Selective Autophagy of RIPosomes Maintains Innate Immune Homeostasis during Bacterial Infection (2022)
Figure 3

Figure 3. NODs, RIPK2, and RIPosomes are degraded by p62-dependent selective autophagy.

(A-B) Western blot analysis with cell lysate of uninfected and S. flexneri infected (MOI 1:25, 8 h) (A) control and ATG5 siRNA knockdown THP-1 cells, (B) wild type (WT) and ATG5 knockout (ATG5-/-) MEF cells with indicated antibodies. Densitometric analysis was performed using Image J software. FC, fold change.

(C) Left panel, representative high-content microscopy images of RIPosomes in HEK293T cells that are control cells or cells treated with rapamycin (500 nM, 4h) or cells treated with rapamycin (500 nM, 4h) and bafilomycin A1 (BafA1, 300 nM). Right panel, the graph depicts the average number of RIPosomes/cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), **p < 0.005 and, ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test).

(D) Left panel, representative high-content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP-1 cells infected with S. flexneri (8 h). Right panel, the graph depicts average number of RIPosomes/cell. About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.

(E) The soluble and insoluble fractions of S. flexneri infected (MOI 1:25, 8 h) control and ATG5 siRNA knockdown THP-1 cells were subjected to Western blot analysis with indicated antibodies.

(F) Representative confocal images of HEK293T cells transfected with GFP-RIPK2 and immunostained with anti-FK2 antibodies. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm.

(G) Autophagy receptor screen using co-immunoprecipitation (Co-IP) assay to analyze the interaction between GFP-RIPK2 and Flag-p62 or Flag-NBRI or Flag-NDP52 or Flag-TAX1BP1 or Flag-OPTINEURIN in HEK293T cell lysate.

(H) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S. flexneri infected THP-1 cells (MOI 1:25, 8 h).

(I) Representative confocal images of HEK293T cells expressing GFP-RIPK2 (6 h) immunostained with p62 and LC3B antibodies. Zoom panels are digital magnifications.

(J) GST pull-down assay using purified GST or GST-RIPK2 proteins with in-vitro translated 35S radiolabeled myc-ULK1 or myc-ATG16L or myc-BECLIN1.

(K-M) Representative confocal images of HEK293T cells transfected with GFP-RIPK2 or mcherry-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (K) ULK1, (L) ATG16L (M) BECLIN1. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.

(N) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32 NFκB-RE, GFP-RIPK2, and Flag-NOD1 plasmids. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).

(O) Luciferase assays performed with HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32NFκB-RE, GFP-RIPK2, and Flag-NOD1 plasmids followed by treatment with rapamycin (500 nM, 4 h) alone or in combination with bafilomycin A1 (300 nM, 5 h). Mean ± SD, n=3 (biological replicates), **p < 0.005 and ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test). The control conditions and readings for Figure 3N and 3P are the same.

(P) The qRT-PCR analysis with total RNA isolated from the uninfected and S. flexneri infected (MOI 1:25, 6 h) control or ATG5 knockdown or ATG5 and RIPK2 double knockdown THP-1 cells. Mean ± SD, n=3 (biological replicates), **p < 0.005, and ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).

(Q) Pictorial representation of results obtained in this section where we found that p62-dependent selective autophagy degrades NODs, RIPK2, and RIPosomes to suppress NF-κB cytokine response.