(D) Real-time qPCR analysis for as a positive control

(D) Real-time qPCR analysis for as a positive control. evaluated using the luciferase assay. Levels of acrolein-conjugated protein, N-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), and hydrogen peroxide were measured. Results SMOX was localized in glial cells in fibrovascular tissues. Hypoxia induced SMOX production in TR-MUL5 cells, which was suppressed by silencing of hypoxia-inducible factor-1 (but not was regulated through HIF-1 binding to hypoxia response elements 2, 3, and 4 sites in the promoter region of 5-AGCAGATGTGAATGCAGACCAAAGA-3 (forward) and 5-TGGCTCACCGCCTTGGCTT-3 (reverse) for as the internal control. Enzyme-Linked Immunosorbent Assay (ELISA) TR-MUL5 cells were cultured under normoxic or hypoxic condition for 24 hours. Levels of SMOX protein in the cell lysate were analyzed using ELISA packages for rat SMOX (MyBioSource, San Diego, CA, USA) following the manufacturer’s protocol. Absorbance was read at 450 nm on a microplate reader (Tecan Sunrise; Tecan, Inc., M?nnedorf, Switzerland). SMOX concentration was normalized by total protein concentration of cell lysates measured by bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Cell Viability Assay TR-MUL5 cells were seeded into a 96-well Broussonetine A plate and incubated for 24 hours at 33C in the atmosphere of 95% air flow and 5% CO2. Subsequently, the cells were cultured under normoxic or hypoxic condition for 6 or 24 hours, and cell viability was assessed using CellTiter-Glo 2.0 (Promega), according to the manufacturer’s training. Luminescence was measured by an Infinite 200 PRO microplate reader (Tecan Sunrise; Tecan, Inc.). RNA Interference TR-MUL5 cells were transfected with a Broussonetine A 5-nM final concentration of Broussonetine A various Dicer-substrate siRNA (DsiRNA) for suppressing the gene expression of hypoxia-inducible factor-1 (siRNA-1, rn.Ri.Hif1a.13.1; siRNA-2, rn.Ri.Hif1a.13.2; siRNA-1, rn.Ri.Hif2a.13.1; siRNA-2, rn.Ri.Hif2a.13.2) (IDT, Coralville, Iowa, USA), and negative control siRNA (Ctrl-siRNA, Mission SIC-001; Sigma-Aldrich Corp., St. Louis, MO, USA). Transfections were performed using the Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific). The composite transfection combination was replaced with 10% FBS/DMEM 24 hours after the transfection. Subsequently, real-time PCR and ELISA for SMOX were performed after 6 and 24 hours of hypoxic activation, respectively. Transient Transfection and Luciferase Assay TR-MUL5 cells were seeded in a 96-well plate at 1.5 104 cells/well containing 65 L of 10% FBS/DMEM. After incubation for 24 hours, cells were cotransfected with the X-tremeGENE HP DNA transfection reagent (Sigma-Aldrich) made up of the pGL4.10 luciferase vector (Firefly-expressing plasmid; Promega), with the promoter (C1067 to +122 bp from transcriptional start site of promoter region. Subsequently, the promoter reporter with each of the six mutant sites was altered into a pGL4.10 luciferase vector using PrimeSTAR Mutagenesis Basal Kit (Takara Bio, Shiga, Japan). The HRE wild-type or mutated constructs, together with pRL-CMV, were transiently cotransfected into TR-MUL5 cells, followed by treatment with hypoxia, and the luciferase activity was measured. Measurement of Hydrogen Peroxide and FDP-Lys Production TR-MUL5 cells were cultured with or without 50 M SMOX inhibitor (MDL72527; Sigma-Aldrich) Broussonetine A for 24 hours with or without hypoxia activation. Subsequently, cells were incubated in phosphate buffered saline at 37C for 3 hours, and the concentration of hydrogen peroxide in the supernatant was measured using the Hydrogen Peroxide Nrp1 Detection Kit (Cell Technology, Inc., Fremont, CA, USA), according to the manufacturer’s protocol. FDP-Lys concentration in the supernatant was evaluated using the ELISA kit (MK-150; Takara Bio) and normalized by protein concentration measured using the Quick Start Bradford 1 Dye Reagent (Bio-Rad, Hercules, CA, USA). Statistical Analyses Data are expressed as mean standard error of the mean for three to six individual experiments. Differences between two groups were compared using the Student’s value <0.05 was considered statistically significant. Results Localization of SMOX, SAT1, and PAOX in Fibrovascular Tissues To investigate the tissue localization of polyamine catabolic enzymes in fibrovascular tissues of patients with PDR, we performed immunofluorescent staining for polyamine oxidase enzymes, that is, SMOX, SAT1, and PAOX. Immunofluorescence staining showed that SMOX signals were intensely localized in the nucleus of GFAP-positive cells of the fibrovascular tissues (Fig.?1A). However, SAT1 and PAOX signals were.