Figure 3. PfAP2-G2 is expressed throughout the gametocyte stages and is essential for maturation of gametocytes.

(A) Images obtained by live fluorescence microscopy performed on Stage I through Stage V gametocytes. The DIC image was merged with the DRAQ5 nuclear stain imaging to confirm the nuclear localization of the signal. The results indicate that PfAP2-G2 was expressed at all stages of gametocyte development and was localized to the nucleus. (B) Nuclear and cytoplasmic fractions from stage III gametocytes were subjected to western blot analysis using anti-GFP antibodies (1:1000). The GFP-tagged AP2-G2 of expected size (~250 kDa) was detected in both the nuclear and cytoplasmic fractions of the parasite lysate. Anti-histone H3 (1:3000) and anti-aldolase (1:1000) were used as the loading controls for the nuclear and cytosolic fractions, respectively. (C) Giemsa-stained smears of gametocytes from the WT and PfAP2-G2 KO lines showed that the morphology of the KO gametocytes appeared normal until Stage III. Most pfap2-g2 KO parasites committed to sexual development did not progress beyond stage III. Morphological staging was quantified by counting ≥ 100 parasites for each time point during gametocyte development from Giemsa-stained smears. The reported number of counts are based on the single replicate.