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. 2020 May 28;16(5):e1008091. doi: 10.1371/journal.ppat.1008091

Fig 2. Mutant pclag::rab11a and pama-1::rab11a do not make ookinetes and down-regulate PbRab11A.

Fig 2

A/B. Microscopy images of immunofluorescence on fixed parasites using anti-RAB11A and/or anti-P25 antibodies as indicated and DAPI as a nuclear stain. B. Fertilized pclag::rab11a spherical ookinetes and Activated Unfertilised Female Gametes (AUFG) i.e. 24h post-activation showing enlarged nucleus (Images taken on Axioplan). Scale bars 5 μm. C. Plot of pclag::rab11a and pama-1::rab11a ookinete development (n = 3, mean +/-SD, two tailed student t test, p-value 0.0001). D. Western blot for Rab11A in pclag::rab11a and pama-1::rab11a parasites at various life cycle stages probed with anti-PbRab11A, anti-c-MYC antibody. GFP is a loading control. Asterisks show the presence of extra bands in schizont and ookinete stages when the blot is probed with anti-PbRab11A antibody. A: Asexual mixed blood stage; S: Schizonts; G: enriched Gametocytes; O: ookinetes. E. Western blots showing the expression of Rab11A in zygotes and ookinetes produced by pclag::rab11a at 2, 4, 6, 8 and 24hpa as well as in AUFG 6 and 24hpa. F-I. Data from live parasites stained with anti-P25-Cy3 antibody, run on an Imagestreamx MkII imaging flow cytometer and analysed in IDEAS software. All figures show analysis of >750 activated cells after gating for activated parasites based on area focus, GFP and P25-Cy3 intensity (See S5 Fig) F. Imaging flow cytometry (IFC) density plots showing the aspect ratio of the masked GFP image (X axis) (AspectRatio_AdaptiveErode(M02, Ch02, 84)) against the circularity of the masked GFP image (Circularity_AdaptiveErode(M02, Ch02, 84)). Percentage values show proportion of parasites falling into each gate as described. G. Left panel: Dot plot of ookinete development (Circularity_AdaptiveErode(M02,Ch02,84)/AspectRatio(M02,Ch02,84) against P25 intensity (Intensity_MC_Ch03) for the WT-GFPCON (blue) pclag::rab11a (red) and pama1::rab11a (green). Right panel: Bar graph representation of data in left panel split into 4 gates as indicated below. Mean +/-SD and P value 2-tailed t test compared to the control line for each promoter swap line. H. Left panel: Dot plot of the ookinete development against a measurement of the periphery of the ookinete using the perimeter measurement feature in IDEAS software for WT-GFPCON (blue) pclag::rab11a (red) and pama1::rab11a (green). Right panel: Graphical representation of the data in the left panel for four gates as indicated below. Bars show median and interquartile range. I. Representative images from gates i-iv for each line showing overlay of channel 2 GFP (green) and channel 3 antiP25-Cy3 antibody (magenta).