Table 1
Real time PCR Primers.
| Gene | Species | Genebank accession | Amplicon length |
|---|---|---|---|
| Per2 | Mouse | NM_011066.3 | 73 |
| HPRT | Mouse | NM_013556.2 | 131 |
| Reverba | Mouse | NM_145434.4 | 62 |
| BMAL | Mouse | NM_001243048.1 | 87 |
| PER3 | Mouse | NM_001289877.1 | 73 |
| RoRa | Mouse | NM_001289916.1 | 68 |
| STRA13 | Mouse | NM_016665.2 | 65 |
| TuBB | Mouse | NM_023279.217 | 58 |
| Hes 5 | Mouse | NM_010419.412 | 73 |
| Neurod1 | Mouse | NM_010894.224 | 94 |
Table 2
Primary and Secondary Antibodies used in this study.
| Antibody | Source | Dilution | Company | Cat. Num. |
|---|---|---|---|---|
| Primary Ab | ||||
| Beta-Actin | Mouse | 1:1000 | Cell Signalling | 3700S |
| Beta-3-Tubulin | Mouse | 1:1000 | Cell Signalling | 4466S |
| SEAA1 | Mouse | 1:1000 | Cell Signalling | 4744S |
| Secondary Ab | ||||
| Goat-Anti-Mouse | Mouse | 1:3000 | Bio-Rad | 170–6516 |
Figure 1
Characterization of P19 cells used in the study. P19 cells were grown in differentiation media containing 1 μM retinoic acid (RA). Images were taken using an Evos microscope after 4 days of aggregation (A), and after P19 cells had formed neurons after differentiation treatment for 4 days (B). To further validate the neuronal differentiation, immunoblot (E) analysis was performed to show expression levels of the stem cell marker SSEA1, and the neuron-specific marker β-3-tubulin in control (untreated) and differentiated (treated with 1 μM RA) P19 cells. β-actin was used as a control or housekeeping protein, and was run on a separate gel, as shown in the supplementary information. SSEA1 and β-3-tubulin were run together in another gel, as shown in the supplementary information. Immunostaining with SSAE1 was also performed (C) and (D). The red color is SSEA1 staining (C), and the blue color in both (C) and (D) shows nuclear Hoechst staining. The gene expression levels of Hes5, Neurod1 and Tubb3 were analyzed in untreated and differentiated P19 cells (F). Results show that the expression profile of the differentiated cells is visibly higher than that of the control.
Figure 2
Gene expression analysis of circadian clock genes in P19 embryonic stem cells. P19 cells were grown as aggregates for 2 and 4 days, and then differentiated for 2 and 4 days in the absence (–) and presence (+) of 1 μM RA. Expression of Per2 (A), ARNTL (Bmal) (B), Rev-erb-α (C), Per3 (D), ROR-α (E), and Stra13 (F) were analyzed by qPCR. Data show that expression of clock genes is dynamic during the different phases of aggregation and differentiation by P19 cells, and the expression of Stra13 increases in the presence of RA.
Figure 3
Oscillation of the expression of circadian clock genes in P19 embryonic stem cells. Blue line shows undifferentiated P19 cells (control); red line shows P19 cells differentiated by treatment with 1 μM retinoic acid (RA) for 4 days (differentiated). Cells were synchronized by temperature change and then collected after 0, 6, 12, 18, 24 and 30 hours for RNA extraction. Gene expression was analyzed by qPCR of Per2 (A), ARNTL (Bmal) (B), Rev-erb-α (C), Per3 (D), ROR-α (E) and Stra13 (F) genes. Each time point was performed in triplicate. Data show means ± standard deviation. Per2 and Rev-erb-α show weak oscillation patterns, while ARNTL (Bmal), Per3 and RoR-α show significant oscillation patterns in both the control and differentiated cells. Stra13 also shows a strong oscillation in the presence of RA.
Figure 4
Oscillation of neuronal differentiation markers in differentiated P19 embryonic stem cells. P19 cells were grown for 4 days in the presence of 1 μM retinoic acid (RA). Cells were synchronized by temperature change and then collected after 0, 6, 12, 18, 24, and 30 hours before RNA extraction. Gene expression was analyzed by qPCR of Hes5 (A), Neurod1 (B), and Tubb3 (C). Each time point was performed in triplicate. Data shows means ± standard deviation. Differentiated P19 cells show clear oscillation of Hes5 and Tubb3, while no oscillation of Neurod1 was detected.
Figure 5
Expression of clock genes in P19 cells treated with clock-associated drugs. Using P19 cells, we tested several ligands of known factors associated with the biological clock. DMSO was used as a control; other drugs used were Sirtinol, EX527, SR8278, and GSK4112. The effect of these drugs on the expression of Per2 (A), ARNTL (Bmal) (B), Reverb-α (C), Per3 (D), ROR-α (E), and Stra13 (F).
Figure 6
Gene expression analysis of circadian clock genes in differentiated P19 cells in the presence of dimethylase and kinase inhibitors. Differentiated P19 cells were seeded in a 6-well plate, then each well was treated with 10 μM of the indicated compound, or 1 μl of DMSO as control. After overnight culture, cells were collected for RNA extraction and qPCR analysis of the clock genes Per2 (A), ARNTL (Bmal) (B), Rev-erb-α (C), Per3 (D), ROR-α (E) and Stra13 (F). Data shows that TCS-ERK IIe elevates the expression of ARNTL (Bmal) and Rev-erb-α, but has no effect on ROR-α. Expression of Per3 and ARNTL (Bmal) increased more than Rev-erb-α and ROR-α, and by LY294002. 5-Azacytidine increased the expression of all clock genes at almost the same level.
Figure 7
Effect of clock gene-modulating compounds on neuronal differentiation of P19 embryonic stem cells. P19 cells were differentiated using 1 μM retinoic acid (RA) for 5 days in the absence (DMSO) or presence of 2 μM final concentration of indicated compounds. After 5 days of differentiation, cells were collected for RNA extraction and qPCR analysis of the neuronal markers Hes5 (A) and Neurod1 (B). The expression of Hes5 and Neurod1 increased by 3–4 times when treated with 2 μM Sirtinol during differentiation, while TCS-ERK IIe changed the expression of either gene in cells.