Inhibition of IRGM Establishes a Robust Antiviral Immune State to Restrict Pathogenic Viruses (2021)
Figure 3

Figure 3. IRGM-depleted cells can restrict infection with CHIKV, HSV-1, JEV, VSV, ZIKV,

WNV, and SARS-CoV2.

(A) Total RNA was isolated from mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with CHIKV specific primers to quantitate total viral load (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).

(B) The graph depicts quantification of CHIKV plaque assays (plaque-forming units/ml) in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cell (n=3, mean ± SE, ****p < 0.00005, Student's unpaired t-test).

(C) Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.

(D) Total RNA was isolated from mock and HSV-1 (MOI 1, 24 h) infected control and IRGM knockdown HT-29 cells and subjected to qRT-PCR with HSV-1 specific primers to quantitate total viral load (n=3, mean ± SE, ***p < 0.0005, Student's unpaired t-test).

(E) The graph depicts quantification of HSV-1 plaque assays (plaque forming unit/ml) in Vero cells performed from culture supernatant of HSV-1 (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cell (n=3, mean ± SE, ***p < 0.0005, Student's unpaired t-test).

(F) Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV-1 (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.

(G). Total RNA was isolated from mock and JEV (MOI 1, 48 h) infected control and IRGM knockdown HT-29 cells subjected to qRT-PCR with JEV specific primers to quantitate total viral load (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).

(H) The graph depicts quantification of JEV plaque assays (plaque-forming unit/ml) in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cell (n=3, mean ± SE, ***p < 0.0005, Student's unpaired t-test).

(I) Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT-29 control and IRGM knockdown cells.

(J) The graph depicts the percentage of WNV (MOI 0.3, 16 h) infected control and IRGM knockdown Huh7 cells stained with 4G2 antibody analyzed by flow cytometry (n=3, mean ± SE, ***p < 0.0005, Student's unpaired t-test).

(K) The graph depicts quantification of WNV plaque assays (plaque-forming units/ml) in Vero cells performed from culture supernatant of WNV (MOI 0.3, 16 h) infected Huh7 control and IRGM knockdown cell (n=3, mean ± SE, ***p < 0.0005, Student's unpaired t-test).

(L) Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV (MOI 0.3, 16 h) infected HuH7 control and IRGM knockdown cells.

(M) The graph depicts the percentage of ZIKV (MOI 5, 48 h) infected control and IRGM knockdown HuH7 cells stained with 4G2 antibody analyzed by flow cytometry (n=3, mean ± SD, *p < 0.05, Student's unpaired t-test).

(N) The graph depicts quantification of ZIKV plaque assays (plaque-forming units/ml) in Vero cells performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells (n=3, mean ± SD, *p < 0.05, Student's unpaired t-test).

(O) Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells.

(P) Total RNA was isolated from mock and SARS-CoV2 (MOI 1, 24 h) infected control and IRGM knockdown THP-1 cells and subjected to qRT-PCR with envelope specific primers of SARS-CoV2 to quantitate total viral load (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).

(Q) Total RNA was isolated from mock and SARS-CoV2 (MOI 1, 24 h) infected control and IRGM knockdown THP-1 cells and subjected to qRT-PCR with nucleocapsid specific primers of SARS-CoV2 to quantitate total viral load (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).

(R) Total RNA was isolated from the supernatant of mock and SARS-CoV2 (MOI 1, 10 h and 20 h) infected control and IRGM knockdown THP-1 cells and subjected to qRT-PCR with nucleocapsid specific primers of SARS-CoV2 to quantitate total viral load (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).

(S) Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS-CoV2 (MOI 1, 24 h) infected THP-1 control and IRGM knockdown cells.

(T) The graph depicts quantification of SARS-CoV2 plaque assays (plaque-forming units/ml) in Vero E6 cells performed from the culture supernatant of SARS-CoV2 (MOI 1, 24 h) infected THP-1 control and IRGM knockdown cells (n=3, mean ± SE, **p < 0.005, Student's unpaired t-test).