Figure 2.

Thalidomide specific inhibition of TBX5 binding and activity. Interactions of TBX5 and GATA4 with the NPPA TBE and GATA sites was assessed by electrophoretic mobility shift assays using nuclear extracts from HEK293T cells overexpressing HA-TBX5 or Flag-GATA4 (A). The binding of TBX5 (arrow) is suppressed by 42% in the presence of 4 µM of thalidomide, while that of GATA4 (arrow) was not affected. (+2 µM, ++4 µM). DMSO treatment was used as a negative control. Immunofluorescence of JR1 cells transfected with the plasmid overexpressing a HA-tagged TBX5 protein and then treated with either 4 µM DMSO or thalidomide. The localization of TBX5 was visualized using an anti‐ HA antibody followed by a fluorescent secondary antibody (B). Nuclei of cells were visualized using the Hoechst dye (blue color). TBX5 showed nuclear localization (green color) in both conditions. (Magnification X40). Same results were obtained for Hela and Hek293 cells. Transcriptional activity was assessed by luciferase assay. Increasing doses of TBX5 (100 to 500 ng) were transiently co-transfected with the rat NPPA (C), or mouse VEGF (D) promoters coupled to luciferase in HEK293 cells. Treatment with thalidomide (4 µM) or its equivalent amount of DMSO was done after 24 hours, and cells were harvested for luciferase assay. Relative luciferase activities are represented as fold changes. The data are the means of 3 independent experiments done in duplicates ± SE. Significance (*P < 0.05) was assessed using the one‐way ANOVA test.