Fig. 5: NPD827 alters membrane dynamics and perturbs multi-drug efflux. | Nature Communications

Fig. 5: NPD827 alters membrane dynamics and perturbs multi-drug efflux.

From: Targeting fungal membrane homeostasis with imidazopyrazoindoles impairs azole resistance and biofilm formation

Fig. 5: NPD827 alters membrane dynamics and perturbs multi-drug efflux.The alternative text for this image may have been generated using AI.

a FM4-64 staining of C. albicans (SN95) following treatment as indicated. Assays were performed in biological triplicate. Scale bar, 5 µm. b Fluorescent microscopy of Cdr1-GFP cells following treatment as indicated. Assays were performed in biological triplicate. Scale bar, 5 µm. c Quantification of cultures shown in b by flow cytometry. Bars represent median fluorescence intensity, normalized to untreated cells, as quantified by flow cytometry for at least 20,000 events. Data are expressed as mean ± SD for biological triplicates. p-value, compared to untreated: 0.0003 (NPD827 5 µg/mL); 0.0005 (NPD827 10 µg/mL); 0.0002 (Combination). Significance determined by by one-way ANOVA with Bonferroni’s multiple comparisons test, where all conditions were compared to wild-type, untreated control, p-value: **<0.01. d For Rhodamine-6G assays, C. albicans cells were treated with NPD827 (10 µg/mL) before staining with rhodamine-6G (1 µg/mL). Assays were performed in biological duplicate. Bars represent median fluorescence intensity, normalized to untreated cells, as quantified by flow cytometry for at least 20,000 events. Data are expressed as mean ± SD for biological triplicates. p-value: 0.0382 (NPD827 10 µg/mL). Significance determined by a two-tailed unpaired t test with Welch’s correction, p-value: *<0.05. e Relative intracellular concentrations of FLC were measured after treatment with NPD827 for 1 h. Data are presented as mean ± SD of technical triplicates (n = 3). Significance was determined by one-way ANOVA with Bonferroni’s multiple comparisons test, where all conditions were compared to wild-type, untreated control. p-value: ***<0.0005. Significance between the drug combo and FLC by one-way ANOVA, p-value <0.0001. p-value, compared to untreated: <0.0001 (FLC 0.25 µg/mL, 16 µg/mL, Combination). p-value, compared to NPD827 5 µg/mL: <0.0001 (Combination). p-value, compared to FLC 0.25 µg/mL: <0.0001 (Combination). f FRAP experiments with GFP-Ras1 C. albicans. GFP-Ras1 cells were incubated without or with 5 μg/mL NPD827 for the indicated duration before performing laser scanning confocal microscopy. Images were taken prior to and after photobleaching, and fluorescence recovery observed (10–90 s). Fluorescence recovery curves are shown for >25 cells per condition. g Total net isotherms from ITC interrogating amphotericin B (AmpB) and NPD827 binding to cholesterol, lanosterol, or ergosterol-containing, or sterol-free POPC LUVs. Data are presented as mean ± SD of technical triplicates. Significance was determined by unpaired t-test between each sterol-containing preparation and the sterol-free preparation and also for NPD827 between lanosterol- and ergosterol-containing conditions, p-value: *<0.05, **<0.005. p-value, compared to POPC only: AmpB, 0.0016 (Cholesterol); 0.0037 (Ergosterol); NPD827, 0.0243 (Cholesterol); 0.0137 (Ergosterol); 0.0065 (Lanosterol). p-value, compared to lanosterol: NPD827, 0.0471 (Ergosterol). Source data are provided as a Source Data file.

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