Urve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each reaction, GAPDH was simultaneously processed in the same sample as an internal control. The level of Apo J, CTGF and fibronectin mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Apo J, CTGF and fibronectin mRNA by the level of the GAPDH housekeeping gene in the same samples. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.13655-52-2 Statistical analysisResults for the analyses of RPE cell death, lipid peroxidation, SA-?Gal activity, real-time PCR, western blot and ELISA experiments are expressed as the mean 6 s.d. For comparison of means between two groups, an unpaired t-test was employed. Statistical significance was defined as P,0.05.Results ZO-1 expression in cultured human RPE cellsThe expression and localization of ZO-1 was used to define the tight junction structure of the cultured human RPE cells. Each RPE cell was outlined by the expression of ZO-1 (Figure 1).Cigarette smoke extract induced cell deathTo determine the cytotoxic effects of cigarette smoke extract (CSE), primary cultured human retinal pigment epithelial (RPE) cells were treated with 2, 4, 8 and 12 of CSE (Fig. 2). In this cell viability assay, untreated control cells demonstrated almost no dead cells staining red by propidium iodide (Fig. 2B). Incubation of cultured human RPE cells with 2, 4, and 8 of CSE led to elevated proportions of non-viable cells with 5.1+/22.4 , 12.0+/ 21.7 , and 14.0+/22.4 of total cells (Fig. 2E). The most pronounced effect was seen after treatment with 12 of CSE, which significantly increased the proportion of non-viable RPE cells to 86.2+/211.4 of total cells (Figs. 2D, 2E). Based on these results, only concentrations of 2, 4, and 8 of CSE were used in the subsequent experiments.Protein extraction and western blot analysisFor nuclear extracts, cells were washed twice with ice-cold PBS, collected, and lysed in three times packed cell volumes of low-salt hypotonic cell lysis buffer [20 mM HEPES pH 7.5, 10 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.1 TritonX-100, 10 glycerol, protease inhibitor cocktail (Roche)] for 10 min on ice. After centrifugation (19,000 g for 30 minutes at 4uC) in a microfuge, the supernatants were transferred to fresh tubes and stored at 270uC for Madecassoside future use. The protein content was measured by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Denatured proteins (2 mg) were separated under reducing conditions by electrophoresis using 10 SDS-polyacrylamide gels. Thereafter, the proteins were transferred with tank blotting onto a nitrocellulose membrane (Protran Ba-183; Whatman, Dassel, Germany) and probed with a mouse monoclonal anti-human ApoJ antibody (Abcam) and rabbit polyclonal anti-human CTGF antibody (Abcam) as described previously [38]. These antibodies were used at a dilution of 1:1000, respectively. Secondary alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Sigma-Aldrich) or AP-conjugated goat anti-rabbit IgG antibodies (Sigma-Aldrich) were incubated for 30 minutes at a dilution of 1:2500 at room temperature. After substrate incubation (CDP-star; Roche) the signals were visualized by exposure to light sensitive films (Hyperfilm ECL; GE Healthcare, Munich, Germany), which were digitized and densitometrically quantified with the Multi Gauge V3.1 software.Urve was obtained from probes of three different untreated human RPE cell cultures. To normalize differences of the amount of total RNA added to each reaction, GAPDH was simultaneously processed in the same sample as an internal control. The level of Apo J, CTGF and fibronectin mRNA was determined as the relative ratio (RR), which was calculated by dividing the level of Apo J, CTGF and fibronectin mRNA by the level of the GAPDH housekeeping gene in the same samples. All experiments were run in triplicate in RPE cultures from three donors and repeated three times.Statistical analysisResults for the analyses of RPE cell death, lipid peroxidation, SA-?Gal activity, real-time PCR, western blot and ELISA experiments are expressed as the mean 6 s.d. For comparison of means between two groups, an unpaired t-test was employed. Statistical significance was defined as P,0.05.Results ZO-1 expression in cultured human RPE cellsThe expression and localization of ZO-1 was used to define the tight junction structure of the cultured human RPE cells. Each RPE cell was outlined by the expression of ZO-1 (Figure 1).Cigarette smoke extract induced cell deathTo determine the cytotoxic effects of cigarette smoke extract (CSE), primary cultured human retinal pigment epithelial (RPE) cells were treated with 2, 4, 8 and 12 of CSE (Fig. 2). In this cell viability assay, untreated control cells demonstrated almost no dead cells staining red by propidium iodide (Fig. 2B). Incubation of cultured human RPE cells with 2, 4, and 8 of CSE led to elevated proportions of non-viable cells with 5.1+/22.4 , 12.0+/ 21.7 , and 14.0+/22.4 of total cells (Fig. 2E). The most pronounced effect was seen after treatment with 12 of CSE, which significantly increased the proportion of non-viable RPE cells to 86.2+/211.4 of total cells (Figs. 2D, 2E). Based on these results, only concentrations of 2, 4, and 8 of CSE were used in the subsequent experiments.Protein extraction and western blot analysisFor nuclear extracts, cells were washed twice with ice-cold PBS, collected, and lysed in three times packed cell volumes of low-salt hypotonic cell lysis buffer [20 mM HEPES pH 7.5, 10 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.1 TritonX-100, 10 glycerol, protease inhibitor cocktail (Roche)] for 10 min on ice. After centrifugation (19,000 g for 30 minutes at 4uC) in a microfuge, the supernatants were transferred to fresh tubes and stored at 270uC for future use. The protein content was measured by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Denatured proteins (2 mg) were separated under reducing conditions by electrophoresis using 10 SDS-polyacrylamide gels. Thereafter, the proteins were transferred with tank blotting onto a nitrocellulose membrane (Protran Ba-183; Whatman, Dassel, Germany) and probed with a mouse monoclonal anti-human ApoJ antibody (Abcam) and rabbit polyclonal anti-human CTGF antibody (Abcam) as described previously [38]. These antibodies were used at a dilution of 1:1000, respectively. Secondary alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Sigma-Aldrich) or AP-conjugated goat anti-rabbit IgG antibodies (Sigma-Aldrich) were incubated for 30 minutes at a dilution of 1:2500 at room temperature. After substrate incubation (CDP-star; Roche) the signals were visualized by exposure to light sensitive films (Hyperfilm ECL; GE Healthcare, Munich, Germany), which were digitized and densitometrically quantified with the Multi Gauge V3.1 software.
