Journal: bioRxiv

Article Title: Extra-ciliary role for polycystins in regulation of Ezrin and renal tubular morphology

doi: 10.1101/2025.11.05.684799

Figure Lengend Snippet: (A) Immortalized mouse Pkd1 fl/fl Pax8rtTA, TetO-Cre medullary renal epithelial cells = Pkd1 (M) (clone #313) treated with or without doxycycline then cultured in Matrigel for 14 days, with additional cultures of wildtype Pkd1 + (M) cells treated with 2μM of pan PKC inhibitor GO6983. (B-E) Semi-automated quantification from three separate experiments of tubuloid circularity (B) and (C) size (DMSO= Pkd1 +(M) n=273; PKC-inhib = Pkd1 +(M)+ 2μM GO6983 n=242) found significant differences in circularity and area in the structures treated with GO6983; +/− SEM, P<0.05, two tailed Student’s T-Test. (D,E) Comparison of the largest structures (e.g. structures indicated with black box) reveals a further enhanced difference in circularity (D) for the structures treated with GO6983; DMSO= Pkd1 +(M) n=88; PKC-inhib = Pkd1 +(M)+ 2μM GO6983 n=103; +/− SEM, P<0.05, two tailed Student’s T-Test. (F) The relationship between size and circularity in the largest structures ( Pkd1 +(M) and Pkd1 +(M) treated with 2μM GO6983), were analyzed with linear regression. Treatment with PKC-inhibitor abolished the relationship between circularity and size (PKC-inhib = Pkd1 +(M)+ 2μM GO6983 n=103; p<0.1517, R2=0.02024) from tubuloids similar to what was previously observed after Doxycycline treatment and treatment with Ezrin inhibitor NSC supporting a common targeted pathway. (G) At the completion of the 14 day experiment structures were removed from the Matrigel and Western blot was performed to confirm the effectiveness of the GO6983 to reduce the amount of phosphorylated PKC. Blot representative of three separate experiments confirmed a significant decrease in the levels of phospho-PKC in the 3D structures after treatment with GO6983. L.C. = loading control. (H-K) Representative merged images or separated montage of Immortalized mouse Pkd1 fl/fl Pax8rtTA, TetO-Cre renal epithelial cells, clone #313 ( Pkd1 (M)), cultured with vehicle (H, DMSO = Pkd1 +), doxycycline (K; DOX = Pkd1 -), or 2μM GO6983 (I,J; PKC-inhib = Pkd1 + 2μM GO6983) labeled with Ezrin 3145 (active form, green), Ezrin 3c12 (inactive, red), and ZO1 (purple). Repeated in three separate experiments, scale bar 50μm. Treatment with the PKC-inhibitor reduces apical active ezrin like treatment with DOX, but unlike DOX, the PKC-inhibitor causes accumulation of the active Ezrin near the junctions (K, inset, enlarged in J, white arrow), mirroring the inactive Ezrin and ZO1 localizations.

Article Snippet: In some experiments we used the ERM phosphorylation inhibitor compound NSC668394 (Sigma 341216) and the Pan PKC inhibitor GO6983 (Med Chem Express HY-13689) which were added directly to the media.

Techniques: Cell Culture, Inhibition, Two Tailed Test, Comparison, Western Blot, Control, Labeling