Fig. 3: PfCERLI2 knockdown inhibits merozoite invasion.
a GFP-expressing PfCERLI2HAGlmS/GFP parasites were treated with a range of concentrations of GLCN from early trophozoite stages until early rings the following cycle (~24 h), with invasion assessed by flow cytometry as the number of newly invaded rings expressed as a percentage of an untreated media control. n = 4, box represents the 25th to 75th percentile, median and maximum value are shown. X-axis presented as log 2 scale for viewing purposes. b PfCERLI2HAGlmS parasites were either treated with GLCN from ring-stage to schizont stage or left untreated. Early schizonts were treated with the egress inhibitor E64 for ~5 h. Following treatment, cultures were smeared and the number of merozoites per schizont was determined by blinded microscopy analysis of Giemsa-stained blood smears. Each data-point represents the mean number of merozoites per schizont from 20 schizonts. n = 3, error bars = SEM, ns = p > 0.05 by unpaired t-test. c Percentage of schizonts that ruptured over a 6-h window in either the presence or absence of GLCN, as determined by flow cytometry. n = 3, error bars = SEM, ns = p > 0.05 by unpaired t-test. d During the invasion assay described in (a), the number of free merozoites was also quantified by flow cytometry, with results presented as % of total RBCs. n = 4, error bars = SEM, *p < 0.05 by unpaired t-test. e PfCERLI2HAGlmS schizonts, either in the presence or absence of GLCN, were matured in the presence of the schizont rupture inhibitor E64 before being fixed, Giemsa-stained and imaged by light microscopy. Scale bar = 2 µm. PfCERLI2HAGlmS parasites were treated with 2.5 mM GLCN or left untreated, with Giemsa smears made following schizont rupture (f) and the number of merozoites bound to RBCs (g) and newly invaded rings (h) quantified, Scale bar = 5 µm, n = 5, error bars = SEM, *p < 0.05, **p < 0.01 by unpaired t-test. i Representative IFAs of merozoites, across three defined invasion stages scored 1–3, stained with DAPI, anti-AMA1 and anti-MSP1-19 antibodies invading CellTrace-stained RBCs. Scale bar = 1 µm. j Number of merozoites blind-assessed for each invasion score. n = 20 merozoites in a single biological replicate. k AMA1 signal diameter was measured under blinded assessment—measurements were taken as shown on the double arrows in (i). N = 20 merozoites in a single biological replicate, error bars = SEM, *p < 0.05, ns = p > 0.05 by unpaired t-test.
