FIGURE 6.

Effects of NKX6.1 overexpression or knockdown on HNF1α. A, EMSA. EMSAs were conducted with βTC3 and NIT1 cell lysates and biotin-labeled HNF1α promoter oligonucleotide (Probe) as indicated. Arrows indicate NKX6.1 binding and supershift. In competition assays, DNA binding reactions were preincubated with 10-fold (10×) or 100-fold (100×) unlabeled HNF1α oligonucleotide or nonspecific (NS) oligonucleotide as indicated. Ab, antibody. B, RT-PCR. Total RNA was isolated from the indicated cell lines, and expression of pancreatic genes was measured by RT-PCR. C, effect of NKX6.1 overexpression and knockdown on mRNA levels. NIT1 mouse beta cells were transfected with pCMV-NKX6.1 expression plasmid (upper panel) or Nkx6.1 siRNA (lower panel) for 48 h. Total RNA was isolated and subjected to reverse transcription. Target gene expression was quantified by real-time PCR and expressed as -fold over the control as indicated. Empty vector plasmid (pcDNA3) transfection was used as a negative control and arbitrarily set to 1.0 for all tested genes.