Fig. 8: Spindle localization of EB1 is independent of NDC80.

a Schematic of CRISPR/Cas9-mediated promoter swap in the ndc80 gene locus for depleting the ndc80 gene expression in gametocytes. The promoter (801 bp) of ndc80 gene in the DTS1 parasite was replaced with the promoter (1,826 bp) of the clag gene, generating the ndc80kd mutant clone. b Immunoblot of Myc-tagged Ndc80 and HA-tagged EB1 in gametocytes of the DTS1 and ndc80kd parasites at 0 and 8 mpa. BiP and α-Tubulin as loading control. Right panels show the quantification of Ndc80 and EB1 protein level after normalizing with that of BiP. Mean ± SD from three independent experiments, two-tailed t-test. c IFA confirming the depleted expression of Ndc80 in the ndc80kd male gametocytes. Asexual blood stage (ABS) and gametocytes at 0 and 8 mpa of the DTS1 and ndc80kd parasites were stained with Hoechst 33342 and antibodies against HA and Myc. Scale bar = 5 μm. Right panel shows the quantification of fluorescence intensity for Ndc80 protein. n is the number of gametocytes measured. Mean ± SD from three independent experiments, two-tailed t-test. d U-ExM of EB1 in male gametocytes at 8 mpa. The parasites were stained with Hoechst 33342 and anti-HA antibody. Scale bar = 5 μm. Right panel shows the percentage of parasites with spindle localizing of EB1. Mean ± SD from three independent experiments, two-tailed t-test. e Chromosome segregation in male gametocytes at 8 mpa. The parasites were co-stained with Hoechst 33342 and anti-Myc antibody. Scale bar = 5 μm. Right panel indicates the percentage of parasites with chromosome clustering. Mean ± SD from three independent experiments, two-tailed t-test. f Quantification of the male gamete containing chromosomes in DTS1 and ndc80kd parasites. Mean ± SD from three independent experiments, two-tailed t-test. g Midgut oocyst formation of the DTS1 and ndc80kd parasite in mosquitoes at day 7 post blood feeding. Red horizontal lines show mean value of oocyst numbers, Mann–Whitney U test. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence. Two independent experiments with similar results.