BMI-1 depletion sensitized cells to cisplatin treatment through PI3K/AKT pathway.

<p>After infection, SAOS-2 cells were treated with 10 µg/ml cisplatin for 24 h and subjected to Annexin V-PI Apoptosis analysis (A) and measurement of caspase-3 and caspase-9 activities (B). (C) Western blot analysis of p-AKT, AKT, BCL-2 and Bid protein. *: <i>P</i><0.01, compared to control cells. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant, Annexin V⁻/PI⁻: viable cells, Annexin V⁺/PI⁻: cells in early apoptosis, Annexin V⁺/PI⁺: cells in late apoptosis, Annexin V⁻/PI⁺: cells in necrosis.</p

Targeted depletion of BMI-1 through lentivirus-mediated siRNA.

<p>(A) Representetive graphs of SAOS-2 cells infected with indicated lentivirus at MOI of 10 were shown (×400). Following infection of cells with indicated lentivirus for 5 days, BMI-1 mRNA levels were measured with real-time PCR (B), and protein levels were detected by Western blot analysis (C). *: <i>P</i><0.01, compared to control cells. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant.</p

Downregulation of BMI-1 suppresses osteosarcoma cell migration.

<p>(A) Statistical plots of haptotactic migration assay. Columns, mean of three individual experiments; bars, SD;*: <i>P</i><0.01, compared to indicated cells. (B) Representative photos of haptotactic migration assay. Con: non-infected, NC: non-silencing, KD: BMI-1 knock down, Rescue: BMI-1 wobble mutant.</p

Immunohistochemical staining of BMI-1.

<p>BMI-1 immunoreactivity was localized in both the nucleus and cytoplasm. Images of positive BMI-1 staining in nucleus of osteosarcoma, osteochondroma and chondrosarcoma, and in cytoplasm of Ewing's sarcoma were shown (×400). Negative BMI-1 staining in a non-cancerous tissue sample served as control.</p