Arpe 19

Human RPE cell line, ARPE-19 (China Center for Type Culture Collection), was incubated in an incubator with 95% O₂ and CO₂ at 37°C and cultured in DMEM/F-12 (DF-12; Gibco, Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, USA). Cells were divided into three groups: low glucose normal control (5.5 mM, NG; DMEM, 11885092, Thermo Fisher Scientific, USA), high glucose (25 mM, HG; DMEM, 11965092, Thermo Fisher Scientific, USA), and mannitol negative control (5.5 mM glucose plus 20 mM mannitol, MG). To inhibit NRF2 expression, cells were exposed to 5 μM ML385 for 24 h.
To study the role of CKIP-1 in HG-induced ARPE-19 cells, CKIP-1 was overexpressed in ARPE-19 cells by transfection with pcDNA3.1-CKIP-1 using Lipofectamine 3000 (Invitrogen) for 48 h. The ORF sequence of human CKIP-1 (GenBank No. NM_016274) cDNA was amplified by RT-PCR and subsequently cloned into vector pcDNA3.1 (Invitrogen, USA). The pcDNA3.1-empty vector plasmids were transfected as negative control.

The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (Manassas, VA, USA). ARPE-19 cells were cultured in a 1:1 mixture of Dulbecco’s Modified Eagle Medium (DMEM) and nutrient mixture F-12 Ham (Invitrogen-Gibco, Grand Island, NY, USA), which contained 4 mM L-glutamine, to which 10% fetal bovine serum (FBS; Invitrogen-Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St Louis, MO, USA) were added. The cells were kept in a humidified environment of 5% CO2 at 37°C, and the medium was exchanged twice a week.
ARPE-19 cells were transfected with miR-4516 or miR-negative control (miR-NC; Life Technologies, Carlsbad, CA, USA) for 24 hours, and then with or without 10 ng/mL human recombinant TGF-β2 (PeproTech, New York, NY, USA) for 24 hours extra. Unless otherwise stated, all experiments were performed under serum-free conditions.

A human RPE cell line, ARPE-19, was obtained from the American Type Culture Collection (Manassas, VA, USA). The ARPE-19 cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium (DMEM) and Nutrient Mixture F-12 Ham (Invitrogen-Gibco, Grand Island, NY, USA) with 4 mM L-glutamine supplemented with 10% fetal bovine serum (FBS; Invitrogen-Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St Louis, MO, USA). The cells were maintained at 37°C in a humidified environment with 5% CO₂, with media exchange performed twice a week.
The miRNA mimics [miR-1285 and negative control (NC)] and inhibitors (miR-1285 and NC) were purchased from Life Technologies (Carlsbad, CA, USA). ARPE-19 cells were transfected with miRNA mimics or miRNA inhibitors for 24 hours, followed by treatment with or without 10 ng/mL human recombinant TGF-β2 (PeproTech, New York, NY, USA) for an additional 24 hours. The transfection of either siRNA or miRNA (mimic/inhibitor) was accomplished using Lipofectamine 2000 (Thermo Fisher Scientific), according to the manufacturer’s instructions.