Fig 1: AP4S1 expression in ARPE-19 cells. AP4S1 relative mRNA levels, reported as 2^−ΔΔCt ± SD and quantized by using GAPDH as control along with AP4S1 protein levels, reported as pg/mL ± SD. N = 9 observations per group (three independent experiments, each performed in triplicate). ** p < 0.01 vs. NG; NG, normal glucose (5 mM) + vehicle; M, mannitol (30 mM); HG, high glucose (35 mM) + vehicle; OTX 2.5, OTX 2.5 µM; OTX 5, OTX 5 µM; OTX 10, OTX 10 µM.The up-regulation of AP4S1 was paralleled by a significant increase of Gal-1 mRNA (LGALS1) in HG cells (3.2 ± 0.3 2^−ΔΔCt, p < 0.01 vs. NG), which was not reverted by OTX treatments (2.5 µM: 3.0 ± 0.4 2^−ΔΔCt; 5 µM: 3.1 ± 0.2 2^−ΔΔCt; 10 µM: 3.4 ± 0.4 2^−ΔΔCt) (Figure 3A). Accordingly, Gal-1-positive cells in ARPE-19 exposed to HG (66 ± 10%; p < 0.01 vs. NG), which was absent in ARPE-19 cells exposed to NG alone (13 ± 4%) or with mannitol (19 ± 7%). HG cells stimulated with OTX008 5 µM and 10 µM exhibited a significant reduction in Gal-1-positive stained cells (OTX 5: 45 ± 4% and OTX 10: 41 ± 7%; both p < 0.01 vs. HG) (Figure 3B). Accordingly, Gal-1 protein levels were significantly increased in HG cells (Gal-1/β-actin = 1.01 ± 0.1, p < 0.01 vs. NG), while they were reduced in HG cells exposed to OTX008 5 µM (Gal-1/β-actin = 0.39 ± 0.04, p < 0.01 vs. HG) and 10 µM (Gal-1/β-actin = 0.42 ± 0.04, p < 0.01 vs. HG) (Figure 3C). OTX alone did not modify the Gal-1 expression in NG-treated ARPE-19 even at the highest concentration of 10 µM.