Rtegm bulletkit

Human foetal retinal pigment epithelium (fRPE) cells were acquired from Lonza (Ref. 00194987, lot number 0000465423) and cultured according to the manufacturer’s protocol in RtEGM^®Bulletkit^® (Ref. 00195409, Lonza). Human iCell induced pluripotent stem cell-derived RPE (iCell RPE) cells were acquired from StemCell (Line 01279, Ref. C1047, lot103261) and cultured in RDMsA. For any assay performed on fRPE or iCell RPE, the cells were used at P3 and grown on MRF-coated transwells (CLS3460, Sigma-Aldrich) for 42 days in RDMsA (unless stated otherwise). All tests were performed with the same fRPE or iCell RPE line and, for fRPE, several maturation tests were done independently.

293T cells and 3T3-L1 preadipocytes were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and Penicillin (10,000 units/ml)/ Streptomycin (10,000μg/ml, 1:100 dilution of stock, Gibco #15140–122). After reaching confluency, 3T3-L1 pre-adipocytes were cultured for 48 hours in DMEM + 10% FBS. The culture medium was then replaced by DMEM + 10% FBS + 5 μg/ml insulin (Sigma, I0516) + 1 μM dexamethasone (G-bioscience, API-04) + 0.5 mM isobutylmethylxanthine (Sigma, I5879)] to induce adipocyte differentiation. After 48 hours, the medium was replaced by DMEM + 10% FBS + 5 μg/ml insulin for an additional 48–72 hours to achieve complete differentiation. To induce lipid droplet formation, cells were treated with 200 μM of oleic acid-albumin from bovine serum (Sigma, O3008). Human fetal retinal pigment epithelial cells (hfRPE, Lonza, #00194987) were cultured in RtEGM with supplement medium as indicated by the manufacturer’s protocol (RtEGM bullet kit, Lonza, #00195409). HfRPE cells were cultured to high confluence on coverglass culture plates (Thermo, 155411) for three weeks to obtain polarized RPE monolayers. After differentiation, hfRPE cells were treated with 20μM A2E (N-Retinylidene-N-Retinylethanolamine, 20mM stock dissolved in DMSO, Gene and Cell Technologies) for 24 hours.

Human primary retinal pigmented epithelial (RPE) cells (Lonza, Walkersville, MD) were cultured in RtEGM^™Bulletkit^® media as per manufacturer’s instruction (Lonza). Vero cells (ATCC CCL-81) and Aedes albopictus clone C6/36 cells (ATCC CRL-1660) were cultured in DMEM, and EMEM media supplemented with 10% FBS, 10 µg/ml L-glutamine, and 1% penicillin and streptomycin solution as per manufacturer’s recommendation. ZIKV Virus (ZIKV) strain PRVABC59, NR-50240, originally isolated from human blood in Puerto Rico in December 2015, and DENV type 2 strain NR12216 were obtained through BEI Resources, NIAID, NIH. This ZIKV strain is 97–100% genetically similar to the current ZIKV strain circulating in Brazil^{57 (link)}. ZIKV and DENV were propagated in ATCC CCL-81 Vero cells and ATCC CRL-1660 C6/36 cells respectively, and titers were determined by plaque assay. RPE cells were infected with ZIKV and DENV at MOI of 1 for the indicated time points as described previously⁷ . For ABCG1 functional studies, Pr. RPE cells were treated with a pharmacological inhibitor of ABCG1, Benzamil (50 µM) (Tocris Biosciences, Minneapolis, MN), and cholesterol-water soluble (10 µM) (Sigma Aldrich, St Louis, MO) 1 h before ZIKV challenge.

RPE cells from VMD2-Cre;Vhl^fl/fl and control Vhl^fl/fl littermates were isolated as previously described (Krohne et al., 2012 (link)). Cells were maintained at 37° and 5% CO₂ in DMEM/F12 from Thermo Fisher Scientific (Waltham, MA) with 2% FBS from Jackson ImmunoResearch (West Grove, PA). 2 μg/μl doxycycline was added to all cultures daily for three days to induce Vhl ablation. Human RPE cells (Lonza) were maintained either on plastic surfaces or on transwell filters (Corning) depending on the application in RtEGM Retinal Pigment Epithelial Cell Growth Medium fortified with RtEGM BulletKit from Lonza (Basel, Switzerland). Glucose, lactate, and glutamine levels were measured from the media in apical and basal compartments using a 2900 Biochemistry Analyzer from YSI (Yellow Springs, OH). A concentrated stock of DMOG from Cayman Chemical (Ann Arbor, MI) was made with DMSO, and added directly to the media in different concentrations to induce pseudo-hypoxia. RPE cells were maintained in low oxygen and metabolic changes were analyzed using Seahorse Flux Analysis (see below) in a Coy Dual Hypoxia Chamber from Coy Lab Products (Grass Lake, MI) (Grassian et al., 2014 (link)).

ARPE-19 cells (CRL-2302) were purchased from ATCC (Manassas, VA, USA) and primary human fetal retinal pigment epithelial cells, H-RPE (00194987) were from Lonza (Basel, Switzerland). DMEM:F12 (11320–033) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). RtEGM BulletKit (00195409) which is a culture system containing RtEBM Basal medium (00195406) and RtEGM Single Quots Supplements (00195407) was purchased from Lonza. ARPE-19 cells were cultured in DMEM:F12 medium containing 100 U/ml penicillin and 100ug/ml streptomycin supplemented with 10% FBS. H-RPE cells were cultured in RtEBM containing Single Quots supplements with the addition of 2% FBS for the first 24hrs or serum free thereafter. Cells were cultured as described until they reach confluent state. Upon confluence, serum was depleted for 24 hours and cells were treated appropriately for another 24 hours. Acadesine was added 1hour before TNF-α and 5-IODO or DPY were added 1 hour prior to Acadesine. Experiments were performed on ARPE-19 at passage 6–9 and on H-RPE at passage 3–4.

H9 human embryonic stem cells (hESCs -WiCell, Madison, WI) were differentiated to RPE cells using the previously published protocol^{76 (link)} and frozen for future use. To generate H9-RPE cell lysate, cells were thawed and plated as 5 million cells per well of a 6 well plate in RPE media (RtEGM™ BulletKit®, Cat # 00195406 and 00195407, Lonza, Basel, Switzerland). Cells were fed with fresh media daily until they formed a confluent monolayer and then they were fed with RPE media without FBS every other day for 3 months until cell lysis for protein analysis.