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. 2018 Mar 1;34(1-2):40–51. doi: 10.1089/jop.2017.0023

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© Cheng-Wen Lin et al. 2018; Published by Mary Ann Liebert, Inc.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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FIG. 2. — Disruption of actin stress fibers and focal adhesions by netarsudil versus other ROCK inhibitors. (A) Netarsudil dose-response in actin stress fiber assay. Primary PTM cells were incubated for 6 h in the presence of 0, 0.015, 0.138, or 1.2 μM netarsudil then fixed and stained with Alexa Fluor-488 phalloidin and Hoechst 33342 to reveal actin fibers and nuclei, respectively. Top panel: fluorescence images of stained cells. Bottom panel: False color images created by an automated, custom algorithm to identify stress fibers and calculate mean stress fiber length. (B) Netarsudil dose-response in focal adhesion assay. Immortalized HTM cells (TM-1) were incubated for 6 h in the presence of 0, 0.015, 0.138, or 1.2 μM netarsudil then fixed and stained with mouse anti-paxillin antibody/Alexa Fluor^®488 goat-anti-mouse IgG and Hoechst 33342 to reveal focal adhesions and nuclei, respectively. Top panel: fluorescence images of stained cells. Bottom panel: False color images created by an automated, custom algorithm to identify focal adhesions and calculate the mean number of focal adhesions per cell. (C) Dose–response curves (n = 4) for netarsudil, netarsudil-M1, AR-12286, Y-27632, and fasudil in the PTM actin stress fiber length assay. Mean stress fiber length is presented as a percentage of the mean length of stress fibers measured in untreated control cells. (D) Dose–response curves (n = 4) for netarsudil, netarsudil-M1, AR-12286, Y-27632, and fasudil in the HTM focal adhesion assay. Mean number of focal adhesions per cell is presented as a percentage of the number of focal adhesions per cell measured in untreated control cells. HTM, human trabecular meshwork; PTM, porcine trabecular meshwork; ROCK, Rho-associated protein kinase.