Fig. 9.

Shld1 dose-dependent increase in GDV1 protein levels. a Immunoblot of saponin-treated Pfgdv1.gfp.dd or wild type NF54 schizonts extracted in 8 M urea/5% SDS after 24 h incubation in the indicated amount of Shld1. The immunoblot was first probed with anti-GFP mAb and imaged, then reprobed with rabbit anti-Histone 3 (anti-H3) antibody as a loading control as described in Methods. The anti-GFP and anti-H3 positive bands are shown and the complete gel is shown in Supplementary Fig. 6A. b The density of the upper (dot) and lower (x) bands was determined after background subtraction using Image J software, normalized to the anti-histone 3 antibody signal and plotted. c–e Shld1 dose-dependent increase in gametocyte production and ap2-g, msrp1 & gexp5 transcription. c After growth in the indicated Shld1 concentration for ~50 h, NAG was added and gametocyte production (average ± s.d.) was determined 9 days later by counting Giemsa-stained smears. d, e The average relative abundance (2^−ΔΔC_T) of ap2-g (blue), msrp1 (red), gexp5 (purple) and gdv1 (black) transcripts in RNA obtained from schizonts (46 ± 2 hpi) d and 14 h later from ring stage parasites (e) grown in the indicated concentration of Shld1 are plotted using early schizonts (36 ± 2 hpi) as the reference. The experiment was run twice with duplicate flasks and a representative data set is shown with the trend lines indicated. The second experiment is shown in Supplementary Fig. 6B-D. f–h Gametocyte conversion rate is positively correlated with ap2-g, msrp1, gexp5 mRNA expression. The gametocyte conversion rate and the relative abundance (2^−ΔΔC_T) of f ap2-g, g msrp1 or h gexp5 RNA in ring stage (14 ± 2 hpi) Pfgdv1.gfp.dd clone T1 parasites cultured in the absence (open circle) or presence (black dot) of 500 nM Shld1 are plotted. The data are from 3 independent experiments performed in triplicate or quadruplicate. The trend line and correlation coefficient (R²) is shown