Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: TAOB-CMs increases lung cancer migration and EMT. TAOB-CMs enhanced the migratory ability of A549 and CL1–0 lung cancer cells. A and B, TAOB-CMs enhanced cell migratory ability, as determined by scratch wound healing assay (A) and Transwell system (B). C, TAOB-CMs enhanced cell invasion ability. D, TAOB-CMs caused EMT in cancer cells. E, the expression of BMP-2 in OB-CM, For A, the migration ability of lung cancer cells was assessed by wound healing assay. OB-CM (control group) and TAOB-CMs (20%) act as a chemoattractant of cancer migration. Quantification of cell migration was carried out by measuring the distance between the migratory fronts of cells in four random selected microscopic fields for each condition and time point. The degree of cell movement is expressed as the percentage of wound closure as compared with the zero time point. For B and C, the invasiveness and migration ability of A549 and CL1–0 cells were quantified by QCMTM 24-well cell migration and invasion assay. The cells were seeded in the upper inset, and the OB-CM (control group) and TAOB-CMs (20%) acted as the chemoattractant for cancer migration and invasion. For D, A549 and CL1–0 cells were treated with TAOB-CMs (20%) for 24 h, and then the expression of various proteins was assessed by immunoblot assay. For E, primary osteoblasts were treated RPMI 1640 (20%), A549-CM (20%), and CL1–0-CM for 24 h. The BMP-2 levels were assessed by BMP-2 ELISA kits. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Wound Healing Assay, Expressing, Invasion Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: BMP-2 is involved in TAOB-CM-mediated enhancement of migration and EMT in lung cancer. A and B, BMP-2 increased migratory ability, as determined by scratch wound healing assay (A) and Transwell system (B). C and D, BMP-2 increased the invasion ability (C) and EMT (D) of A549 and CL1–0 cells. E and F, noggin decreased TAOB-CM-mediated cell migration (E) and EMT (F). The migration ability of lung cancer cells was assessed by wound healing assay, in accord with the description under “Experimental Procedures.” BMP-2 (20 ng/ml for EMT assay) acts as the chemoattractant for cancer migration. For E and F, A549 and CL1–0 cells were pretreated with or without noggin for 1 h, and then OB-CM and TAOB-CMs were added for another 24 h. Cell migration was assessed by wound healing assay, and the expression of various proteins was determined by immunoblot assay. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Wound Healing Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: Sera from lung cancer patients increase lung cancer migration. A, the levels of BMP-2 in lung cancer patient sera. B, lung cancer sera enhance the migratory ability of lung cancer cells. C, depletion of BMP-2 decreased lung cancer patient serum-mediated cell migration. The levels of BMP-2 were assessed by ELISA. Horizontal bars represent means. The cells were treated with or without noggin for 1 h, and then culture medium containing healthy donor sera (15%) or lung cancer patient sera (15%) was added for another 24 h. Cell migration was assessed by wound healing assay. For C, BMP-2 depleted from lung cancer patient serum was performed using anti-BMP-2 and antibodies (4 μg/ml) and Sepharose A/G beads, following regular immunoprecipitation techniques. The migration ability of A549 and CL1–0 cells were quantified by QCMTM 24-well cell migration assay kit. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Immunoprecipitation, Cell Migration Assay

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: TAOB-CMs and BMP-2 increase the activation of MAPK and elevate the expression of Runx2 and Snail. A and B, TAOB-CMs (A) and BMP-2 (B) increase the phosphorylation of SMAD, ERK, and p38. C and D, TAOB-CMs (C) and BMP-2 (D) enhance the expression of Runx2 and Snail protein. Cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for the indicated times. The expressions of various proteins were determined by immunoblot assay. E, TAOB-CMs and BMP-2 enhance the expression of Runx2 and Snail mRNA. The cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for a specific time (3 h for snail and 12 h for E-cadherin). The expressions of mRNA were determined by quantitative PCR. F, noggin decreases TAOB-CM-mediated MAPK activation and Runx2 and Snail up-regulation. The cells were treated with OB-CM (20%), TAOB-CMs (20%), or BMP-2 (20 ng/ml) for the indicated times. The expressions of mRNA and various proteins were determined by quantitative PCR and immunoblot assay. For F, A549 and CL1–0 cells were pretreated with or without noggin for 1 h and then treated with BMP-2 (20 ng/ml) for 6 h. The expression of various proteins was then assessed by immunoblot assay. The data shown are representative of three independent experiments. Each value is the mean ± S.D. of three independent experiments. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05).

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Activation Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway *

doi: 10.1074/jbc.M111.256156

Figure Lengend Snippet: Runx2 is the upstream regulatory factor of Snail. A and B, inhibition of Runx2 decreases BMP-2-mediated Snail up-regulation and E-cadherin down-regulation (A), as well as cell migration (B). Cells were transfected with pLKO-AS2 or pLKO-AS2-RUNX2 shRNA. Stable clones were created by puromycin selection, and the efficacy of shRNA was assessed by RT-PCR. Cells were treated with BMP-2 (20 ng/ml) for the specified times (cell migration, 24 h; Runx2 and Snail, 6 h; E-cadherin, 24 h). Then the expression of various proteins was then assessed by immunoblot assay. C, overexpression of Snail reversed the inhibitory effect of Runx2 shRNA on BMP-2-mediated cell migration. Runx2-transfected A549 and CL1–0 cells were transected with pCMV or pSnail plasmid, and stable clones were established by G418 and puromycin. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett's test (p < 0.05). The data shown are representative of three independent experiments.

Article Snippet: Runx2-transfected A549 and CL1–0 cell were transected with pCMV or pSnail plasmid (Origene, Rockville, MD), and stable clones were established by G418 and puromycin.

Techniques: Inhibition, Migration, Transfection, shRNA, Clone Assay, Selection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Plasmid Preparation