IFA Sample Preparation and Structured Illumination Microscopy:

PyDOZI::GFP and PyNOT1-G::GFP expressing female gametocytes were imaged by indirect immunofluorescence microscopy on the BioVision Technologies VT-iSIM super-resolution microscope on 100X (oil) objective for high-resolution image capture of Z-slices every 1μm with MetaMorph software. Images were deconvolved using the Microvolution plug-in on ImageJ, and colocalization was measured using the JACoP ImageJ plug-in (⁷²).

Cells were prepared for IFA as previously described (^{22, 40}). Briefly, the transgenic gametocytes were pelleted at 1,400 xg for 3 minutes and fixed in 4% v/v paraformaldehyde (VWR, Cat# PI28908) and 0.0075% v/v glutaraldehyde (VWR, Cat#AAAA17876-AE) in 1xPBS for with rocking for 30 minutes at room temperature. Cells were washed once in 1x PBS and permeabilized in 1% v/v Triton X-100 (Fisher Scientific, Cat # AC327372500) for 10 minutes at room temperature. Cells were well washed with 1x PBS to remove permeabilization solution before blocking with 3% w/v BSA (Sigma-Aldrich, Cat # A7906–100G) in 1x PBS overnight at 4°C. Blocked cells were stained with primary antibody at 1:1000 dilutions in the 3% w/v BSA blocking solution for one hour at room temperature with rocking. Primary antibodies used were against GFP (Mouse mAb 4C9, DSHB, Cat# DSHB-GFP-4C9), HsDDX6 (DOZI homolog, rabbit pAb) (^{20, 80}), or PyPABP1 (rabbit pAb, Pocono RF&L, custom antibody) (⁷¹). Cells were washed after incubation with the primary antibody twice with 3% w/v BSA blocking solution, then resuspended in the blocking solution and incubated with the secondary antibodies for 1 hour, all at 1:1000 dilutions. Secondary antibodies used were against mouse (Donkey anti-mouse, AF488; Invitrogen Cat#A21202) or goat (Donkey anti-goat AF488, Invitrogen Cat# A11055) or rabbit (Donkey anti-rabbit AF594, Invitrogen, Cat# A11012). Cells were washed in 1x PBS, then stained with 1 μg/mL DAPI (4′,6-diamidino-2-phenylindole) in 1x PBS for 5 min at room temperature. Cells were washed once in 1x PBS, then resuspended in 1xPBS before mounting on glass slides in a 1:1 ratio with ProLong Gold Antifade Mountant (Invitrogen, Cat# {"type":"entrez-protein","attrs":{"text":"P36930","term_id":"1248281091","term_text":"P36930"}}P36930), and covered with a glass cover slip before imaging.