Fig. 5

Alteration of ureter markers and calcium imaging in Ahnak KO mice at PN1. (A) p63 marks basal cell layer and multilayered basal cell layer is detected in WT ureter. (D) In Ahnak KO ureter, p63 positive mono basal cell layer and expanded lumen is observed at PN1. (B) Uroplakin 1b (Upk1b), a urothelium marker, and E-cadherin double-staining results show that multilayered urothelium is developed at PN1 in WT. (E) Upk1b and E-cadherin are weakly localized in Ahnak KO at PN1. (C) Thick α-SMA-positive muscularis layer is observed in WT ureter at PN1. c-kit, interstitial cells of Cajal-like cells (ICC-LCs) marker, is localized adjacent to the smooth muscle layer in WT ureter. (F) c-kit and α-SMA are weakly or not localized around thin smooth muscle layer in Ahnak KO mice. (G) Expression level of p63 is reduced in Ahnak KO ureter compared to WT at PN1. (H) Upk1b expression level is decreased in Ahnak KO ureter compared with WT. (I) Relative expression level of c-kit is down-regulated in Ahnak KO ureter compared to WT at PN1. (J) Adrb1 expression level is reduced in Ahank KO compared to WT. (K) Expression level of Cacna1d is decreased in Ahank KO ureter compared with WT. (L) α-SMA expression level is lower in Ahnak KO ureter compared to WT ureter at PN1. (M) Upk1b and α-SMA protein level is down-regulated in Ahnak KO mouse ureter at PN1. (N) Volcano plot presenting down-regulated genes related to muscle tissue development, such as Adrb1 and Cacna1d in Ahnak KO ureter. (O, P) The green fluorescent protein (GFP) response by Fluo-4 is significantly increased in WT ureter smooth muscle cells after CaCl₂ treatment. (Q, R) GFP response by Fluo-4 is not altered in Ahnak KO ureter smooth muscle cell after CaCl₂ treatment. (S) GFP intensity is dramatically up-regulated in WT but not Ahnak KO ureter smooth muscle cells until 100 s after CaCl₂ treatment. (T) GFP intensity of ureter smooth muscle cells ((max-min)/max) is much higher in WT compared to Ahnak KO (n = 10). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant; Scale bar, A-F, 100 μm, O-R, 20 μm