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

Electrospun Microfiber Membranes Embedded with Drug-Loaded Clay Nanotubes for Sustained Antimicrobial Protection

Guided tissue regeneration/guided bone regeneration membranes with sustained drug delivery were developed by electrospinning drug-loaded halloysite clay nanotubes doped into poly(caprolactone)/gelatin microfibers. Use of 20 wt % nanotube content in fiber membranes allowed for 25 wt % metronidazole drug loading in the membrane. Nanotubes with a diameter of 50 nm and a length of 600 nm were aligned within the 400 nm diameter electrospun fibers, resulting in membranes with doubling of tensile strength along the collector rotating direction. The halloysite-doped membranes acted as barriers against cell ingrows and have good biocompatibility. The metronidazole-loaded halloysite nanotubes incorporated in the microfibers allowed for extended release of the drugs over 20 days, compared to 4 days when directly admixed into the microfibers. The sustained release of metronidazole from the membranes prevented the colonization of anaerobic Fusobacteria, while eukaryotic cells could still adhere to and proliferate on the drug-loaded composite membranes. This indicates the potential of halloysite clay nanotubes as drug containers that can be incorporated into electrospun membranes for clinical applications

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

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

Inhibition of HIF-1α by siRNA results in upregulations of Cyclin D1 and c-Myc expressions in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

HIF-1α inhibits Wnt pathway in vitro.

<p>(A) HIF-1α inhibited Topflash reporter activity in a dose-dependent manner. HEK293 cells were transfected with a Topflash reporter along with 50 ng β-catenin without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ±S.D. (B) HIF-1α cooperated with Osx to inhibit Wnt pathway activity. HEK293 cells were transfected with a Topflash reporter along with 25 ng β-catenin without or with different groups of HIF-1α expression plasmid and Osx plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values were presented as the mean ±S.D.</p

Inhibition of HIF-1α by siRNA results in downregulation of Sost expression in osteoblasts.

<p>MC3T3 osteoblasts were transfected with siRNA control or siRNA against HIF-1α. RNA was isolated 24 hr post-transfection and quantitated by quantitative real-time RT-PCR for HIF-1α and Sost, and HSP90 was used as a negative control. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Hypoxia inhibits osteoblast proliferation.

<p>(A) Osteoblast number counts in the growth medium. MC3T3 osteoblastic cells were cultured in Alpha Minimum Essential Medium, and maintained for different time points as indicated from 4 hr to 72 hr in normoxic (20%O₂) or hypoxia (1%O₂) condition. (B) Inhibition of HIF-1α expression by siRNA resulted in an increase of osteoblast growth. MC3T3 osteoblastic cells were transfected by siRNA, and cultured under hypoxia for 48 hr. si-control: si-RNA control; si-HIF-1α: si-RNA against HIF-1α. A paired <i>t</i>-test was performed comparing si-control group and si-HIF-1α group. *: A star indicates statistical significance compared to control group.</p

Effect of HIF-1α on Sost promoter activity.

<p>(A) HIF-1α activates the <i>Sost</i> promoter in a dose-dependent manner. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of an HIF-1α-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D. (B) Jab1 does not activate <i>Sost</i> promoter activity. HEK293 cells were transfected with a 1 kb <i>Sost</i> promoter-luciferase reporter gene without or with increasing amounts of a Jab1-expression plasmid as indicated. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.</p