It also indicated that the active site of the enzyme that recognizes

negative cross talk between p53 and C EBP b may ultimately affect the expression of their targeted genes such as miR 145. On the other hand, although the level of LAP 2 is relatively Hedgehog inhibitor low compared with LIP, this band was readily detectable in these cells except for MCF 10A cells. No LAP 1 was detected in any of them, consistent with the previous finding. Of note, the phosphorylation at Thr 235 has been shown to activate the transcription of C/EBP b, this band was very weak in MCF 10A cells, compared with that in other three cancer cell lines, which may highlight the importance of phosphorylation of C/EBP b in cancer cells. Therefore, LAP 2 and LIP were further investigated for their role in miR 145 expression in this study. We first ectopically expressed LAP 2 and LIP, respectively, and then tested the effect of each of them on suppression Skin infection of the miR 145 promoter activity as well as the endogenous miR 145. Although it was reported that the LAP isoforms have active transcription activity whereas the LIP form has repressive activity, our study indicated that both LAP 2 and LIP suppressed the promoter activity by 60%. To further determine the role of C/EBP b isoforms in the suppression of miR 145, we knocked down both isoforms in MDAMB 231 and BT 549 cells, and demonstrated that this knockdown increased miR 145 expression as well as miR 145 promoter activity. These provide further evidence that C/EBP b is a negative regulator of miR 145. C/EBP b represses the p53 mediated induction of miR 145 To determine the role of C/EBP b in the suppression of miR 145 in relation to p53, we first examined their effect on the miR 145 promoter activity. As in the case for the endogenous miR 145, both LAP 2 and LIP suppressed the miR 145 promoter activity. In contrast, p53 induced the miR 145 promoter activity by about a 5 fold. This finding was further supported by the miR 145 promoter GFP reporter. Moreover, suppression of C/EBP b by RNAi caused canagliflozin the upregulation of miR 145 promoter activity, especially with co transfection of p53, suggesting that C/EBP b can counteract with the ability of p53 to induce miR 145. This result is consistent with the previous finding that C/EBP b can functionally interact with and suppress p53 activity. We then generated a miR 145 promoter reporter containing mutations in the C/EBP b binding site. Mutations of the C/EBP b binding site caused further upregulation of miR 145 promoter activity, i. e. over four relative units higher than that of miR 145p WT in the presence of p53. To further determine the role of C/EBP b in the suppression of p53 mediated miR 145 promoter activity, we transfected cells with p53 along various amounts of LAP 2 expression vector and demonstrated that an equal amount of LAP 2 expression vector was sufficient to suppress its ability to induce miR 145 promoter activity, consistent with the previous report that there is a negative cross talk between p53 and C/EBP b.