Autotaxin and IOP Figure 8. Suppression of ATX expression in human TM cells induces changes in actin cytoskeletal organization and myosin II phosphorylation. A. Primary cultures of human TM cells treated with ATX-specific siRNA revealed significant suppression of ATX expression (different isoforms) relative to cells treated with FITC-conjugated scrambled siRNA control. b-tubulin was used as a loading control. B. Protein levels of one of the variants (,125 kDa) of ATX were significantly reduced (by .75%) in ATX siRNA treated HTM cells based on densitometric semi- quantification. C. The ATX siRNA treated human TM cells (after 72 hrs) maintained under serum starved conditions for 24 hrs revealed a marked decrease in actin stress fibers (labelled using rhodamine-phalloidin) relative to cells treated with scrambled siRNA (FITC-siRNA). D. ATX siRNA treated human TM cells (for 72 hrs) maintained under serum starved conditions for 24 hrs showed a significant decrease in the levels of phosphorylated myosin light chain (pMLC), a regulatory subunit of myosin II, as compared to cells treated with scrambled siRNA (FITC-siRNA). b-tubulin was blotted as a loading control. E. Changes in the levels of phospho-MLC in the ATX siRNA treated cells compared to the scrambled siRNA treated control, based on densitometric semi-quantitative analysis. doi:10.1371/journal.pone.0042627.g008 intracameral injections. Importantly, in this case, drug was dissolved initially in DMSO and subsequently diluted in phosphate buffered saline, pH 7.4. Using this method, there was no evidence for drug precipitation or ocular hyperaemia, and IOP was found to be significantly decreased in drug treated eyes by more than 5 mm Hg, with the ocular hypotensive response lasting for more than 48 hrs in response to a single injection of 5 ml of 100 mM S32826. The final concentration of drug in the AH was estimated be in the range of 2 to 2.5 mM based on the volume of AH (approximately 200 to 250 ml) in the adult rabbit eye. Although this experiment provided convincing evidence for the ocular hypotensive effect of S32826, and the ability of S32826 to inhibit LysoPLD activity in rabbit AH was confirmed using an in vitro assay, we could not validate that the ocular hypotensive response of S32826 was indeed associated with decreased levels of LPA. Our preliminary attempts to determine levels of LPA in the limited amounts of AH obtained from rabbit eyes by mass spectrometric analysis were not successful. Further work is needed to establish protocols for determining LPA levels in AH. However, S32826 has been shown to decrease LPA levels produced by adipocytes [29]. Finally, siRNA-mediated suppression of ATX expression in TM cells resulted in a decrease in actin stress fibers and myosin light chain phosphorylation, indicating a role for ATX in cell contractile properties, cell tension and cell adhesive properties of TM cells. Consistent with these observations, our previous studies demonstrated that LPA, which is a direct product of ATX activity, PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42627