Figure 1.

The indication of autophagy, and localization of autophagosomes/lysosomes and pathogens in C. jejuni infection. (A, C, E) Representative immunofluorescence images of HeLa cells infected with S. Enteritidis (SE) for 1 h or C. jejuni (Cj) for 6 h. S. Enteritidis and C. jejuni was stained with 10 ng/ml 5(6)-Carboxyfluorescein diacetate (CFDA) solution, and infected to HeLa cells. Cells were immunostained by anti-LC3 (A), p62 (C), and LAMP2 (E) antibody and DAPI, and observed by Keyence BZ-X700 fluorescence microscope. (B) Intracellular bacteria which co-localized with LC3 were quantified. Co-localization rate was determined at 1 h p. i. (Cj-1 h), 6 h p. i. (Cj-6 h), and 12 h p. i. (Cj-1 hr) in (C) jejuni infection, and 1 h p. i. (SE) in S. Enteritidis infection. Cj-1 h: 15.93 ± 14.33% of colocalization; n = 36, Cj-6 h: 7.88 ± 4.30% of colocalization; n = 55, Cj-12 h: 6.54 ± 2.38% of colocalization; n = 94, and SE: 62.40 ± 4.16% of colocalization; n = 186. Sample number indicated the sum of 3 independent experiments. (D) Intracellular bacteria which co-localized with p62 were quantified. Cj-1 h: 3.03 ± 5.25% of colocalization; n = 37, Cj-6 h: 7.35 ± 3.35% of colocalization; n = 59, Cj-12 h: 15.13 ± 5.05%; 12 h p. i. of colocalization; n = 117, and SE: 64.25 ± 2.50% of colocalization; n = 191. Sample number indicated the sum of 3 independent experiments. (F) Intracellular bacteria which co-localized with LAMP2 were quantified. Cj-1 h: 16.67 ± 16.67% of colocalization; n = 35, Cj-6 h: 11.21 ± 10.22% of colocalization; n = 55, Cj-12 h: 15.13 ± 5.05%; 12 h p. i. of colocalization; n = 96, and SE: (61.72 ± 6.99% of colocalization; n = 115. Sample number indicated the sum of 3 independent experiments. (G) HeLa cells were infected with C. jejuni NCTC 11168 up to 12 h and detected autophagy-related proteins, NDP52, p62/SQSTM, LC3 by Western blotting. (H). Caco-2 cells were infected with C. jejuni and detected autophagy-related protein levels.