Fig. 5. The mechanisms of RAC1- and FOS-mediated cystogenesis.

(A) Scatterplots comparing expression profiles of PKHD1^−/− and PKHD1^+/− organoids under flow. Red and blue dots represent >1.5-fold up-regulated and down-regulated genes, respectively, in PKHD1^−/− organoids when compared to PKHD1^+/− under flow conditions. (B) Schematic illustration of RAC1- and FOS-mediated signals. (C) Heatmaps of microarray datasets for “negative regulation of cell population proliferation” (left) and “actin cytoskeleton” (right) in PKHD1^−/− and PKHD1^+/− organoids, cultured under flow, on day 35 of differentiation. (D) Schema of the culture and treatment protocol. (E) qPCR for RAC1 genes (ARFIP2 and BAIAP2) in green color and FOS genes (DUSP1, NPTX2, and PRDM1) in red color in PKHD1^−/− organoids treated with R-naproxen or T-5224 under flow conditions on day 23 of differentiation. Values are normalized against the control, PKHD1^−/− organoids cultured in static conditions. (F) Immunocytochemistry for ARFIP2 and PRDM1 in PKHD1^−/− organoids with and without flow and PKHD1^−/− organoids treated with R-naproxen or T-5224 treatment under flow conditions on day 23 (left). Scale bars, 20 μm. Percentage of ARFIP2⁺CDH1⁺ tubules/CDH1⁺ tubules in PKHD1^−/− organoids without flow, with flow, and with flow and R-naproxen (top right). Percentage of PRDM1⁺CDH1⁺ tubules/CDH1⁺ tubules in PKHD1^−/− organoids without flow, with flow, and with flow and T-5224 on day 23 of differentiation (bottom right). (G) Immunohistochemistry for F-actin and CDH1 in organoid tubules under static (left), flow (middle), and R-naproxen–treated flow conditions (right) on day 23. Scale bars, 20 μm. The plot profiles at the bottom show the intensity of F-actin across the tubule denoted by yellow arrows. a.u., arbitrary units. (H) Schematic illustration of RAC1- and FOS-mediated disease mechanism. Values shown are means ± SD. P values were determined by Student’s t test. *P < 0.05, **P < 0.01.