Reduced activity of PP2A is the primary cause of impaired gene expression.

(A) HIT cells were transfected with the indicated amounts of PP2A Cα siRNA for 48 h. RNA was harvested and subjected to RT-PCR analysis or proteins subject to western analysis. Scrambled RNA was employed to validate the specificity of PP2A Cα-specific siRNA. (B) HIT cells grown in 5.5 mM glucose were transfected with 50 nM siRNA specific for PP2A Cα or with scrambled sequences. Prior to harvesting, 30 µM forskolin was added to the culture for 1–6 h. Western analysis was performed with anti-phospho CREB (pCREB Ser133) or PP2A Cα antibody, followed by antibody against total CREB. (C) RT-PCR analysis with HIT cells grown in 5.5 mM glucose indicated that PP2A Cα-specific siRNA altered the basal and forskolin-induced gene expression of ICER, NeuroD, SUR1, and insulin as shown in hyperglycemic conditions. (E) RT-PCR analysis with HIT cells stably overexpressing PP2A Cα. Overexpression of PP2A in HIT cells grown in 25 mM glucose restored the ICER, NeuroD, SUR1, and insulin to the values obtained in normoglycemic (5.5 mM) condition. (D, F) Data from three independent experiments shown in C, E are presented as fold ratios with respect to the value of scrambled siRNA or mock-transfected HIT cells (^#, P<0.05; ^##, P<0.05), and to the value of without forskolin (*, P<0.05; **, P<0.01).