A list http://www.selleckchem.com/products/ganetespib-sta-9090.html of SAP dependent genes with published cancer related functions, whose transcripts were downregulated more than 3 fold in HC11 SAP compared to HC11 FL control cells, is pre sented in Table 1. To confirm that these transcripts are indeed differentially expressed in the different HC11 cell strains, qRT PCR analysis was performed using cDNA from three different batches Inhibitors,Modulators,Libraries of the respective HC11 strains. Differences in gene expression between HC11 SAP and control cells are presented in Table 1 and in more detail in Additional file 4 Figure S1. The qRT PCR results agreed with the data obtained by tran script profiling. We also tested the SAP dependent gene expression in the HC11 strains when grown in the pres ence of serum.

It is interesting to note that in the presence of 3% FCS, these transcripts remained Inhibitors,Modulators,Libraries strongly reduced in HC11 SAP compared to control cells. Thus, the induction of these genes seems to depend mainly on whether the SAP domain is present in the transfected Mkl1 construct. In addition, we monitored changes in the expression Inhibitors,Modulators,Libraries of some of the SRF independent SAP dependent Mkl1 targets on a protein level. In agreement with the changes seen at the transcript level, we confirmed the reduction of tenascin C, Wisp1 and Nox4 proteins in cells overex pressing the SAP Mkl1 construct compared to the HC11 FL control and HC11 mutB1 cells. Using zymography, we found that Mmp2, a gene that was not affected by Mkl1 overexpression at the transcript level was highly expressed in all three cell strains, whereas Mmp3 and or 12, which belonged Inhibitors,Modulators,Libraries to the SRF dependent SAP dependent gene set, were al most completely lacking in HC11 mutB1 as well as HC11 SAP cells, corresponding to the data obtained by transcript profiling.

SRF independent SAP dependent transcripts represent direct Mkl1 target genes Since we have previously shown that the SAP domain of Mkl1 interacts with the proximal promoter of tenascin C to induce its Inhibitors,Modulators,Libraries transcription, we tested whether this was also the case for other transcripts of the same group. The promoters of the SRF independent SAP dependent genes listed in Table 1 encompassing at least 500 bp up stream of the transcription start site were fused to the secreted alkaline phosphatase reporter gene of pSEAP2 Basic. We tested the induction of each promoter reporter construct by co transfection with FL Mkl1.

This revealed that the majority of the new promoters tested were induced at least 2 fold by Mkl1 in comparison to co transfection with an inactive Mkl1 devoid of the transactivation domain, indicating Pacritinib phase 3 that these are indeed direct Mkl1 target genes. The promoter constructs that did not respond to Mkl1 overexpression may represent genes that are in directly regulated by Mkl1, or the relevant promoter regions were not contained in the constructs tested.