Inhibition of IRGM Establishes a Robust Antiviral Immune State to Restrict Pathogenic Viruses (2021)
Figure 2

Figure 2. Depletion of IRGM induces key viral restriction factors and anti-viral mechanisms.

(A) Pictorial representation of stages (black font) of a typical life cycle of RNA viruses with host viral restriction factors (red font) induced in IRGM/Irgm1 depleted cells. Created using Biorender.com

(B, C) Heatmaps representing the expression pattern of viral restriction factors from RNA sequencing data in (B) control and IRGM knockdown shRNA stable HT-29 cells (3 biological replicates) and (C) irgm1+/+ and irgm1-/- mice BMDMs (2 biological replicates).

(D) RNA was isolated from irgm1+/+ and irgm1-/- BMDMs and subjected to qRT-PCR with indicated viral restriction factor genes (n=3, mean ± SD, *p < 0.05, **P ≤ 0.005, ***p < 0.0005, Student's unpaired t-test).

(E) Western blot analysis with lysates of control and IRGM+/- HT-29 cells with indicated antibodies of viral restriction factors.

(F) Western blot analysis with lysates of control or IRGM+/- HT-29 cells or Flag-IRGM complemented IRGM+/- HT-29 cells with indicated antibodies of viral restriction factors.

(G) RNA isolated from control or Flag-IRGM transfected or Flag-IRGM S47N transfected THP-1 cells subjected to qRT-PCR with indicated genes. (n=3, mean ± SD, **p < 0.005, ***p < 0.0005, Student's unpaired t-test).

(H-J) RNA isolated from control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system. (n=3, mean ± SD, *p < 0.05, **p < 0.005, ***p < 0.0005, Student's unpaired t-test).

(K) Antigen uptake assay shown by representative flow cytometry analysis of control and IRGM siRNA transfected THP-1 cells treated with OVA conjugate AF488 (5 µg/ml, 30 min). The graph depicts the mean fluorescence intensity of control and IRGM knockdown THP-1 cells treated with OVA conjugate AF488 (n=3, mean ± SD, **p < 0.005, Student's unpaired t-test).

(L, M) Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP-1 cells treated with DQ-OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP-1 cells with DQ-OVA puncta's (n=3, mean ± SD, ***p < 0.0005, Student's unpaired t-test).

(N, O) SIINFEKL based Antigen presentation assay shown by representative flow cytometry analysis of H-2Kb-SIINFEKL on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h). (O) The graph depicts the mean fluorescence intensity of H-2Kb- SIINFEKL on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h) (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).

(P) Representative confocal images of H-2Kb-SIINFEKL (red) on the surface of Irgm1+/+ and irgm1-/- mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated).

(Q) Western blot analysis with lysates of control and IRGM siRNA knockdown HT-29 cells with indicated antibodies of stress granules signaling pathway.

(R) Representative Immunofluorescence confocal images of irgm1+/+ and irgm1-/- mouse BMDMs immunostained with dsRNA (green) and TIA-1 (red) antibody. Scale bar 3 µm.