Fig-8. Alterations of GSK3β / β-catenin are associated with the development of the glomerulocystic phenotype.

Fig-8 Representative pictures of renal cortex from 30 (A, B) and 50 days old (C, D) mice. Ctr (A, C) and Dicer cKO (B, D) sections were stained with anti β-catenin antibody. In Dicer cKO, β-catenin expression is limited to the basolateral domain in both tubules and parietal cells of the Bowman’s capsule, at both P30 and P50 (B-D). (A-D) Magnification63X, zoom1.5. Panel E and F show immunoblotting of Cortex/OSOM samples of 30 (E) and 50 (F) days old mice. GSK3β expression is already upregulatedcat the pre-cystic stage (P30) in Dicer cKO mice (E) and this is persistent at P50 (F) n power is 5 vs 5. Glomerular cyst formation is associated with loss of cytosolic β-catenin expression and general downregulation of cortical protein level (F). No significant changes were observed in the fraction of their main regulating phosphorylated forms. Panel G represent qPCR analysis of mRNA of β-catenin in 30 (right) and 50 days old mice (left). mRNA level of β-catenin is downregulated in Dicer-cKO mice at P50. Data are expressed as mean ± sem; * is for p value < 0.05; ** is for p-value <0.01.

图8 展示30日龄（A、B）与50日龄（C、D）小鼠肾皮质的代表性图像。对照组（A、C）与Dicer条件性敲除（Dicer cKO）组（B、D）的肾皮质切片均采用抗β-连环蛋白（β-catenin）抗体进行免疫染色。结果显示，在出生后30日龄与50日龄的Dicer cKO小鼠中（B-D），β-连环蛋白的表达均仅局限于肾小管与肾小囊壁层细胞的基底外侧区域。（A-D）图像的放大倍数为63倍，变焦系数为1.5。
图E与F分别展示30日龄（E）与50日龄（F）小鼠肾皮质/外髓质外带（OSOM）样本的免疫印迹结果。Dicer cKO小鼠在囊前阶段（出生后30日龄，图E）即出现糖原合成激酶3β（GSK3β）表达上调，且该上调趋势在出生后50日龄时仍持续存在（图F），两组样本量均为n=5。肾小球囊状形成与胞质β-连环蛋白表达缺失以及肾皮质总蛋白水平普遍下调相关（图F），而其主要调控性磷酸化形式的占比未出现显著变化。
图G为实时定量聚合酶链反应（qPCR）分析结果，其中右侧为30日龄小鼠样本，左侧为50日龄小鼠样本的β-连环蛋白mRNA检测数据。在出生后50日龄时，Dicer cKO小鼠体内β-连环蛋白的mRNA水平出现下调。
实验数据以均值±标准误（standard error of the mean, SEM）表示；*表示p值<0.05，**表示p值<0.01。