Oleic acid

The human RPE cell line, ARPE‐19 that was purchased from Procell Life Science&Technology Co.,Ltd (CL‐0026; China), was cultured in DMEM/Ham's F‐12 containing 10% FBS and 100 U/mL penicillin in a humidified incubator with a 5% CO₂ atmosphere at 37°C. After reaching 80% confluence, ARPE‐19 cells were dissociated using 0.25% trypsin for subsequent experiments. Oleic acid (HY‐N1446; MedChemExpress, China) was dissolved in DMSO at 1000 mM and stored at −80°C. For working medium preparation, an Oleic acid solution was added into the prewarmed medium and was incubated under shaking for 6 h at 37°C. ARPE‐19 cells were treated with Oleic acid (100, 250, 500, 1000 µM) for 24 h to induce a lipid‐overload model (Chang et al., 2020 (link)). The condition of 250 µM Oleic acid for 24 h was selected for treating ARPE‐19 cells. ARPE‐19 cells were washed with PBS to remove Oleic acid before adding EVs to the medium. Different concentrations of EVs (1 × 10⁸ EVs/mL, 1 × 10⁹ EVs/mL, 1×10¹⁰ EVs/mL) were used to treat the injured ARPE‐19 cells.

TCN was provided by Kunming Institute of Botany, the Chinese Academy of Sciences (purity > 99%, HPLC analysis) as previously described [41 (link)] (Fig. 4a). Dimethyl sulphoxide (DMSO, Sigma) was used to dissolve TCN. The final concentration of DMSO in the culture media was kept less than 0.1% v/v which had no significant effect on the cell growth. Elaidic acid and Oleic acid were purchased from MedChemExpress (NJ, USA).
The antibodies against β-actin and CDK4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies against RB, pRB and Ki67 were obtained from Cell Signaling Technologies (Danvers, MA, USA). The antibody to detect DHRS2 (PA5–25258) was from Thermo Fisher Scientific. Anti-cyclin D1 was from Bioworld Technology.
pEZ-Lv105-DHRS2 was from GeneCopeia. The open reading frame of DHRS2 gene was inserted into the lentivirus vector pEZ-Lv105 using Gateway® recombination technology.