doi:10.1006/bbrc.2000.4169. -catenin. Western blot analysis of lysates isolated from Natural 264.7 Arctigenin cells treated with COB-187 revealed a dose-dependent reduction in -catenin phosphorylation at Ser33/37/Thr41 and consequent increase in -catenin accumulation [ 0.05 were significantly different relative to 0.1% DMSO control. -Actin was used as a loading control. COB-187 enhances -catenin localization to Rabbit Polyclonal to RPS2 the perinuclear and nuclear region. Another hallmark of GSK-3 inhibition, and consequent -catenin stabilization, is definitely build up of -catenin in the nucleus (1, 11, 19, 42). Therefore the effect of COB-187 on -catenin localization was evaluated using immunocytochemistry. As demonstrated is definitely Figs. 6 and ?and7,7, confocal microscopic analysis of THP-1 and Natural 264.7 macrophages, respectively, subsequent to immunostaining, revealed an increase in total -catenin as well as accumulation of -catenin in the perinuclear and nuclear regions with increasing concentration of COB-187. Translocation of accumulated -catenin from your cytoplasm to the nucleus is definitely more pronounced in Natural 264.7 cells (Fig. 7) relative to THP-1 Arctigenin macrophages (Fig. 6). These findings compliment the findings in the section above and further support that COB-187 inhibits phosphorylation of -catenin. Open in a separate windowpane Fig. 6. Treatment of THP-1 macrophages with COB-187 results in an apparent dose-dependent translocation of -catenin to the perinuclear and nuclear region. The effect of COB-187 on -catenin localization in THP-1 macrophages was identified using immunocytochemistry. Each image, acquired by confocal microscopy, shows nuclear staining with DAPI (blue) and -catenin staining (green). panel of each set of data shows the merged composite image. Results are representative of two independent experiments. Nuclear and perinuclear build up is definitely indicated by brightening of the blue area and accumulation of a green transmission (halo) round the nucleus, respectively, in the merged images with increasing concentration of COB-187. Level bars?=?10 m. THP-1 macrophages were treated for 5 h with COB-187 before immunocytochemical analysis. Isotype control image is definitely THP-1 macrophages treated with an isotype control for -catenin (image is definitely representative of what was observed for those COB-187/DMSO/medium treatment organizations). Open in a separate windowpane Fig. 7. Treatment of Natural 264.7 cells with COB-187 effects in an apparent dose-dependent translocation of -catenin to the nucleus. The effect of COB-187 on -catenin localization in Natural 264.7 cells was identified using immunocytochemistry. Each image, acquired by confocal microscopy, shows nuclear staining with DAPI (blue) and -catenin staining (green). panel of each set of data shows the merged composite image. Results are representative of two independent experiments. Nuclear build up is definitely indicated by brightening of the blue area in the merged images with increasing concentration of COB-187. Arctigenin Level bars?=?10 m. RAW 264.7 cells were treated for 5 h with COB-187 before immunocytochemical analysis. Isotype control image is usually RAW 264.7 cells treated with an isotype control for -catenin (image is representative of what was observed for all those COB-187/DMSO/medium treatment groups). Treatment of THP-1 macrophages and RAW 264.7 cells with COB-187 for 5 h does not increase the mRNA level of -catenin. To determine if the increase in the level of -catenin observed in the Western blot (Figs. 4and ?and5)5) and immunocytochemistry (Figs. 6 and ?and7)7) could be due to a change in the level of de novo Arctigenin -catenin production, RT-qPCR Arctigenin was performed on mRNA isolated from THP-1 macrophages and Natural 264.7 cells treated with varying concentrations of COB-187. As shown in Fig. 8, and and and were used as housekeeping genes for THP-1 macrophages and RAW 264.7 cells, respectively. Results are the average of three impartial experiments performed in triplicate. Error bars symbolize the SE. Results were analyzed using a one-way ANOVA coupled with post hoc Games-Howell test. Treatment of THP-1 macrophages with COB-187.
