Figure 1

(C) HeLa GFP-LC3 cells were transfected with indicated siRNAs and infected with DsRed-labeled Salmonella. The percentage of GFP-LC3+ Salmonella at 1 hr post-infection is shown. Data are shown as means ± SD; n = 75 cells per condition from three biological replicates. Data are representative of three independent experiments.

(D) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in (C). The scale bars represent 10 μm.

(E) Western blot of

HeLa

cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments.

(G) HeLa GFP-LC3 cells were transfected with control siRNA, CLEC12A siRNA1, or CLEC12A siRNA2 as well as CLEC12A rescue constructs (R1 or R2) and infected for 1 hr with DsRed-labeled Salmonella. Data are shown as mean ± SD; n = 75 per condition. Data are representative of three independent experiments.

(H) HeLa cells from (G) were lysed, and HA-

CLEC12A

expression levels were assessed by western blot. Data are representative of three independent experiments.

Figure 2

(A) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA. The scale bars represent 10 μm. (B) Percentage of LC3-Salmonella colocalization in WT HeLa cells, ATG16L1 KO HeLa cells, and ATG16L1 KO HeLa cells stably expressing ATG16L1^∗300T or ^∗300A alleles in the presence of control siRNA or CLEC12A siRNA. Data are shown as means ± SD; n = 75. Data shown are representative of three independent experiments.

Figure 3

(A) Confocal micrographs of LC3-Listeria colocalization in WT and Clec12a^−/− BMDMs at 1 hr post-infection. The scale bars represent 10 μm. (B) Quantification of LC3-Listeria colocalization in WT and Clec12a^−/− BMDMs. Data shown represent mean ± SD of n = 3 independent experiments; unpaired t test. ^∗∗p < 0.01.

(C and D) Salmonella cfus quantified per gram of stool (C) and per organ (D) at 4 days post-infection are displayed. Data are shown as mean ± SD; ^∗∗p ≤ 0.01. Data are representative of at least two independent experiments (n = 9 for WT; n = 7 or 8 for Clec12a^−/−).

(E) Clinical score for infected mice at 4 days post-infection. Data are shown as mean ± SD. Data are representative of at least two independent experiments. ^∗∗∗∗p < 0.0001; unpaired t test. (n = 14 for WT; n = 13 for Clec12a^−/−).

(F) Survival curve for infected WT and Clec12a^−/− mice (n = 9 mice per genotype).

Figure 4

(A) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs. Data are shown as mean ± SD; n = 6. Data are representative of four independent experiments.

(B) Quantitation of CLEC12A-bacteria and LC3-bacteria colocalization at indicated time points. Data are shown as mean ± SD; n = 3 independent experiments. (C) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP-CLEC12A at 20, 40, 60, and 80 min post-infection. The scale bars represent 10 μm.

Figure 5

(A-C) Representative epifluorescent images of HeLa cells showing colocalization of (A) CLEC12A (green) and galectin 8 or galectin 3 (red) around intracellular Salmonella (blue), (B) GFP-CLEC12A (green) and NDP52 (red) around Salmonella (blue), and (C) endogenous CLEC12A (red) and GFP-LC3 (green) around Salmonella (blue).

(D) CLEC12A or GFP-CLEC12A (green) and GABARAPL2 or ATG16L1 (red) around intracellular Salmonella (blue). Images are representative of three or more independent experiments.

(E) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed-labeled Salmonella for 1 hr. (F) Quantitation of ubiquitin-bacteria colocalization at 1 hr post-infection in cells shown in (E). Data are shown as means ± SEM from three independent experiments.

(G) Representative epifluorescent images of HeLa cells transfected and infected as in (E). (H) Quantitation of NDP52-bacteria colocalization at 1 hr post-infection in cells shown in (G). Data are shown as means ± SEM from three independent experiments.

(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.

Figure 6

(E) HeLa GFP-LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed-labeled Salmonella for 1 hr. The fraction of GFP-positive intracellular Salmonella is shown. Data are mean ± SD and are representative of three independent experiments.

(F) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1, KLHL9, NEDD8, or KLHL13. Data shown as mean ± SD; n = 4. Data are representative of three independent experiments.