Figure S4.

Insulin Selectively Increases PER Expression, Related to Figure 4

(A) No significant increase in luciferase expression is observed following insulin application to fibroblasts expressing luciferase constitutively under the control of the SV40 promoter (n = 3, mean ± SEM, 2-way ANOVA, Sidak’s multiple comparisons test). (B) Addition of insulin to cells expressing Cry1:LUC produces a phase shift but no acute induction (n = 4, mean ± SEM). (C) Western blotting on whole cell lysate from PER2::LUC fibroblasts shows a significant increase in the abundance of PER1 and PER3 following insulin treatment but no significant increase in the abundance of CRY1 or CRY2 (n ≥ 3). All samples were harvested 3 h after insulin addition. (D) Quantification of western blotting, normalized against relevant loading control (n ≥ 3, mean ± SEM, Welch’s t test). E, F Western blotting on mouse livers harvested 1 h following IP injection with glucose (3 g/kg) and insulin (2.25 IU/kg) shows a significant increase in both PER1 and PER2 abundance (n = 3, mean ± SEM, Welch’s t test), samples were normalized to histone H3 levels. Positive control (OX) in left-hand lane is extract from HEK cells transiently transfected with PER expression constructs. G,H Insulin addition to Bmal1^−/− PER2::LUC fibroblasts elicits a modest but significant PER2::LUC induction (n ≥ 3, mean ± SEM, 2-way ANOVA, Tukey’s multiple comparisons test). WT traces repeated from Figure 4A. I,J Combined addition of phosphodiesterase inhibitor (IBMX) and adenylyl cyclase activator (forskolin) to Bmal1^−/− PER2::LUC fibroblasts increases basal PER2::LUC transcription (first arrow), allowing the effect of acute insulin treatment (second arrow) to be readily observed (n = 4, mean ± SEM, 2-way ANOVA, Sidak’s multiple comparisons test).