Differential impact of EBOV and MARV on cAMP-mediated function in human gut organoids reveals region-specific loss of forskolin-induced swelling in HCOs.
<p>(A) iPSC-derived HIOs were infected with EBOV, EBOV-ZsGreen, or MARV at an MOI of 10 on day 35 of differentiation. At 1 dpi, HIOs were treated with 5 µM forskolin dissolved in DMSO or treated with DMSO and imaged immediately prior to treatment (0 hours, 1 dpi), and then at 24 hours (2 dpi),...
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2025
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| Shrnutí: | <p>(A) iPSC-derived HIOs were infected with EBOV, EBOV-ZsGreen, or MARV at an MOI of 10 on day 35 of differentiation. At 1 dpi, HIOs were treated with 5 µM forskolin dissolved in DMSO or treated with DMSO and imaged immediately prior to treatment (0 hours, 1 dpi), and then at 24 hours (2 dpi), and 48 hours (3 dpi) post-treatment. Time points on the left indicate days post-infection, and time points on the right indicate hours post forskolin treatment. Yellow arrows indicate organoids exhibiting visible swelling responses. (B) iPSC-derived HCOs were infected with EBOV or MARV at an MOI of 10 on day 35 of differentiation. At 1 dpi, organoids were treated with 5 µM forskolin dissolved in DMSO, with DMSO-treated organoids serving as vehicle controls. Organoids were imaged at 0-, 24-, and 48-hours following treatment, corresponding to 1, 2, and 3 dpi, respectively. Time points on the left of the panel indicate days post-infection, while those on the right indicate hours post-forskolin treatment. <b>(C)</b> Automated imaging analysis of iPSC-derived HIOs, before (top) and after forskolin stimulation (bottom), was performed using OrganoSeg software to quantify changes in cross-sectional area (CSA). <b>(D)</b> Quantification of forskolin-induced swelling in HIOs and HCOs. Spheroid number and CSA were calculated using automated analytical software and normalized CSA was used to assess the mean change in organoid size following forskolin treatment. Quantification of forskolin-induced swelling is presented across three independent differentiations and three separate wells per experimental condition. Images were captured using the EVOS M5000 Imaging System, with brightfield and fluorescence imaging at 25 ms exposure. Scale bars represent 100 µm. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed in GraphPad Prism using two-way ANOVA with Tukey’s multiple comparisons test (95% confidence interval). Asterisks indicate statistically significant differences compared to the corresponding mock-infected controls (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.1; ns, not significant).</p> |
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