Overexpression of METRNL suppresses AP-1 transcription factor complex activity in 293T cells.

<p>In cells co-transfected with pCDNA3.1-METRNL, pAP-1, and pRL-TK, METRNL suppressed basal endogenous AP-1 transcription factor complex activity. METRNL also inhibited AP-1 transcription factor complex activation in cells treated with exogenous AP-1 activity stimulator (PMA and ionomycin). Data showing means from three independent experiments are presented after normalization for Renilla luciferase activity. *P < 0.01</p

Stable overexpression of METRNL may inhibit MG63 cell differentiation.

<p>(A) METRNL-EGFP fusion protein was highly expressed in the cytoplasm of MG63 cells. (B) Real-time PCR data suggest that METRNL mRNA levels in the METRNL overexpression group were significantly higher than those in the control. (C) Overexpression of METRNL suppresses mineralized nodule formation in MG63. Mineralized nodule formation is observed in EGFP-overexpressing cells in mineralizing medium. Mineralized nodule formation is absent in cells overexpressing pEGFP-METRNL. (D, E) Quantification of OCN and OPG, detected by RT-PCR after treatment with mineralization condition medium for 7 days and 14 days, revealed remarkable downregulation of these genes in the METRNL overexpression group compared with controls (*P < 0.05).</p

Immunohistochemistry for METRNL protein in rat bone.

<p>(A) In western blotting analyses, METRNL expression is observed in lysates (L) and supernatants (S) of stable CHO cells overexpressing His-tagged METRNL, indicating that METRNL indeed is a secreted protein and the polyclonal antibody was specific to METRNL (N for lysates of CHO cells). (B) METRNL is detected in hypertrophic chondrocytes (→) in the tibial growth plate of three-week-old rats. (C) In the primary spongiosa, METRNL is detected in osteoblasts (→) lining the trabecular bone surfaces, but osteocytes (▼) are absent.(D) In the secondary spongiosa, METRNL protein is detected weakly and diffusely in osteoblasts lining the bone surfaces (→).</p

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

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

Clinical Finding in 50 Patients with LDH.

<p>F, indicates female; LDH, lumbar disc herniation; M, male; P, protrusion; S, sequestration; SE, subligamentous extrusion; TE, transligamentous extrusion; L, lumbar; S, sacral; MRI, magnetic resonance imaging.</p

Malformations in the cervical and thoracic regions of VAD neonates.

<p>(A) Side view of cervical region showing normal skeletal in a control fetus. (B) VAD fetus showing loss of the neural arch in cervical 1. (C) Ventral view of the scapula region showing normal development in a control fetus. (D) VAD fetus showing dysplasias of the scapula. (E) Side view of the sternal elements showing normal development the manubrium, sternebrae and xyphoid process in a control fetus. (F) VAD fetus showing malformation of the sternal elements as well as loss of xyphoid process. (G) Ventral view of the thoraric region showing normal development in a control fetus. (H) VAD fetus showing anomalies of vertebrae in thoraric region as well as rib fusions. (I) Ventral view of the thoraric region showing normal development in a control fetus. (J) VAD fetus showing loss of rib in vertebral 20.</p

HOXD10 is down-expression in degenerative NP tissues.

<p>(A) The protein levels of HOXD10 were down-regulated in four degenerative NP tissues and the idiopathic scoliosis tissues. (B) Western blot analysis of HOXD10 protein expression in three patients whose miR-10b expression was down-regulated in NP tissues compared to the three patients whose miR-10b expression was up-regulated in NP tissues. As compared with control, **p<0.01 and *p<0.05.</p