

(G) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control and IRGM siRNA transfected HT-29 cells. Mean ± SE, n = 3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.

(H) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells. Mean ± SE, n = 3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.

(I) The qRT-PCR analysis with total RNA isolated from control or IRGM shRNA or EtBr treated IRGM shRNA HT-29 (Rho0) cells. Mean ± SD, n = 3 (biological replicates), **p < 0.005, ***p < 0.0005, Student's unpaired t-test.

(J) The qRT-PCR analysis with cytosolic DNA (minus mitochondria) isolated from Irgm1+/+ and Irgm1-/- mouse BMDMs transfected with DNase I (15 μg, 6hr). Mean ± SE, n = 3 mice, ***p < 0.0005, Student's unpaired t-test.

(K) The qRT-PCR analysis with RNA isolated from control and IRGM siRNA knockdown THP-1 cells electroporated with DNase I (5 μg, 1hr) as indicated. Mean ± SD, n = 3 (biological replicates), *p < 0.05, **p < 0.005, Student's unpaired t-test.

(L-M) Representative confocal images of Irgm1+/+ and Irgm1-/- mouse BMDMs processed for IF analysis with (L) dsRNA (green) and Tom20 (red) antibodies. (M) The graph depicts the percentage of Irgm1+/+ and Irgm1-/- mouse BMDMs with dsRNA aggregates. Mean ± SD, n = 3 (biological replicates), ***p < 0.0005, Student's unpaired t-test. Scale bar, 5 µm.

(N) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with RIG-I (red) and dsRNA (green) antibodies. Line profile: Co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications.

(O-P) Representative confocal images of Irgm1-/- mouse BMDMs processed for IF analysis with (O) Mda5 (red) and dsRNA (green) antibodies (Scale bar, 5 µm) or (P) Mda5 (red), dsRNA (green) and Tia1 (blue) antibodies (Scale bar, 3 µm). Line profile: Co-localization analysis using line intensity profiles. Zoom panels are digital magnifications.