Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection

A

Left panel, the domain organization map of NOD1, NOD2, and RIPK2. Right panel, representative confocal images of immunofluorescence assays performed in HEK293T cells transfected with GFP‐NOD1, GFP‐NOD2, and GFP‐RIPK2 plasmids for 9 h. Scale bar 5 μm.

B

Left panel, representative high‐content microscopy images of HEK293T cells transfected with GFP‐NOD1, GFP‐NOD2, and GFP‐RIPK2 plasmids for 9 h. Right panel, the graph depicts the average number of RIPosomes per GFP‐positive cell. About 15,000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n = 3 (biological replicates), ****P < 0.00005, ordinary one‐way ANOVA (Tukey's multiple comparisons test).

C

Western blot analysis of soluble and insoluble fractions of HEK293T cells transfected with the CARD domain‐containing region of NOD1, NOD2, and RIPK2.

D

Representative confocal images of HEK293T cells transfected with GFP‐NOD1^CARD, GFP‐NOD2^CARD, and GFP‐RIPK2^CARD. Scale bar 5 μm.

E

The soluble and insoluble fractions of S. flexneri‐infected THP‐1 (MOI 1:25, 8 h) cell lysates were subjected to Western blot analysis with indicated antibodies.

F

Representative confocal images of immunofluorescence assays conducted with THP‐1 cells infected with RFP expressing S. flexneri (pseudo‐colored, blue) (MOI 1:25, 8 h). Line profile: co‐localization analysis using line intensity profiles. Scale bar, 5 μm. Zoom panels are digital magnifications.

G

Representative confocal images of immunofluorescence assays performed with HEK293T cells transfected with GFP‐RIPK2 and Flag‐NOD2 (upper panel) or Flag‐NOD1 (lower panel). Line profile: co‐localization analysis using line intensity profiles. Scale bar, 5 or 10 μm as indicated in figures. Inset zoom panels are digital magnifications.

H, I

HEK293T cells were transfected with (H) full length and (I) CARD domains of NOD1, NOD2, and RIPK2 followed by Western blot analysis with soluble and insoluble fractions using indicated antibodies.

J, K

Western blot analysis of soluble and insoluble fractions of RIPK2^+/+ and RIPK2^−/− HT‐29 cells infected with S. flexneri (MOI 1:25, 8 h) with indicated antibodies.

L, M

Representative immunofluorescence images of doxycycline‐inducible GFP‐RIPK2 expressing HeLa cells. (L) Upper panel, uninfected. Lower panel, S. flexneri‐infected MOI 1:25, 4 h). Immunostaining was performed with the p65 antibody (red) and DNA stained with DAPI (Blue). (M) The graph indicates % of cells that are RIPosomes positive or negative with nuclear/cytoplasmic p65 (5 fields (each group), Mean ± SD, n = 3). ****P < 0.00005, Student's unpaired t‐test.

Figure 2. RIPK2 oligomers provide a platform for NODs oligomerization.