A down regulation of pro apoptotic genes and an over expression of anti apoptotic genes were also observed. Expression SB1518 of genes involved Inhibitors,Modulators,Libraries in the regulation of the cytoskeleton by qPCR The mRNA level of the 21 genes involved in the regula tion of the cytoskeleton that were identified as differen tially expressed after 24 hrs was confirmed by qPCR. The expression of these genes was also studied after exposure to DEHP for 5 hrs. Out of the 21 genes, four were significantly up regulated by DEHP treatment after 5 hrs of exposure and one was significantly down regu lated. A clear dose response relationship was observed for these 5 genes. After 24 hrs, these changes were con firmed for 3 genes. Nevertheless, the down and up regulation was more pronounced after 24 hrs than after 5 hrs of DEHP exposure, for nrp2 and kif23 respectively.
For instance, in cells exposed to 50 uM of DEHP, Kif23 Inhibitors,Modulators,Libraries was up regulated 17 fold at 24 hrs versus 3 fold at 5 hrs. After Inhibitors,Modulators,Libraries 24 hrs, 5 other genes were significantly up regulated by a factor ranging from 2. 0 to 4. 5 with a dose related effect. Eight other genes were significantly down regulated, with an expres sion ratio between 0. 2 and 0. 5. All these genes were down regulated in a dose dependent manner, except for cdh3, enah, ctnnbip1, lrrc8a and snx6. A threshold was observed with the latter genes. Ctnnbip1 was significantly down regulated only for the lowest dose of Inhibitors,Modulators,Libraries DEHP. Although they had been identified as differentially expressed in DD, five genes were not shown to be significantly over or under expressed by qPCR.
Yet the Inhibitors,Modulators,Libraries expression profiles of these genes indicated a dose related increase for tubb2b, b actin and pleckha5 but below the qPCR 2. 0 fold threshold. As for thy1 and nid2, the dose related decrease was inferior to 0. 5. Expression of apoptosis related genes, PPARs and CYP4 genes after DEHP treatment The expression level of bcl 2 and c myc mRNA was used as controls of DEHP effects. An increased level of bcl 2 after 5 hrs of exposure and a decreased level of c myc after 24 hrs were observed according to qPCR, as expected. p53 was down regulated in a dose and time depen dent manner, a significant decrease of the mRNA level was found after 24 hrs at 50 uM DEHP. None of the PPAR genes was identified as being differ entially expressed by DD after DEHP exposure.
In order to check these results, we measured the mRNA level of PPARa, PPAR b and PPAR g, by qPCR using hamster selleck kinase inhibitor specific primers. No change in the expression of these genes was observed by qPCR after 5 or 24 hrs of exposure with DEHP in our study conditions. The same verification was carried out for CYP4 genes. Neither Dif ferential Display nor qPCR allowed us to identify signifi cant expression changes compared to the control. Discussion The DDRT PCR techni que was used in the present study to identify the differ ential mRNA expression patterns between control and DEHP treated SHE cells.