Eupatilin and ROCK inhibitors rescue ciliogenesis defects in NPHP1 URECs. (a) Control (1-56NC) and NPHP1 (1-12P) URECs grown in ciliogenesis conditions for 5 days at nonpermissive temperature (39 °C) were fixed and stained for primary cilia (ARL13B, green) and basal bodies (γ- tubulin, red). (b) Ciliogenesis in NPHP1 (1-12P and 1-03P) and CTL (1-56NC) URECs were quantified using Harmony software and expressed as percentage of ciliated cells shown using a box-and-whisker plot (n = 5–11 experiments). Mixed linear regression model with quasibinomial penalization taking into account the correlation of observations coming from the same individuals using a random effect on the cell line: ∗∗∗∗P < 0.0001. (c) Cilium length in NPHP1 (1-12P and 1-03P) and CTL (1-56NC) URECs length was quantified as explained in the Methods and expressed in μm using a dot-plot (n = 3 experiments). Unpaired 2-tailed t test: ∗∗∗P < 0.001. (d) NPHP1 URECs (1-12P) treated with either DMSO (0.04%) or 20 μM EUP for 48 hours were fixed and stained as in (a) for primary cilia (ARL13B, green) and basal body (γ-tubulin, red). (e) Ciliogenesis in 1.12P and 1.03P NPHP1 URECs treated for 48 hours with either alprostadil (Alpro), Eupatilin (EUP) or ROCK inhibitor (Y-27632) was expressed as the ratio to control DMSO-treated cells. Mean ± SEM (n = 4–7 experiments); paired 2-tailed t test: ∗∗∗∗ P < 0.0001, ∗∗∗ P < 0.001, ∗∗ P < 0.01, ∗ P < 0.05. (f and g) Ciliogenesis in URECs derived from CTL (1-56NC) and NPHP1 patients (1-12P and 1-03P) treated for 48 hours with DMSO or EUP (20 and 40 μM; n = 4–6 experiments per cell line; (f) or Y-27632 (25 and 50 μM; n = 2–5 experiments per cell line) was quantified similarly as in (b). ∗P < 0.05,∗∗ P < 0.01. Scale bars 10 μm. CTL, control; EUP, DMSO, dimethyl sulfoxide; Eupatilin; EUP, Eupatilin; ns, not significant; UREC, urinary renal epithelial cell.
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