Figure 5
Validation of the microarray data for AMOT-p80 and miR-146a and confirmation of direct effect of HOXD10 on expression. (A) Expression of AMOT-p80 assessed by qPCR and WB, confirming raised expression in the HOXD10-transfected cells, compared with empty vector-transfected controls. β-actin is used as a loading control. (B) Expression of AMOT-p80 assessed using qPCR, confirming reduced expression in the HOXD10 siRNA-transfected cells, compared with scrambled siRNA controls. (D) Expression of miR-146a assessed using qPCR, confirming increased expression in the HOXD10 siRNA-transfected cells, compared with scrambled siRNA controls. (E) pGL3 luciferase reporter vectors containing either WT AMOT promoter, AMOT promoter with either mutated HOXD10 binding sites or AMOT promoter with both mutated binding sites were transfected into D19 cells stably transfected with either pcDNA3.1-HOXD10 or pcDNA3.1 control plasmids. DLR analysis shows that HOXD10 increases luciferase expression when pGL3 vector contains WT promoter. However, luciferase expression decreases when either of HOXD10-binding sites in AMOT promoter were mutated. No significant reduction in luciferase expression was detected when both mutated sites existed in the same pGL3 vector compared with pGL3 vector containing only one mutated binding site. **P<0.01, each assay was conducted in triplicate and repeated three times. Data show the mean of triplicate repeats±s.e.m. (C) Expression of miR-146a assessed using qPCR, confirming reduced expression in the HOXD10-transfected cells, compared with empty vector-transfected controls. (F) pGL3 luciferase reporter vectors containing either WT miR-146a promoter or miR-146a promoter with a mutated HOXD10-binding site were transfected into D19 cells stably transfected with either pcDNA3.1-HOXD10 or pcDNA3.1 control plasmid. DLR analysis shows that HOXD10 increases luciferase expression when pGL3 vector contains WT promoter. However, luciferase expression decreases when either of HOXD10-binding sites in miR-146a promoter was mutated. **P<0.01, each assay was conducted in triplicate and repeated three times. Data show the mean of triplicate repeats±s.e.m.
