Journal: Communications biology

Article Title: A type II protein arginine methyltransferase regulates merozoite invasion in Plasmodium falciparum.

doi: 10.1038/s42003-023-05038-z

Figure Lengend Snippet: Fig. 1 PfPRMT5 expression, localization, and its histone modifications during IDC. a PfPRMT5 expression at different stages of the IDC was analyzed by Western blots with anti-Protein C antibodies (targeting PTP tag) to detect the full-length PfPRMT5-PTP (~ 106 kDa) in the PfPRMT5::PTP parasite line. PfHSP70 was used as a loading control. R: ring, ET: early trophozoite, LT: late trophozoite, and S: schizont. The relative expression level of PfPRMT5 was normalized with PfHSP70. b Identification of PfPRMT5 in the parasite cytoplasmic (Cyto) and nuclear (Nuc) fractions (upper panel). Anti-H3 antibodies (middle panel) and anti-PfHLP antiserum (lower panel) are used as nuclear and cytoplasmic markers, respectively. c IFA with anti-Protein C and FITC- conjugated anti-rabbit IgG as primary and secondary antibodies, respectively, to detect the localization of PfPRMT5 in the PfPRMT5::PTP parasite line. Nuclei were stained with Hoechst 33342. The size of the scale bar is 5 μm. d Endogenous PfPRMT5 was used in the methylation assay with human mononucleosomes, bovine, and P. falciparum core histones as the substrates, and the reactions were separated by SDS-PAGE (15% gel). Left panel: Coomassie blue-stained gel. Right panel: fluorograph. e Western blots with anti-H3R2me2s, H3R8me2s, and H4R4me2s antibodies were conducted to detect PfPRMT5-dependent methylation on H3 and H4 in human mononucleosomes. H3 and H4 were used as loading controls. f The levels of H3R2me2s, H3R2me2a, H3R8me2s, and H4R4me2s in the WT parasite during the IDC were analyzed by Western blots with respective antibodies. H3 was used as a loading control and human mononucleosome (Mono) was used as a negative control without any modifications in the histones. g IFA with anti-H3R2me2s and Alexa fluor 488-conjugated anti-rabbit IgG as primary and secondary antibodies, respectively, to detect the localization of H3R2me2s in the WT 3D7 parasite. Nuclei were stained with DAPI. The size of the scale bar is 5 μm.

Article Snippet: The modified histone H3 peptide (EpiCypher #SKU 12-0235 H3R2me2s, biotinylated) and the unmodified P. falciparum histone H3 peptide (250 ng of each peptide) were spotted on a PVDF membrane followed by blocking with 1% casein in Tris-buffered saline (TBS) and incubating with H3R2me2s and H3 antibodies at 1:2000 dilutions.

Techniques: Expressing, Western Blot, Control, Staining, Methylation, SDS Page, Negative Control

Journal: Communications biology

Article Title: A type II protein arginine methyltransferase regulates merozoite invasion in Plasmodium falciparum.

doi: 10.1038/s42003-023-05038-z

Figure Lengend Snippet: Fig. 2 Alteration of parasite growth and protein modifications upon PfPRMT5 disruption. a Growth curves of two PfPRMT5 disruptants (ΔPfPRMT5-1 and ΔPfPRMT5-2) compared to WT 3D7 parasite. The disruptants grew significantly slower than 3D7 (ANOVA, P < 0.01, n = 3 biological replicates). b The IDC progression profiles of the PfPRMT5 disruptant (ΔPfPRMT5-1) along with 3D7 control showing the proportions of the ring, trophozoite, and schizont stages through a 52 h period. The dynamics of the ring, trophozoite, and schizont stages were very similar between the 3D7 and ΔPfPRMT5-1. c Distribution of merozoite numbers in schizonts from 3D7 and two PfPRMT5 disruptants. The PfPRMT5 disruptants had fewer numbers of merozoites in schizonts than in 3D7 (ANOVA, P < 0.05, n = 3 biologically independent experiments). d The capacity of invasion by merozoites from PfPRMT5 disruptant (ΔPfPRMT5-1). The invasion rate is significantly lower than the 3D7 control (P < 0.01, n = 3 biologically independent experiments). e Western blots show a substantial decrease of H3R2me2s and relatively stable H3R8me2s, H4R3me2s, and H3R2me2a in the schizonts of PfPRMT5 disruptant (ΔPfPRMT5-1). H3 was used as a loading control. f Western blots indicate the pattern of overall protein arginine methylation (MMA, aDMA, and sDMA) between ΔPfPRMT5-1 and WT (3D7) parasites. A considerable reduction in the sDMA level was detected in ΔPfPRMT5-1. PfHSP70 was used as an equal loading control. The relative levels of MMA, aDMA, and sDMA were normalized with PfHSP70. The standard deviation was shown as error bars.

Article Snippet: The modified histone H3 peptide (EpiCypher #SKU 12-0235 H3R2me2s, biotinylated) and the unmodified P. falciparum histone H3 peptide (250 ng of each peptide) were spotted on a PVDF membrane followed by blocking with 1% casein in Tris-buffered saline (TBS) and incubating with H3R2me2s and H3 antibodies at 1:2000 dilutions.

Techniques: Disruption, Control, Western Blot, Methylation, Standard Deviation