Journal: bioRxiv

Article Title: Plasmodium falciparum PP1 phosphatase is a key regulator of malaria parasite transmission

doi: 10.1101/2025.03.06.641796

Figure Lengend Snippet: A. Sorbitol-induced isosmotic lysis of stage GIE from the B10 clone treated for 30 minutes with DMSO or with 50 nM Calyculin A, in the presence or absence of 100 µM NPPB. Bars represent the mean ± SEM from 4 independent experiments (n = 4). Statistical analyses were performed using a one-way ANOVA test. B. Sorbitol-induced isosmotic lysis of stages V GIE from the pfpp1-iKO transgenic line treated with DMSO (-RAP) or rapamycin (+RAP), in the presence or absence of 100 µM NPPB. Bars represent the mean ± SEM from 5 independent experiments (n = 5). Statistical analyses were performed using a one-way ANOVA test. C . Retention in microbeads of stages V GIE from the pfpp1-iKO transgenic line treated with DMSO (-RAP) or with rapamycin (+RAP). Bars represent the mean ± SEM from 3 independent experiments in 3 technical triplicates (n = 3). Statistical analyses were performed using a Mann-Whitney test. D. Membrane shear moduli measured by micropipette aspiration of stages V GIE from the pfpp1-iKO transgenic line treated with DMSO (-RAP) or rapamycin (+RAP). Each dot represents an individual cell measurement. Statistical analyses were performed using a Mann-Whitney test. E. Immunoblotting analysis of stage V gametocytes treated with Streptolysin O (SLO). The pellet (P) corresponds to the parasite fraction while the supernatant (SN) corresponds to the erythrocyte fraction, including parasite proteins exported to the erythrocyte. The quality control of parasite and erythrocyte fractions were checked using anti-Pfg27 (middle panel) and anti- Pf HSP70 antibodies (lower panel), respectively. The upper panel shows the presence of Pf PP1 exclusively in the parasite fraction. F. Immunoblot analysis of phosphorylated PKA substrates in MACS-purified stage V GIE from the pfpp1-iKO transgenic line treated with DMSO (-RAP) or rapamycin (+RAP). Immunoblot was probed with rabbit monoclonal antibody directed against phospho-PKA substrates (RRX*S/*T), anti-Band 3 antibody to control for red cell quantity, anti-Histone H3 antibody to control for parasite quantity and anti-HA antibody to validate Pf PP1 depletion. G. Quantification of Phospho-PKA levels relative to Band III, as shown in G, was performed by densitometry (Quantity one software). Statistical analyses were performed using a Mann-Whitney test.

Article Snippet: A glass micropipette mounted on a hydraulic micromanipulator (Narishige, Japan) was used to extract the GIE membrane shear modulus by micropipette aspiration technique.

Techniques: Lysis, Transgenic Assay, MANN-WHITNEY, Membrane, Shear, Western Blot, Control, Purification, Software