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Fig. 3. MIC8 is required for parasite survival and is essential during the invasion of the host cell. (A) Upper panels: growth of MIC8KOi parasites was compared to RH_wt parasites using plaque assay. Parasites were inoculated on HFF cells in the presence or absence of ATc for 7 days prior to GIEMSA staining. Lower panels: same experiment as above. Parasites were analysed 72 hours post-infection. Note that in the case of MIC8KOi grown in the presence of ATc, freshly egressed parasites did not invade neighbouring host cells. (B) Analysis of non-induced egress by quantification of intact initially infected host cells. HFF cells were inoculated with MIC8KOi-GFP parasites in the presence or absence of ATc and the decrease in initially infected host cells was analysed over time. The depicted quantification is representative of the results derived from three independent experiments. (C) Attachment assay of freshly released parasites grown in the presence or absence of ATc to fixed cells. Equal numbers of parasites were allowed to attach on host cells at 37°C for 15 minutes before non-attached parasites were removed by consecutive washing steps with cold PBS. Data are mean values of three independent experiments ± s.d. (D) Invasion assay of freshly egressed parasites grown in the presence or absence of ATc as in C. After the indicated time, non-invaded parasites were removed by several washing steps. Intracellular parasites were further incubated for 12 hours and the number of parasitophorous vacuoles was compared. ON, over night. Data shown are mean values of three independent experiments ± s.d. (E) Re-expression of MIC8 in extracellular parasites restores invasion capability. Invasion assay was performed similar to as in D. After MIC8KOi grown in the presence or absence of ATc were allowed to attach for 10 minutes, non-attached parasites were removed. Attached parasites were further incubated in the presence or absence of ATc (–/–: parasites constantly kept in the absence of ATc; +/+: parasites constantly kept in the presence of ATc; +/–: parasites kept in the presence of ATc and further incubated in media without ATc). Data represent mean values of three independent experiments ± s.d.

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