Loss of retention after shedding of episome in the SFM⁺ line. (A) Immunofluorescence analysis of the SFM⁺ parasite line cultured during 21 generations in the presence (SFM⁺, top panel) or absence (SFM⁻, bottom panel) of 40nM pyrimethamine. Infected erythrocytes were stained with anti–c-myc mAb followed by anti–rat Alexa-488–conjugated IgG, and parasite nuclei were counterstained with Hoechst 33342. Pictures were taken under identical exposure conditions. Absence of c-myc expression in SFM⁻ line indicates a loss of episomal expression of the epitope-tagged STEVOR protein. (B) Scanning electron micrograph of B3, SFM⁺, and SFM⁻ parasite-infected erythrocytes. Right and middle panels: SFM⁻ and SFM⁺ infected erythrocytes with normal knobs compared with erythrocyte infected with the KAHRP-deficient B3 parasite line (left panel) in which knobs are absent. The bars represent 2 μm. (C) Kinetics of retention in microsphere matrices for erythrocytes infected with B3 (gray line), SFM⁺ (green continuous line), and SFM⁻ (green dotted line) during the parasite life cycle. Measurements were performed at 10, 16, 22, 28, 34, and 40 hpi on 2% hematocrit cultures containing 2% to 10% tightly synchronized parasites.