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

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© 2013 ELL & Excerpta Medica.

This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.

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Figure S3 — RNAi to Validate the Role of SIK1 in Regulating Phase Shifting of the Clock in NIH 3T3 Fibroblasts, Related to Figure 2

(A) Relative expression in NIH 3T3 fibroblasts of Sik1 after transfection siSik1-1 (red) or a nontargeting control siRNA (siNT; white). siSik1-1 is modified (siStable formulation) for in vivo use, resulting in marginally lower efficiency compared with unmodified siRNA.

(B) Relative expression of Crtc1 (light green), Crtc2 (green) and Crtc3 (dark green) mRNA in NIH 3T3 fibroblasts.

(C) Relative expression levels to measure silencing of Crtc1 (light green) and Crtc3 (dark green) after transfection of siRNA against Crtc1 (siCrtc1) and Crtc3 (siCrtc3) respectively.

(D) Relative expression levels of Sik1 (red) Crtc1 (green) and Crtc3 (blue) after transfection with nontargeting control (siNT); Sik1 siRNA (siSik1) or Crtc1 and Crtc3 siRNAs (siCrtc).

(E) Level of CRTC1 phosphorylation, indicated by incorporation of ³²P from ³²P ATP by NIH 3T3 cell lysates. Cells treated with nontargeting siRNA (siNT) show significant increase in CRTC1 phosphorylation after serum shock (siNT 120 min) when compared with before (siNT 0 min). Cells treated with Sik1 siRNA show no increase in CRTC1 phosphorylation after serum shock (siSik1 120 min) when compared with before (siSik1 0 min).

(F–I) Areas under the curve from traces in Figure 2H (Egr1 - F), 2I (Nr4a1- G) and 2D (Sik1- H) showing increased expression of Egr1 (siSik1 versus siNT: p = 0.0006) and Nr4a1 (siSik1 versus siNT: p = 0.046) and decreased expression of Sik1 (siSik1 versus siNT: 0.008) with Sik1 siRNA (siSik1). Crtc siRNA (siCrtc) induced reduced or unchanged expression of all three transcripts as indicated by the area under the curve (siCrtc versus siNT: p = 0.38 for Egr1, p = 0.07 for Nr4a1, p = 7.7 × 10⁻⁷ for Sik1) All p values derived from Students’ t test, error bars = SEM and n = 4. See Table 1 and Table 2 for results of individual comparisons and statistical tests in Figures 2D–2I and S3F–S3H. In order to ensure the increases of Per1, Egr1 and Nr4a1 induction seen after Sik1 silencing are not due to off-target effects, we confirmed the expression patterns with two separate siRNA sequences against Sik1 (siSik1-2 and siSik1-3) (I).

(J–L) Per1 (J) Nr4a1 (K) and Egr1 (L) in NIH 3T3 fibroblasts treated with either nontargeting control siRNA (siNT; blue); siRNA sequence 2 against Sik1 (siSik1-2; red) or siRNA sequence 3 against Sik1 (siSik1-3; orange) all show increased induction following a serum shock after silencing of Sik1. Error bars = SEM, n = 4.