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. 2013 Aug 29;154(5):1100–1111. doi: 10.1016/j.cell.2013.08.004

Figure 2.

Figure 2

The Role of Sik1 in Phase-Shifting Responses in NIH 3T3 Embryonic Fibroblasts

(A) CRTC1 shows nuclear translocation after serum shock (before serum, top; after 10 min serum, bottom).

(B and C) Bioinformatic analysis of the Sik1 promoter region showing conservation of the CRE across several mammalian species (B, top). Sik1 was induced after serum shock (B, bottom, n = 4), leading to (C) increased phosphorylation of CRTC. A CRTC peptide is phosphorylated by purified SIK1, measured by the incorporation of 32P from γ32P ATP (+SIK1). When incubated with cell lysates (lysate, 0 min), serum shock (lysate, 120 min) increased significantly phosphorylation of CRTC (p = 0.006, Student’s t test, n = 8). Experiments containing no lysate are shown as a negative control (− lysate). Representative blots are shown (left).

(D) Sik1 induction following serum shock was attenuated significantly following silencing of Sik1 or Crtc compared with nontargeting control siRNA (siNT: p = 1.5 × 10−6, siSik1: p = 4.1 × 10−1, siCrtc: p = 0.01, n = 4, area under curve [AUC] analysis shown in Figure S3).

(E) Sik1 silencing enhanced Per1 induction following serum shock (siNT: p = 2.2 × 10−5, siSik1: p = 9.3 × 10−5).

(F) Following Crtc silencing, no significant induction of Per1 expression was observed (siCrtc: p = 0.06). AUC analysis (normalized to NT control) for Per1 expression following silencing of Sik1 showed a significant increase (p = 0.00047 siSik1 versus siNT, Student’s t test), not seen with Crtc silencing (p = 0.799 siCRTC versus siNT, Students’ t test).

(G) Normalised data for Per1 induction following Sik1 and Crtc knockdown as measured by Area Under Curve (AUC) from (E) and (F).

(H and I) Egr1 (H) and (I) Nr4a1, both CRE-regulated transcripts, show enhanced expression with Sik1 silencing and attenuated or no induction with Crtc silencing. AUC analysis shown in Figure S3. All mRNA levels normalized to housekeeping controls and t = 0, change over time analyzed by one-way ANOVA, ∗∗∗ = p < 0.001, ∗ = p < 0.05, n.s. = p > 0.05. Error bars = SEM; n = 4. See also Figure S3 and Tables 1 and 2.