Selective Autophagy of RIPosomes Maintains Innate Immune Homeostasis during Bacterial Infection (2022)
Figure 5

Figure 5. IRGM mediates the degradation of NODs, RIPK2, and RIPosomes to suppress NF-κB-dependent cytokine response

(A) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR-Cas9 mediated IRGM partial knockout (IRGM+/-) HT-29 cells (2 clones were tested). Densitometric analysis was performed using Image J software. FC, fold change.

(B) Left panel, soluble and insoluble fractions of S. flexneri (MOI 1:25, 8 h) infected mouse BMDMs from Irgm1+/+ and Irgm1-/- mice were subjected to immunoblot analysis with antibodies as indicated. Right panel, Western blot analysis with the colon lysates from Irgm1+/+ and Irgm1-/- mice with indicated antibodies. The graph indicate ratio of band intensity (measured using ImageJ) and actin (n=5, Mean ± SD, *p < 0.05, **p < 0.005, and ****p < 0.00005, Student's unpaired t-test)

(C) Doxycycline-inducible stable GFP-RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S. flexneri (MOI 1:25, 4h). The cells were fixed and subjected to high-content microscopy to quantitate the number of RIPosomes formed. The graph depicts an average number of RIPosome/cell. About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test.

(D-E) Western blot analysis with the cell lysate of (D) HT-29 clones stably expressing Flag-vector control or Flag-IRGM (E) THP-1 cells transiently transfected with GFP or GFP-IRGM for 6 h. Densitometric analysis was performed using Image J software. FC, fold change.

(F) Left panel, representative high-content microscopy images of NODo-RIPosomes in HEK293T cells transfected with GFP-RIPK2 and myc-NOD2 (upper panel) or GFP-RIPK2, myc-NOD2, and Flag-IRGM (lower panel). Right panel, the graph depicts average number of NODo-RIPosome/cell. About 15000 cells plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=5 (biological replicates), ****p < 0.00005, Student's unpaired t-test.

(G) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies.

(H) Representative confocal images of HEK293T cells transfected with mCherry and GFP-RIPK2CARD or mCherry-IRGM and GFP-RIPK2CARD for 9 h. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.

(I-K) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin, Flag, Myc, and, GFP antibodies as indicated. Densitometric analysis was performed using Image J software. FC, fold change.

(L) Left panels, representative high-content microscopy images of HEK293T cells transfected with GFP-RIPK2 or GFP-RIPK2 and Flag-IRGM or GFP-RIPK2 and Flag-IRGM (S47N). The graph depicts the average number of RIPosome/cell. The details are mentioned in the legends of Figure 1F. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).

(M) Luciferase assays performed with HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32NFκB-RE, along with plasmids as indicated, followed by treatment with MDP (10 μg/ml, 4 h) or iE-DAP (10 μg/ml, 4 h) as indicated. Mean ± SD, n=3 (biological replicates), **p < 0.005, ***p < 0.0005 and ****p < 0.00005, Student's unpaired t-test.

(N-O) The qRT-PCR analysis with total RNA isolated from THP1 cells transfected with indicated siRNA and treated with (N) L-18 MDP (1 μg/ml, 6 h) for indicated genes (O) L-18 MDP (1μg/ml, 6h) and GSK583 (1μM ). Mean ± SD, n=3 (biological replicates), *p < 0.05, **p < 0.005, ***p < 0.0005 and, ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).