Figure 1
Circadian oscillation profiles of clock genes in wild-type and GSK3β-/- MEFs. A, wild type and B, GSK3β-/- cells were synchronized, harvested, processed, and the gene products were amplified as described in the Materials and Methods. The resulting transcriptional profiles of murine GAPDH, mPer2, mCry1, RevErbα, and Bmal1 were analyzed by reverse-transcription PCR. The subjective time points (TP) of peak expression are designated in white above the corresponding bands for each transcript examined. Panel C is a graphical depiction of mPer2 transcriptional oscillation based on relative values derived from densitometric measurements of PCR-amplified DNA bands in panels A and B expressed as percentages of the highest recorded value in each respective data set.
Figure 2
Analysis of GSK3α knockdown efficacy in a GSK3β nullizygous background. Puromycin-resistant, GSK3α-knockdown MEF lines were generated from GSK3β-/- MEFs as described in the Materials and Methods. A total of 13 individual clones (A1.3-A1.15) were subsequently isolated, expanded, and tested by Western blotting to determine the level of GSK3α knockdown, as well as any concomitant increase in cytosolic levels of β-Catenin protein. GAPDH levels were used as a loading control.
Figure 3
Circadian oscillation profiles of mPer2 following pharmacological or genetic GSK3 inhibition. The transcriptional profile mPer2 (A) was analyzed by reverse-transcription PCR in: wild-type, GSK3β-/-/GSK3αRNAi (clones A1.4 and A1.6); as well as 20 mM Lithium or 25 μM kenpaullone treatment in a wild-type background. The subjective time points of peak expression are designated in white above the corresponding bands for each transcript examined. The time intervals where these effects are most visible (TP0-4, TP24-32, and TP44-52) are isolated in white boxes. The effects of genetic (B) and pharmacological (C) interference of GSK3 activity on mPer2 transcriptional oscillation are graphically depicted based on relative values derived from densitometric measurements of PCR-amplified DNA bands in panel A expressed as percentages of the highest recorded value in each respective data set.
Figure 4
Analysis of total GSK3 expression prior to and following serum-shock. To verify that effects observed at the level of transcription in clones A1.4 and A1.6 corresponded to an expected level of GSK3 knockdown, at the indicated time points, protein and RNA samples were simultaneously isolated from harvested wild-type, A1.4, and A1.6 cells in order to monitor levels of GSK3 expression prior to and following serum shock. RNA samples were subsequently used for reverse-transcription PCR analysis (as seen in Fig. 3), while the protein samples were subjected to Western blot analysis. Protein samples were harvested at TP0, TP4, TP24, TP32 and TP48, and blotted for total GSK3. β-Actin levels were used as a loading control.
Figure 5
Circadian oscillation profile of mPer2 in wildtype and GSK3β(-/-); GSK3α(flox/-) MEFs. The transcriptional profile of mPer2 and GAPDH were analyzed by reverse-transcription PCR in the A6(wt) and c2.1(3/4 DKO) cell lines, as depicted in panels A and B, respectively. Protein samples harvested from whole-cell lysates in parallel to RNA samples harvested for transcriptional analysis at corresponding time points were analyzed by SDS-PAGE electrophoresis. Western blot analysis of these protein samples for total-GSK3 and GAPDH is depicted in Panel C for TPs 0, 4, 12, 24, 30, and 36. Panels D and E are graphical depictions of relative levels of mPer2 expression in A6 and c2.1 based on three separately harvested A6 RNA sample sets and six c2.1 RNA sample sets. Relative values derived from densitometric measurements of PCR-amplified DNA bands are expressed as percentage values of mPer2 at TP1.