Journal: bioRxiv

Article Title: Febrile temperature enhances Plasmodium falciparum cytoadhesion by disrupting the endothelial glycocalyx

doi: 10.1101/2025.09.07.674757

Figure Lengend Snippet: Immunofluorescence z-stack of microvessel cross sections (left) and MFI normalised by number of cells quantified by DAPI (left) of a , sialic acids labelled with FITC-conjugated Wheat Germ Agglutinin (WGA), b, heparan sulfate and c , syndecan-4; box plots show mean ± SD, n = 4 microvessels per condition, each dots is a region of interest. Scale bars: 50 µm. d, Syndecan-4 shedding quantified by ELISA of 3D brain microvessel supernatants following exposure to 37 °C or 40 °C. e , Syndecan-1 shedding quantified by ELISA of 3D pulmonary microvessel supernatants. Graphs show mean ± SD, which each dot is from a different microvessel batch. Immunofluorescence MFI quantification of f , sialic acids labelled with WGA, and g , heparan sulfate in 3D pulmonary microvessels; box plots show mean ± SD, n = 4 microvessels per condition, each dots is a region of interest. Conditions in a-g include 37 °C or 40 °C incubation for 1 hour, and NA treatment for 30 min at 37 °C (1 U/mL). Statistical analysis was done by one-way ANOVA with Tukey’s multiple comparisons or pairwise comparisons were performed using the two-sided Mann-Whitney U test. h-i) HB3var03 and IT4var19 binding to 3D brain microvessels treated with NA (n = 5 microvessels) compared to 37 °C and 40 °C (n = 6 microvessels). Medians are shown as dots; error bars represent interquartile range. Statistical analysis by Kruskal-Wallis test for binned WSS regions (< 1 dyn/cm² vs ≥ 1 dyn/cm²; dotted line).

Article Snippet: The following antibodies were used: goat anti-human EPCR (5 μg/ml; R&D Systems, AF2245), followed by donkey anti-goat Alexa Fluor 647-conjugated secondary antibody (1:400; Invitrogen, A32849); PE-conjugated anti-human ICAM-1 (2 μl per 106 cells; Miltenyi Biotec, REA266, 130-120-711) and relative isotype control PE-conjugated human IgG1 (Miltenyi, 130-113-471); FITC-conjugated anti-human CD31 (5 μl per 105 cells; BD Pharmingen, 560984) and relative FITC-labelled mouse IgG1 (Abcam, ab18447); FITC-labelled anti-human CD36 (10 μl per 105 cells; Abcam, ab39022) and relative FITC-conjugated mouse IgG1 isotype control (2 μg/ml; Abcam, ab106163).

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Incubation, MANN-WHITNEY, Binding Assay