Fig. 6

The restoration of SELENBP1 leads to the suppression of c-Jun and STAT1 phosphorylation, both of which are required for SELENBP1-mediated transcriptional induction of p21 protein. a Cell lysates of stable UMUC3 (Vector) and UMUC3 (HA-SELENBP1) cells were subjected to immunoblotting assay with indicated antibodies that might be involved in transcriptional regulation of p21 expression. b UMUC3 cells stably transfected with dominant-negative mutant form of c-Jun (TAM67) or pcDNA3.1(Vector) were extracted and then subjected to immunoblotting analysis with indicated antibodies. Densitometric quantification of p21 (relative to GAPDH) is shown. c UMUC3 cells were stably transfected with dominant-negative mutant STAT1 (DN-STAT1) or pEGFP-C1 construct (Vector), and were then extracted for immunoblotting assay with indicated antibodies. Densitometric quantification of p21 and SOCS1 (relative to GAPDH) is shown under each blot. d TAM67 (1 μg) and pcDNA3.1 (1 μg) plasmids were transiently transfected into stable UMUC3 (DN-STAT1) and UMUC3 (pEGFP-C1), respectively. Following 48 h of transient transfection, cells were collected and then subjected to immunoblotting assay. Densitometric quantification of p21 (relative to GAPDH) is shown. e Indicated amounts of empty vector (pcDNA3.1) and TAM67 was transiently co-transfected into stable UMUC3 (DN-STAT1) or UMUC3 (Vector) cells, together with 1.3 Kb of p21 promoter-driven luciferase reporter and pRL-TK, as an internal control. Thirty-six hours post transfection, luciferase reporter activity was determined. Luciferase activities of UMUC3 (Vector) and UMUC3 (DN-STAT1) groups are normalized to those of corresponding control group that transfected with 0.6 μg of pcDNA3.1, respectively. The asterisk (*) indicates a significant difference as compared to UMUC3 (DN-STAT1) group transfected with 0.6 μg of pcDNA3.1 (p < 0.05). The symbol (#) indicates p < 0.05 when compared with UMUC3 (Vector) group transfected with 0.6 μg of pcDNA3.1. The values are shown as mean ± standard deviation from four independent experiments. f Following 48 h of transient transfection with indicated plasmids, UMUC3 cells were collected and then subjected to flow cytometry assay