The Interaction between Bmal1 and Per2 in Mouse BMSC Osteogenic Differentiation

Figure 1

The decreased expression of Bmal1 and/or Per2 in mRNA and protein levels after virus transfection in BMSCs. (a) Expression of GFP in BMSCs infected by GFP-expressing lentivirus and Bmal1 shRNA lentivirus. After the BMSCs were infected for 48 h, we can observe abundant GFP. Magnification is 200x. (b) The expression level of Bmal1 mRNA 48 h after transfection. (c) Expression of RFP in BMSCs infected by RFP-expressing adenovirus and Per2 shRNA adenovirus. After the BMSCs were infected for 48 h, we can observe abundant expression of RFP. Magnification is 100x. (d) The expression level of Per2 mRNA 48 h after transfection. (e) Expression of GFP and RFP in the Bmal1 shRNA lentivirus- and Per2 shRNA adenovirus-transfected BMSC group (group 6, Lenti-Bmal1 shRNA-Adeno-Per2 shRNA) and the GFP-expressing lentivirus- and RFP-expressing adenovirus-transfected BMSC group (group 7, Lenti-GFP-Puro- Adeno- RFP-Puro). After the BMSCs were infected for 48 h, we can observe abundant expression of GFP and RPF. Magnification is 100x. (f–h) The expression level of BMAL1 and PER2 protein of BMSCs in each group 48 h after transfection. 1, 2, 3, 4, 5, 6, and 7 represented the Vacant, Lenti-Bmal1 shRNA, Lenti-GFP-Puro, Adeno-Per2 shRNA, Adeno-RFP-Puro, Lenti-Bmal1 shRNA-Adeno-Per2 shRNA, and Lenti-GFP-Puro-Adeno-RFP-Puro groups, respectively. The Bmal1 shRNA lentivirus (in group 2) weakened the expression of BMAL1 protein compared with the GFP-expressing lentivirus (in group 3), and it also downregulated the expression of PER2. The Per2 shRNA adenovirus (in group 4) weakened the expression of PER2 protein compared with the RFP-expressing adenovirus (in group 5), and it also downregulated the expression of BMAL1. In addition, the Bmal1 shRNA lentivirus and Per2 shRNA adenovirus (in group 6) weakened the expression of BMAL1 and PER2 protein compared with the GFP-expressing lentivirus and RFP-expressing adenovirus (in group 7). β-actin served as the endogenous reference gene. Densitometry by Quantity One software was used to quantify the protein level, and the protein level was displayed as the ratio of BMAL1 and PER2 relative to β-actin. Data represent the mean ± SD (); the asterisk indicates the group compared to the control groups, that is, group 1, group 3, group 5, or group 7. , , and . The pound key indicates a significant difference between the two groups (^##, ^###).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)