Figure 5.

CARD3 is Involved in F. nucleatum-Mediated Autophagy. (A) Domain architecture of CARD3 and alignment of the LIR motifs (pink) between humans and rodents. (B) Western blot analysis was performed with HCT116 cells transfected with CARD3-targeting siRNA and cocultured with either PBS or F. nucleatum (F01). (C-D) Representative electron micrographs of autophagosomes (red arrows) in HCT116 cells infected with F. nucleatum (F01, blue arrows) or transfected with CARD3-targeting siRNA (C; scale bar, 0.5 µm). Quantification of cells containing autophagosomes (from C) was conducted with five random fields, and the data are presented as the means ± SDs (D; *P < 0.05, and **P < 0.01; unpaired Student's t-test). (E-F) Representative immunofluorescence images of LC3-Ⅱ in HCT116 cells (E; scale bar, 10 µm). The positive cells were counted in five random fields, and the data are presented as the means ± SDs (F; *P < 0.05, **P < 0.01, and ***P < 0.001; unpaired Student's t-test). (G) Schematic of the experimental setup. (H-J) Representative colorectal tumors (red arrows) and H&E staining of tumors from CARD3^wt and CARD3^-/- mice (I; 200× magnification). Statistical analysis of the tumor numbers (H) and sizes (J) in the different groups of mice (n = 5 mice/group; *P < 0.05; unpaired Student's t-test; the error bars indicate the SDs). (K) Western blot analysis was performed with CARD3^wt and CARD3^-/- mouse tissues.