Overexpression of IRE1α correlates with reduced Insulin mRNA in cultured cells.

<p>(A) COS-7 cells were transfected with mouse Insulin 2 and cultured for 24 hr. Cells were then split onto 3 plates and transfected again with wild-type human Ire1α; IRE1α WT, a kinase/endoribonuclease inactive mutant human Ire1α; IRE1α KA; or pcDNA3 control. They were then cultured for 24 hr. Protein and RNA were collected from the same plates. Total IRE1α, phosphorylated IRE1α, and actin were measured by immunoblot. Expression levels of human IRE1α and mouse Insulin 2 were measured by real time PCR (n = 3; values are mean±SEM). (B) INS-1 832/13 cells were transfected with human IRE1α WT or pcDNA3 control and cultured for 24 hr. Expression levels of human IRE1α, endogenous rat insulin 1, insulin 2, and glucose transporter 2 (glut 2) were measured by real time PCR (n = 3; values are mean±SEM). (C) INS-1 832/13 cells were transfected with either pcDNA3 control or increasing concentrations of human IRE1α WT and cultured for 24 hr. Expression levels of human IRE1α, endogenous rat insulin 1, insulin 2, and glucose transporter 2 (glut 2) were measured by real-time PCR (n = 3; values are mean±SEM).