PDZK1 Protects Against RPE Senescence by Targeting the 14-3-3ε-mTOR Axis to Attenuate Early Diabetic Retinopathy

In diabetic retinopathy (DR), hyperglycemia induces retinal pigment epithelium (RPE) cell senescence via downregulation of PDZK1. PDZK1 overexpression counteracts senescence by binding 14-3-3ε to modulate mTOR signaling, reducing oxidative stress and enhancing autophagy. Both genetic PDZK1 restoration and pharmacologic senolysis (Nutlin-3a) attenuate retinal senescence and ameliorate early DR pathology, establishing the PDZK1-14-3-3ε-mTOR axis as a therapeutic target Cells are maintained in DMEM with 10 % fetal bovine serum and 1 % penicillin–streptomycin at 37 °C in a humidified 5 % CO₂ incubator. At approximately 50 % confluence, cultures are transduced with PDZK1-overexpressing or control lentivirus (Tsingke) at MOI 10. After 48 h of transduction, 4 μg/ml puromycin is added for stable selection. Control and PDZK1-overexpressing cells are then exposed to 37.5 mM d-glucose for 48 h, after which total RNA is extracted for downstream analysis.