Herein, we addressed the question whether miR-133b is also downregulated in prostate cancer

the cells were treated with either Tgfb or 14192894 vehicle, and they were harvested 48 hours later for western blotting or RT-PCR. Western Blotting and b-Gal Assay Cells were collected, lysed, separated by SDS-PAGE and transferred to PVDF membrane with 50100 mg total protein per sample. The membrane was incubated with primary antibody for two hours, washed trice in Tris-Buffered Saline Tween-20 for 15 minutes each time; and then incubated with horseradish peroxidase -labeled secondary antibody for one hour. After washing in TBST, the membrane was incubated with 2 ml ECL for 5 minutes and visualized by exposure to film. b-galactosidase assays were performed in Arf lacZ/lacZ MEFs as previously described using a commercial kit. For western blotting, antibodies directed against the following proteins were utilized: C/ebpb, and Hsc70; phospho-p38 Mapk, and phospho-Smad2; and p19Arf. Experimental findings were Statistical Analysis Quantitative data are presented as the mean6S.D. from three or more representative 481-53-8 chemical information experiments. Statistical significance was calculated using Student’s t test. Results Recognizing the substantial delay between Smad binding to the Arf promoter and increased synthesis of Arf primary transcript, we considered potential roles for other transcription factors whose function might be influenced by Tgfb. Among those, C/ Sp1 and C/ebpb Mediate Arf Induction by Tgfb ebpb was an attractive candidate because previous work had implicated it as an Arf repressor in primary epidermal keratinocytes, and putative consensus DNA binding elements are found within 500 bp 59 to the Arf translation initiation codon. Utilizing chromatin immunoprecipitation, we demonstrated that C/ebpb was bound to this region in cultured mouse embryo fibroblasts at passage 3. We next investigated whether Tgfb influenced the binding of endogenous C/ebpb to the Arf promoter. We previously established that Smad 2/3 binding to elements in the proximal Arf promoter is enhanced within 1.5 hours following the addition of Tgfb2 to the culture medium, whereas RNA polymerase II binding is not increased until 24 hours, after which Arf mRNA increases. Paralleling the delayed RPolII binding, C/ebpb localization to a proximal promoter element in the Arf promoter was diminished at 24 hours following an initial increase at 1.5 hours. Interestingly, Tgfb stimulation diminished C/ebpb mRNA and protein between 24 and 72 hours. The effect on C/ebpb protein expression was evident when it was ectopically expressed, implying that the decreased repression was not simply due to decreased transcription of the native mRNA. Of note, the fact that p19Arf level did not strictly inversely correlate with C/ebpb indicates that other factors, such as cell “culture shock”that has been described for cultured mouse fibroblasts, must play a role in expression of this 22634634 tumor suppressor and these other factors maybe be independent of Tgfb signaling. We confirmed that ectopically expressed C/ebpb blunted Arf transcription by showing that b-galactosidase activity was repressed in cultured Arf lacZ/lacZ MEFs infected with retrovirus encoding the liver-enriched activator protein isoform of C/ ebpb, which includes a transactivation domain . Schematic diagram showing potential C/ebpb, Smad, Sp1 and E2F binding sites at the Arf promoter.. Tgfb decreases C/ebpb binding to the Arf locus in MEFs. Quantitative analysis of representative chromatin immunoprecipitation assays of using wild type MEFs exposed to vehicle