Supplementary Materialsijms-18-02447-s001. that CDX2 knockdown decreased cell proliferation and inhibited Saracatinib inhibitor both pathways. Furthermore, the mTORC1 and Wnt/-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H Cthiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/-catenin signaling pathways. mRNA abundance (= 6) and protein expression (= 3) in the control and overexpression group. AU: arbitrary unit; (d,e) the OD value and cell number were assessed by MTT assay (= 20) and cell counting (= 6), respectively. Control: control group; Overexpression: CDX2 overexpression group. Representative results of three independent experiments are shown as the mean SEM; * 0.05. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and cell counting were used Saracatinib inhibitor to evaluate the effect of CDX2 overexpression Saracatinib inhibitor on IPEC-J2 cell proliferation. OD values (Figure 1d) and cell numbers (Figure 1e) were increased in the overexpression group. 2.2. CDX2 Overexpression Activated Both the mTORC1 and Wnt/-Catenin Pathways in IPEC-J2 Cells To measure the effect of CDX2 overexpression on the mTORC1 and Wnt/-catenin pathways, Western blot analysis was used. Compared with the control group, levels of p-mTOR (Ser2448), p-S6K1 (Thr389), p-S6 (Ser235), p-4EBP1 (Thr70) and eIF4E had been improved in the overexpression group (Shape 2a,b). Degrees of Axin2 and GSK-3 had been decreased, Rabbit polyclonal to PBX3 and degrees of -catenin, Cyclin D1 and c-Myc had been improved in the overexpression group (Shape 2c,d). Open up in another windowpane Shape 2 CDX2 overexpression activated both Wnt/-catenin and mTORC1 pathways. (a,b) Traditional western blot analysis from the mTORC1 pathway activity after CDX2 overexpressed in IPEC-J2 cells with Saracatinib inhibitor quantification (= 3); (c,d) traditional western blot of Wnt/-catenin pathway related protein after CDX2 overexpressed with quantification (= 3). AU: arbitrary device. Control: control group; Overexpression: CDX2 overexpression group. Representative outcomes of three 3rd party experiments are demonstrated. Data are indicated as the mean SEM; * 0.05. 2.3. CDX2 Knockdown in IPEC-J2 Cells Reduced Cell Proliferation By calculating CDX2 mRNA great quantity at 48 h post-transfection using the three CDX2-siRNA, we discovered that siRNA-002 created an optimal disturbance impact (Shape 3a). We also discovered CDX2 mRNA great quantity in IPEC-J2 cells Saracatinib inhibitor to become the cheapest at 36 h post-transfection with siRNA-002 (Shape 3b). Weighed against the adverse control, Traditional western blot evaluation also showed a decrease in CDX2 manifestation in the knockdown group (Shape 3c). Open up in another window Shape 3 CDX2 knockdown in IPEC-J2 cells decreased cell proliferation. (a) The result of three siRNAs on mRNA great quantity was assessed by real-time PCR 48 h post-transfection. Empty: control group; NC: adverse control group; siRNA-001: CDX2-siRNA-001 group; siRNA-002: CDX2-siRNA-002 group; siRNA-003: CDX2-siRNA-003 group; (b) the effect of siRNA-002 transfection time on mRNA abundance was measured by real-time PCR. Data are expressed as the mean SEM (= 6). The bars without same letters indicate a significant difference ( 0.05); (c) siRNA-002 treatment reduced CDX2 protein expression compared with the negative control group. AU: arbitrary unit; (d,e) OD values and cell numbers were assessed by MTT assay (= 20) and cell counting (= 6), respectively. Negative Control: negative control group; Knockdown: CDX2-siRNA-002 group. Representative results of three independent experiments are shown. Data are expressed as the mean SEM; * 0.05. The results of the MTT assay and cell counting showed that CDX2 knockdown decreased OD values (Figure 3d) and cell numbers (Figure 3e), respectively. 2.4. CDX2 Knockdown Inhibited Both the mTORC1 and Wnt/-Catenin Pathways in IPEC-J2 Cells Western blot was used to evaluate the effect of CDX2 knockdown on the mTORC1 and Wnt/-catenin pathways. The result showed that levels of p-mTOR (Ser2448), p-S6K1 (Thr389), p-S6 (Ser235), p-4EBP1 (Thr70) and eIF4E were decreased in the knockdown group (Figure 4a,b). Levels of Axin2 and GSK-3 were increased, and levels of -catenin, Cyclin D1 and c-Myc were decreased in the knockdown group (Figure 4c,d). Open in a separate window Figure 4 CDX2 knockdown inhibited both the mTORC1 and Wnt/-catenin pathways. (a,b) Western blot analysis of the mTORC1 pathway activity after.