Figure 4.

Effect of constitutive clusterin expression on TM actin cytoskeleton. Adenovirus‐mediated constitutive expression of clusterin (AdCLU) in serum‐starved HTM cells compared to control (AdMT) significantly increased ‐ (a) clusterin mRNA expression, (b) clusterin protein levels in WCL, (c) in CM, and (d) cellular clusterin distribution (Alexa Flour 568/red) (bottom panel). GAPDH was used as a loading control for WCL, and Ponceau S‐band at ~63 kDa was used for the CM. The nucleus was stained with DAPI in blue. Images were captured in z‐stack in a confocal microscope, and stacks were orthogonally projected. Scale bar 20 microns. (e) Changes in the levels of actin cytoskeleton‐associated proteins due to AdCLU. Western blot analysis showed significant decrease in the actin‐associated proteins such as PRK2 and LIMK1, tight‐junction protein Zo‐1, membrane receptors‐ integrin‐ INTαV, INTβ5 in AdCLU compared to AdMT. GAPDH was used as a loading control for immunoblotting analysis. Values represent the mean ± SEM, where n = 4 (biological replicates). *p ≤ 0.05 was considered significant. CM, conditioned media; mRNA, messenger RNA; SEM, standard error of mean; TM, trabecular meshwork; WCL, whole‐cell lysate