Cell culture and transfection

Normal retinal epithelial ARPE-19 cells (control) and the human Rb cell line Y79 were purchased from BeNa Culture Collection; Beijing Beina Chuanglian Biotechnology Research Institute. The human Rb cell line SO-Rb50 was obtained from Qincheng Biotechnology, the human Rb cell line Weri-Rb-1 was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and the human Rb cell line RBL-13 was purchased from the American Type Culture Collection. All cells were cultured in RPMI-1640 medium (American Type Culture Collection), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and maintained in a humidified incubator at 37°C with 5% CO₂.

Synthetic sequences of short hairpin RNA (shRNA) targeting FEZF1-AS1 (shRNA-FEZF1-AS1; Shanghai GenePharma Co., Ltd.) and non-targeting shRNA (shRNA-NC) were inserted into pGPU6/Neo vector (Shanghai GenePharma Co., Ltd.). 2×10⁴ cells/well were seed into a 24-well plate, then 0.8 µg shRNA was added into each well at confluence of 40–60% using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The miR-1236-3p mimic and negative control (NC; miR-NC mimic), miR-1236-3p inhibitor and miR-NC inhibitor were generated from Shanghai GenePharma Co., Ltd., 1×10⁵ cells/well were seed into a 6-well plate, 100 nM miR-1236-3p mimic/miR-NC mimic or 200 nM miR-1236-3p inhibitor/miR-NC inhibitor was transfected into Y79 cells at confluence of 40–60% using Lipofectamine^® 2000 reagent. Following 48 h of transfection at 37°C, the transfection efficiency was validated using reverse transcription-quantitative PCR (RT-qPCR). All sequences are listed in Table I.

Oligonucleotide sequences used for the transfection experiments.

miR, microRNA; NC, negative control; shRNA, short hairpin RNA; F, forward; R, reverse.