Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT + Bmi-1.
Bmi-1 antibody was from Zymed, CA, USA.
However, mechanisms regulating Bmi-1 chromatin association are still unclear.
It was demonstrated that BMI-1 protein level was significantly downregulated following BMI-1 knockdown treatment (Figure 2C).
As shown in Figure 2B, endogenous BMI-1 mRNA was obviously reduced in BMI-1 knockdown group.
Our results demonstrate that Akt signaling causes Bmi-1 phosphorylation in NSCs resulting in up-regulation of Bmi-1.
However, inhibition of the Akt-dependent Bmi-1 stabilizing process by p38 MAPK signaling reduces the levels of Bmi-1.
These results suggest that although Bmi-1 contains no known p38 phosphorylation motif (http://www.scansite.edu), p38 may also phosphorylate Bmi-1 when Akt is unable to phosphorylate Bmi-1 under oxidative stress conditions.
Here we show that Bmi-1 may be a substrate of Akt and up-regulation of Akt signaling coincides with up-regulation of Bmi-1 phosphorylation and that phosphorylated Bmi-1 is more stable.
Bmi-1 was originally isolated as a collaborator with Myc in tumorigenesis [46], [47], and Myc has been shown to directly activate Bmi-1 [48].
To suppress BMI-1 expression in osteosarcoma cells, short harpin RNA (shRNA) targeting BMI-1 gene was designed and inserted into the recombinant lentivirus plasmid.
