Fig 2.

CD40 stimulation enhances autophagic activity in nonhematopoietic cells. (A) HBMVEC, hmCD40-mHEVc, or hCD40-HRPE cells stimulated with CD154 for indicated times were lysed and analyzed by immunoblotting for expression of Beclin 1, LC3 II, or actin. (B) hmCD40-mHEVc or hCD40-HRPE cells stimulated with or without CD154 in the presence of bafilomycin A1 (Baf A1; 100 nM) for 6 h were lysed and analyzed by immunoblotting for expression of LC3 II or actin. (C) hmCD40-mHEVc or hCD40-HRPE transfected with tfLC3 were stimulated with or without CD154 for 5 to 6 h and monitored by fluorescence microscopy for the number of autophagosomes (yellow; arrowheads) or autolysosomes (red; arrow). (D) hmCD40-mHEVc or hCD40-HRPE cells transfected with LC3-EGFP were stimulated with or without CD154 and infected with T. gondii expressing RFP. At 5 h postinfection, cells were analyzed and quantitated by fluorescence microscopy for accumulation of LC3 around the parasite (arrowheads). Results are shown as means ± SEM and are representative of 3 or 4 independent experiments. *, P ≤ 0.05; **, P ≤ 0.01.