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. 2021 Dec 6;10:e70451. doi: 10.7554/eLife.70451

Figure 3. MEDLE2 is expressed by trophozoites and passes through the secretory pathway.

Figure 3.

(A) Schematic representation of hypothetical patterns of MEDLE2 export in C. parvum. (B) Immunofluorescence assay (IFA) of wild type (WT) and MEDLE2-HA sporozoites fixed on poly-L-lysine-treated coverslips. We note that MEDLE2-HA is not observed in sporozoites. Red, hemagglutinin (HA)-tagged protein; green, sporozoite antigen Cp23; blue, Hoechst. (C) HCT-8 cells infected with MEDLE2-HA parasites were fixed in 30 min increments and processed for IFA. Data shown are representative images from a time-course bridging 3 hr (no observed MEDLE2-HA) and 6 hr (MEDLE2-HA abundant in host cell). White arrowheads denote parasite nuclei. Red, HA-tagged protein; blue, Hoechst. (D) MEDLE2-HA parasites were excysted and used to infect HCT-8 and after 3 hr media were supplemented with brefeldin A (BFA) (10 µg/mL). 10 hr post infection, cells were fixed and processed for IFA. Red, HA-tagged protein; green, parasites (VVL); blue, Hoechst (D). (E) The impact of BFA treatment on MEDLE2-HA export was quantified showing a significant reduction in MEDLE2 export when comparing BFA-treated (red) and untreated cells (blue) (n = 191 untreated, n = 98 treated; mean ± SEM; p<0.0001; unpaired t test with Welch’s correction).

Figure 3—source data 1. Numerical data used for the quantification of HA positive host cells in the absence and presence of Brefeldin A (BFA).