The presence of LAMP1 in fractions 9 and 10 indicates the autophagosome fusion with lysosome and a potential role of autophagy in bacterial delivery and clearance (Fig 7C). is shown. (C) MLE-12 cells and Primary AMs were infected with Pa as above, and then lysed for pulldown assay. GST-Lyn 1C230 containing both SH3 and SH2 domains shows association with Atg5-Atg12, LC3 and Pa in AMs. (D) Quantification of Atg12-Atg5, LC3 and Pa protein levels in Fig 3G and S4C Fig is shown. (E) Quantification of pLyn and LC3-II level in Fig 3J is shown. (F) MH-S cells were pretreated with PP2 (5 nM, 30 min). Cells were then infected with PAO1 as above and then lysed BTT-3033 for immunoblotting to detect pLyn, Lyn and LC3. (G) Quantification of pLyn and LC3-II level in S4D Fig is shown. Data are representative and shown as means+SD from three independent experiments. One-way ANOVA (Tukeys post hoc); *, p<0.05; **, p<0.01.(TIF) ppat.1005363.s004.tif (538K) GUID:?4CE92CD7-5A7B-450A-BBC4-F309EA0CF845 S5 Fig: TLR2 is involved in Lyn-mediated autophagy. (A) Quantification of pLyn and LC3-II level in Fig 4A is shown. (B) Quantification of pLyn and LC3-II level in Fig 4I is shown. (C) BTT-3033 Quantification of pLyn and LC3-II level in Fig 4J is shown. (D) MH-S cells were pretreated with Pam3CSK4 (300 ng/ml), and infected with PAO1 (1 h). Cells viability was determined using MTT assay. (E) MH-S cells were transfected with LC3-RFP for 24 h and then treated as BTT-3033 above. CLSM imaging was used to detect LC3 puncta. (F) Quantification BTT-3033 of TLR2, pLyn and LC3-II level in Fig 4N is shown. Data are representative and shown as means+SD from three independent experiments. One-way ANOVA (Tukeys post hoc); *, Mouse monoclonal to CD10 p<0.05. Scale bar = 5 m.(TIF) ppat.1005363.s005.tif (387K) GUID:?3E33EED0-07A6-4DC0-B719-C9B17A0C95C6 S6 Fig: Lyn affects canonical phagocytosis through Rab5 and Rab7. (A) MH-S cells were infected with PAO1 (MOI = 10, 1 h). Cells were lysed for Co-IP to detect the interaction of Lyn with Rab5 and Rab7. (B, C) MH-S cells were co-transfected with Rab7-RFP and Ctrl or Lyn siRNA for 24 h. The cells were then infected with PAO1-GFP (MOI = 10, 8 h). CLSM imaging was used to detect related pores and the number of puncta in BTT-3033 each cell was shown. Data are derived from 100 cells in each group. Scale bar = 5 m. (D, E) MH-S cells were transfected with Rab5-RFP or Rab5-DN-RFP plasmid for 24 h. Then the cells were infected with PAO1-GFP. Colocalization between Rab5 and Pa was monitored. Arrows indicate the colocalized puncta and quantification was performed over time. Data are derived from 100 cells in each group. Scale bar = 25 m. (F, G) MH-S cells were transfected with Rab7-RFP or Rab7-DN-RFP plasmid for 24 h and infected with PAO1-GFP. The internalized bacteria in each cell were counted in the lasting 12 h. Data are derived from 100 cells in each group. Scale bar = 25 m. (H, I) MH-S cells were infected with PAO1 (8 h) and were homogenized. Cell lysates were immunoblotted with antibodies against phosphorylated Cofilin-1 (pCofilin-1), Cofilin-1, Flotillin-1, and Actin. The protein levels of pCofilin-1 and Flotillin-1 were quantified. (J) Whole cell lysates were immunoprecipitated (IP) with beads coated with Lyn antibody and immunoblotted with Cofilin-1 and Flotillin-1 antibody. Data are representative and shown as means+SD from three independent experiments. One-way ANOVA (Tukeys post hoc); *, p<0.05.(TIF) ppat.1005363.s006.tif (2.5M) GUID:?A5E25690-F65F-4AA3-9A44-B615B55CB7E8 S7 Fig: also induces autophagy in macrophages. (A) Quantification of Pa and Flagellin level in Fig 7G is shown. (B) Quantification of Flagellin level in Fig 7J is shown. (C) MH-S cells were transfected with Ctrl or Lyn siRNA for 24 h. Cells were pretreated with rapamycin (500 nM, 12 h) or 3-MA (5 mM, 3 h), then infected with Kp (MOI =.