Figure 6
From: Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness
Treatment with ROCK inhibitor Fasudil amiliorates the cystic phenotype. (a) Phosphorylation of MLC in activatory sites Thr18/Ser19 were tested by Western blot on kidney lysates of Pkd1flox/−: KspCre mice at P8 after boost treatment with ROCK inhibitor Fasudil or vehicle. Quantification of pMLC/MLC ratio in three independent experiments is in Supplementary Fig. 1. Bands were cropped from different parts of different gels. Full-length blots are presented in Supplementary Fig. 2. (b) IHC staining for YAP on Pkd1flox/−: KspCre kidney at P8 after boost treatment with ROCK inhibitor Fasudil or vehicle. Aperio image analysis software mask for the semi-automated quantification of YAP staining. Scale bar = 50 μm. (c) Percentage (%) of nuclear YAP localization quantified as strong positive by the semi-automated Aperio image analysis software on IHC slides of Pkd1flox/−: KspCre kidneys at P8 after boost treatment with ROCK inhibitor Fasudil or vehicle (n = 3 cystic fasudil and n = 3 cystic vehicle treated mice). Values are mean ± SEM of forty five cysts (1715 nuclei counted for vehicle; 2003 nuclei counted for Fasudil). Statistical analysis: Student’s unpaired one-tailed t-test. ****p < 0.0001. (d) qRT-PCR analysis for YAP targets: Ctgf, Ankrd1 and Cyr61 mRNA normalized towards Hprt1 on Pkd1flox/−: KspCre kidneys at P8 after boost treatment with ROCK inhibitor Fasudil or vehicle (n = 5 cystic fasudil and n = 5 cystic vehicle treated mice). Values are mean + SEM. Statistical analysis: ordinary One-way ANOVA; **p < 0.01, ****p < 0.0001 (e) Kidney/body weight of Pkd1flox/−: KspCre mice and Pkd1flox/+: KspCre controls after three day-treatment with ROCK inhibitor Fasudil or vehicle. Values are mean ± SEM of at least 6 animals. Statistical analysis: Student’s paired two-tailed t-test; **p < 0.01. (f) Percentage (%) of both normal tubules and cysts on total stuctures on sildes of Pkd1flox/−: KspCre kidneys at P8 after 3-day treatment with ROCK inhibitor Fasudil or vehicle (n = 3). Values are mean ± SD of at least 1000 cortical structures counted per kidney. Statistical analysis: Student’s paired one-tailed t-test; *p < 0.05. (g) Percentage (%) of Ki67 staining quantified by semi-automated Aperio image analysis software on IHC slides of Pkd1flox/−: KspCre kidneys at P8 after boost treatment with ROCK inhibitor Fasudil or vehicle (n = 3). Values are mean ± SEM of 30 cysts (1904 nuclei counted for vehicle; 1709 nuclei counted for Fasudil). Statistical analysis: Student’s unpaired one-tailed t-test; **p < 0.01. (h) Percentage (%) of apoptosis (TUNEL positive cells) quantified by immunofluorescence in slides of Pkd1flox/−: KspCre kidneys at P8 after boost treatment with ROCK inhibitor Fasudil or vehicle (n = 3 cystic fasudil and n = 3 cystic vehicle treated mice). Values are mean ± SEM of 58 cysts (2580 nuclei counted for vehicle; 2497 nuclei counted for Fasudil). Statistical analysis: Student’s unpaired one-tailed t-test; ns. non significant.
