Benzopyrans

Cataract surgery was modeled in mice using a modified extracapsular lens extraction technique (ECLE) based on previously published techniques.^{32 (link)–34 (link, link)} Adult mice were anesthetized with 80 mg/kg ketamine and 5 mg/kg Xylazine. One eye of each mouse was dilated using several drops of topical phenylephrine and tropicamide. A 1- to 1.5-mm central corneal incision was made using a disposable ophthalmic knife. Following reinflation of the anterior chamber with an ophthalmic viscoelastic agent, a similarly sized incision was made in the anterior capsule. A viscoelastic cannula was used to instill saline into the capsular space to hydro-dissect the lens fiber mass away from the capsule. Angled jeweler forceps were used to remove the lens mass by applying gentle pressure near the equator of the eye. After the lens mass was expelled, careful irrigation of the capsule was performed to remove any residual lens material (in particular, the lens cortex). A viscoelastic agent was then injected into the capsule and anterior chamber to reinflate the eye and maintain its structural integrity postoperatively. The corneal incision was closed using 10-0 nylon sutures. Animals were euthanized 5 days postoperatively and lenses removed from both the surgical eye as well as the contralateral eye, which served as an experimental control. For analysis, whole eyes from ECLE experiments were preserved for immunofluorescence. In some cases, capsular bags were microdissected from the eye and used for isolation of RNA for PCR measurements. Surgery, euthanasia, and dissection were performed in parallel for each experiment to ensure consistency and comparability. Furthermore, the surgeon was blinded to the genetic strain of each mouse during operations. For experiments involving treatment with Sorbinil ([4S]-6-Fluoro-2,3-dihydro-spiro[4H-1-benzopyran-4,4′-imidazolidine]-2′,5′-dione), mice were injected intraperitoneally with 10 mg/kg at the time of surgery followed by twice daily injections on postoperative days 1 through 5. Sorbinil was provided by Pfizer Central Research (Groton, CT, USA). Vehicle controls contained 1X phosphate buffered saline.

ONIX B04A plates were loaded with medium and pre-warmed to 37 °C. Exponentially growing cells were incubated at 37 °C in fresh LB without shaking for 60 min before transitioning into experimental conditions and imaging. For spent medium assays, cells were perfused with spent LB subsequent to fresh LB. For oscillatory osmotic shock assays, cells were subjected to alternating hyperosmotic shocks of 400 mM sorbitol (Sigma-Aldrich, Cat. #S1876, dissolved in LB) and recovery without sorbitol, with a period of 2 min (1 min shock and 1 min recovery). For plasmolysis/lysis assays to measure OM stiffness, cells were subjected first to hyperosmotic shock with 1 M sorbitol in LB and then treated with 5% N-lauroylsarcosine sodium salt (MP biomedicals, Cat. #190289) to remove the OM. Cells were stained with 300 μM 3-[[(7-Hydroxy-2-oxo-2H-1-benzopyran-3-yl)carbonyl]amino]-D-alanine hydrocholoride (HADA, MedChemExpress, Cat. #HY-131045/CS-0124027) and perfused with 0.85X PBS prior to the osmotic shock.

HT29 cells were
seeded in 24-well plates and treated with chromenes for definite time
points. After the cellular treatments with chromenes, the cells were
visualized in inverted light microscopy (Motic, Hongkong) using a
20× objective, and the morphological changes were captured.