Figure 6. Overexpression of Nkx6.1 inhibits endogenous levels of glucagon mRNA in InR1G9 cells by competing with Pax6 trans-activation.

(A) InR1G9 cells were transiently transfected with an expression vector encoding Nkx6.1 using the TransFectin™ reagent. This approach permitted 75% transfection efficiency as assessed by FACS analysis. Expression of the Nkx6.1 transgene in transfected cells (+) and its absence in untransfected cells (−) was determined by semi-quantitative PCR. Relative abundance levels of endogenous glucagon mRNA were then evaluated by quantitative PCR and normalized to the housekeeping transcript TBP. (B) Two stable clonal derivatives of the InR1G9 cell line expressing either an empty expression vector (control) or a dominant-negative variant of Pax6 (DN-Pax6) were transiently transfected with Nkx6.1. Expression of Nkx6.1 in non-transfected (−) and transfected (+) cells was evaluated by semi-quantitative PCR. Relative abundance levels of endogenous glucagon mRNA were then evaluated by quantitative PCR and normalized to the housekeeping transcript TBP. ** indicates statistical significance with P<0.02.