Fig 2: IRS-1/PI3K/AKT signaling pathway is suppressed by inhibition of NPTX1. (A and B) The expression of p-IRS-1, PI3K and p-AKT was significantly suppressed after transfection with NPTX1 siRNA, which inhibited the expression of NPTX1 at the translational level. However, the expression of IRS-1 and AKT was significantly increased in the NPTX1 siRNA group in comparison with that in the NC group. (C) After NPTX1 siRNA was transfected into the cultured U251 cells, the expression of miR-128-3p in the NPTX1 siRNA group was not significantly different from that in the NC group. *P<0.05, **P<0.01, ***P<0.001. NPTX1, neuronal pentraxin 1; miR, microRNA; siRNA, small hairpin RNA; NC, negative control.

Fig 3: Apigenin and galangin antagonize against blue light by regulating MAPKs and MST-1/2-Akt-FoxO3a signaling. (A,B) Apigenin and galangin regulate phosphorylation of MAPKs in blue light-irradiated HaCaT cells. (A) Phosphorylation levels of MAPKs were determined by Western blotting at 30-, 60-, and 90-min incubation after, pretreatment with apigenin (30 µM) or galangin (20 µM) for 1 h and subsequent blue light irradiation (30 min, 76 W/m2). The total proteins were extracted from the cells immediately after experimental conditions, and ß-actin was used as a loading control. (C,D) Apigenin and galangin inhibited FoxO3a nuclear translocation by FoxO3a phosphorylation. (C) Phosphorylation levels of FoxO3a were determined by Western blotting at 30-, 60-, and 90-min incubation after, pretreatment with apigenin (30 µM) or galangin (20 µM) for 1 h and subsequent blue light irradiation (30 min, 76 W/m2). The total proteins were extracted from the cells immediately after experimental conditions, and ß-actin was used as a loading control. (D) The nuclear translocation of FoxO3a were determined by Western blotting at 24 h incubation after, 24 h apigenin (10, 20, 30 µM) and galangin (1, 10, 20 µM) pretreatment and 2-day repetitive blue light irradiation (30 min, 76 W/m2). The nuclear fraction was extracted from the cells immediately after experimental conditions by using NE-PER™ Nuclear and Cytoplasmic Extraction reagents. Lamin B1 was used as a loading control of nucleus fraction aliquot. (E,F) Apigenin and galangin suppress phosphorylation of MST-1/2. (E) Phosphorylation levels of MST-1/2 were determined by Western blotting at 30-, 60-, and 90-min incubation after, pretreatment with apigenin (30 µM) (E) or galangin (20 µM) (F) for 1 h and subsequent blue light irradiation (30 min, 76 W/m2). The total proteins were extracted from the cells immediately after experimental conditions, and ß-actin was used as a loading control. (G,H) Apigenin and galangin activated Akt in blue light irradiated HaCaT cells. Phosphorylation levels of Akt were determined by Western blotting at 30-, 60-, and 90-min incubation after, pretreatment with apigenin (30 µM) (G), and galangin (20 µM) (H) for 1 h and subsequent blue light irradiation (30 min, 76 W/m2). The total proteins were extracted from the cells immediately after experimental conditions, and ß-actin was used as a loading control.

Fig 4: (A) qPCR analysis of SELE mRNA 4 hours after treatment with human TNF-a at 50 ng/mL. SELE primers amplify a region conserved between human and macaque. Normalized to B2M. (B) Representative phase (top) and DiI (bottom) images of HUVEC, HREC, and RF/6A after treatment with DiI-labeled acetylated LDL. (C) Arteriole/venule pair in Sattler's layer of a normal human eye labeled with PECAM1 to outline cell junctions. Arrows indicate flow direction. (D) Fluorescent micrographs of RF/6A under static (no flow) and shear stress conditions labeled with phalloidin and DAPI. Nuclear orientation was quantified with respect to the flow direction. (E) Immunoblotting of AKT and ERK1/2 phosphorylation in HUVEC, HREC, and RF/6A cells after VEGF-A stimulation for indicated durations.

Fig 5: IRS-1/PI3K/AKT signaling is activated after inhibition of miR-128-3p. (A and B) Following transfection with miR-128-3p inhibitor, the amount of U251 cells was increased compared with that in the NC group. (C) An MTT assay indicated that the proliferation rate of the cells in the miR-128-3p inhibitor group was significantly elevated in comparison with that in the NC group. (D and E) Western blot analysis indicated that the levels of p-IRS-1, PI3K and p-AKT in the miR-128-3p inhibitor group were significantly higher than those in the NC group. In addition, the expression of IRS-1 and AKT was not significantly different between the miR-128-3p inhibitor group and the NC group. *P<0.05, ***P<0.001. p-IRS-1, phosphorylated insulin receptor substrate 1; PI3K, phosphoinositide-3 kinase; miR, microRNA; NC, negative control.