Fig. 2.

IFN-γ–induced liver-stage P. vivax elimination depends on Beclin 1 and PI3K. (A–C) HC04 cells were transfected with scramble control siRNAs or siRNAs against Beclin 1 and cDNAs encoding RFP-GFP-LC3. At 48 h posttransfection, the cells were treated or not with IFN-γ for 4 h and then processed for fluorescence microscopy. RFP⁺GFP⁺-LC3 (autophagosomes) and RFP⁺GFP⁻-LC3 (autolysosomes) were quantified, and the percentage of puncta-containing cells was determined by analyzing at least 100 cells per condition from three independent experiments. Only puncta ≥0.25 µm in size were counted. The number of puncta per cell was also quantified in Z-stack images of at least 30 cells per condition per independent experiment. Data are mean ± SEM relative to the full control. N.S., not significant. (Scale bar: 5 µm.) (D) Beclin 1 can be successfully depleted in HC04 cells. HC04 cells were transfected with scramble control siRNAs or siRNAs against Beclin 1 and then harvested at 48 h posttransfection for immunoblot analysis. Actin served as an internal loading control. (E and F) Beclin 1-deficient or -proficient HC04 cells were infected with P. vivax sporozoites at an MOI of 1, followed by IFN-γ treatment for 4 h. The cells were washed and then maintained in complete medium until being processed for IFAs on day 4. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.) (G and H) HC04 cells infected with P. vivax sporozoites were treated with IFN-γ with or without the PI3K inhibitor 3-MA for 4 h, washed, and maintained in complete medium until being processed for IFA on day 4. Data are mean ± SEM of at least three independent experiments; the results are expressed relative to the full control, defined as 100%. (Scale bar: 5 µm.)