VopQ-mediated autophagy interferes with NLRC4 inflammasome activation.

<p><b>A.</b> BMMs from wild-type or NLRP3-deficient (<i>Nlrp3</i>^−/−) mice were infected with Δ<i>tdhAS</i> or Δ<i>tdhAS</i>Δ<i>vopQ</i> mutants for one hour, and then immunostained with FITC-labeled anti-LC3 antibody (green) and Cy5-labeled anti-<i>V. parahaemolyticus</i> antibody (red). The merged image and differential interference contrast (DIC) were shown. Bar, 10 µm. <b>B.</b> BMMs were infected with bacteria (1 h) or treated with rapamycin as a control (2 h) in the absence or presence of bafilomycin A1. The cells were analyzed by immunoblotting with anti-LC3 antibody to evaluate the conversion of endogenous LC3-I to II or with anti-β-actin antibody. The relative intensity of LC3-II was calculated by normalizing the untreated control to a relative intensity of 1 and then dividing subsequent LC3-II band intensity by actin band intensity using densitometry. <b>C.</b> Knockdown efficiency of Atg5 by shRNA in NLRP3-deficient BMMs was analyzed by immunoblotting with anti-Atg5 antibody. <b>D.</b> Effect of knockdown of Atg5 on rapamycin-mediated LC3-I to II conversion in NLRP3-deficient BMMs. <b>E.</b> Effect of knockdown of Atg5 on caspase-1 activation and IL-1β processing in infected NLRP3-deficient BMMs infected with indicated bacteria. <b>F.</b> Effect of knockdown of Atg5 on IL-1β release from infected NLRP3-deficient BMMs at 3 hpi. was analyzed using an ELISA. Data are presented as the means ± SD of triplicate samples. *p<0.05.