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PLB-985 cells were transduced with either Atg5 shRNA or the control shRNA, differentiated and then infected with LF82 (50 MOI). (A) A defect in autophagy (Atg5 silencing) favours intracellular survival of bacteria. Intracellular bacteria were numbered 1 h and 5 h after addition of gentamycin. Data are means ± SEM of three experiments. *<0.01 and **p<0.003. Silencing of Atg5 mRNA was confirmed by RT-PCR analysis (left). (B) Two inducers of autophagy, nutrient starvation (HBSS) and rapamycin (rapa, 100 nM in complete medium) decreased intracellular LF82 survival (left), rescued the autophagic flux (as shown by detection of LC3-II, upper right), and increased bacterial degradation (lower right, arrowhead, transmission electron micrograph). Modulation of LF82-induced IL-8 production in response to inhibition of autophagy (Atg5 shRNA) or to stimulation of autophagy (rapamycin or starvation) was analyzed by immunoblotting (C) and qRT-PCR (D). 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