*2.6. Cells and Reagents*

ARPE-19 cells stably expressing S179C-TIMP3, wild-type-TIMP3 (WT), or vector alone were reported previously [19]. Cells were expanded in DMEM-F12 with 10% FBS before transfer to polyester inserts coated with mouse laminin (Corning Inc., Corning, NY, USA, #23017). 720,000 cells and 100,000 cells were plated per well in each well of a 12-well plate or 24-well plate, respectively using a previously published protocol [36]. Essentially, ARPE-19 cells were cultured for at least 2 weeks in nicotinamide-supplemented media with 1% FBS. Media were replaced twice per week. Cells were serum-starved for 24 h before treatment with the FGF Receptor inhibitor BGJ-398 (Selleckchem, Houston, TX, USA, #S2183) for 48 h. Similarly, cells were treated with FGF-2 (Gibco from Thermo

Fisher Scientific, #13256-029) with the required cofactor heparin sodium salt (1 μg/mL, Sigma Aldrich, #H3149) for 48 h after serum starving for 24 h.

### *2.7. Quantitation of Immunofluorescence by Integrated Density Analysis*

Fluorescence intensity of HABP staining was quantified using integrated density analysis as previously described [37,38]. For all the RPE cell culture confocal microscopy images, fluorescence was quantitated using a standard measure of integrated density, which is the product of area and mean gray value. A custom written automated image analysis code was developed using Matlab (MATLAB 2019a, The MathWorks, Inc., Natick, MA, USA) for separating the desired color channel from the image, thereby obtaining the total area (in pixels), the mean gray value, and the integrated density.

### *2.8. In Vivo Imaging and Laser Injury Model*

Laser mediated CNV was induced as described previously [28]. Briefly, mice were anesthetized with 65–68 mg/kg sodium pentobarbital delivered intra-peritoneally. Topical 0.5% procaine solution was applied for cornea anesthesia. Following anesthesia, pupils were dilated with 0.5% topical tropicamide/phenylephrine combination drops (Santen Pharmaceuticals, Osaka, Japan).

Four laser spots were placed in the superior, superior-temporal, or superior-nasal quadrants of the fundus using a green solid-state laser (Oculight by Iridex Corp., Mountain View, CA, USA) (532 nm; 2500 mW; 0.50 s pulse duration; 50 μm spot size) using a slit lamp delivery system and a microscope coverslip placed and a ffixed to the cornea with a drop of Systane Ultra artificial tears (Alcon, Ft Worth, TX, USA). All animals were scanned immediately after laser injury with optical coherence tomography (Envisu R2210 UHR Leica Microsystems Inc., Wetzlar, Germany) to confirm successful RPE-Bruch's membrane rupture, an endpoint in laser-induced CNV models.
