Fig 2: ZO-1 staining for BRB integrity. The representative images display ZO-1 staining in the RPE layer at day 3 after rAION induction. The ZO-1 was labeled with FITC (green), and the nuclei were stained with DAPI (blue). Scale bar: 50 µm. BRB, blood-retinal barrier; DAPI, 4, 6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; rAION, rodent model of anterior ischemic optic neuropathy; RPE, retinal pigment epithelium; ZO-1, zona occludens 1.

Fig 3: Metformin counteracts the increased susceptibility to dedifferentiation in L-ORD-iRPE.a, b Representative immunofluorescent images of the membrane marker ZO-1 (green) in healthy (a) and L-ORD-iRPE (b) following 7 consecutive days of POS uptake. Scale bar 20 µm. c The effect of POS uptake on the expression of dedifferentiation-related genes in L-ORD-iRPE compared to healthy-iRPE. A dashed line indicates a fourfold difference. Housekeeping genes: ACTB and GAPDH. d Concurrent treatment of L-ORD-iRPE with metformin (3 mM) on POS-induced increase in cell size (ZO-1, green) after 7 days of POS uptake. Scale bar 20 µm. e Quantification of cell area after 7 days of POS uptake and metformin (3 mM) treatment in L-ORD-iRPE. Cells were labeled with anti-ZO-1 antibody and area was quantified using an AI-based algorithm80, low whisker: 5% of data, low hinge: 25% of data, midline: median, high hinge: 75% of data, high whisker: 95% of data. (n = 6 images). f Expression of 31 dedifferentiation-related genes in metformin-treated (magenta) L-ORD-iRPE (fed POS for 7 days) compared to untreated cells. A dashed line indicates a fourfold difference. Housekeeping genes: ACTB and GAPDH. g Apically secreted beta-hydroxybutyrate (ß-HB) in L-ORD-iRPE after 1 week of metformin treatment. Cells were supplied with a ß-HB metabolic substrate, BSA-palmitate conjugate, for 3 h before measuring ß-HB levels (n = 12). h Secreted NPD1 in untreated (n = 8) and metformin-treated L-ORD-iRPE (n = 9) measured by tandem mass spectrometry lipidomic analysis. Cells were POS-fed for 24 h prior to media collection. *p < 0.05, **p < 0.01, ***p < 0.001.