Fig. 5
WASp deficiency leads to impaired bacterial autophagy. a WT or WAS KO BMDCs were co-cultured with WT EPEC (MOI of 5) for 0.5 or 1.5 h. After co-culture, uninfected control and infected cell lysates were immunoblotted for LC3-I and LC3-II expression. β-Actin was used as a loading control. A representative of three experiments is shown. b WT or WAS KO BMDCs were co-cultured with WT EPEC (MOI of 5) for 3 h. Infected cells were fixed and stained for bacteria (DAPI), F-actin (green), p62 (red) and LC3 (yellow). Co-localization of p62 and LC3 to DAPI-bacteria compartmentalized within an actin-based structure in WT (upper panel) and not in WAS-KO (lower panel) BMDCs. Images were taken at ×63 magnification. Scale bar = 5 µm. c Percentage of BMDCs showing close association between WT EPEC (MOI of 5) and LC3 puncta 3 h after co-culture. Quantification was performed on at least 500 BMDCs. Data presented are from two independent experiments done in triplicate. Data are presented as mean ± SEM. Two-sided Student’s t-test. d WT or WAS KO BMDCs were co-cultured with WT EPEC (MOI of 5) for 3 h, followed by gentamicin treatment for 2 h. After infection, cells were fixed and processed for TEM, followed by image acquisition. The black boxed region of the TEM image is enlarged and shown. The scale bar represents 500 nm (first row, left and right; second row, left; third row, left and middle; fourth row, left) and 100 nm (first row, middle; second row, middle and right; third row, right; and fourth row, middle and right). e BMDCs were treated with rapamycin for 2 h, followed by EPEC infection (MOI of 5) for 3 h. Rapamycin was maintained throughout the period of infection. Culture supernatants were collected for IL-1β quantification by ELISA. Data are presented as mean protein concentration ± SEM from three independent experiments in duplicate. Two-sided Student’s t-test. **p = 0.01; ***p < 0.001
