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

Journal: Oncology Letters

Article Title: Antitumor activity of nimotuzumab in combination with cisplatin in lung cancer cell line A549 in vitro

doi: 10.3892/ol.2018.7923

Figure Lengend Snippet: Cyclin D1 expression in A549 cells in groups A-E following treatment for 48 h. Lanes: A, nimotuzumab; B, cisplatin; C, nimotuzumab followed by cisplatin; D, nimotuzumab and cisplatin simultaneously; and E, untreated control.

Article Snippet: Cell culture The A549 human lung adenocarcinoma epithelial cell line (supplied by the Central Laboratory of Qinhuangdao No. 1 People's Hospital) was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd., Huzhou, China), 1 U/ml penicillin and 1 mg/ml streptomycin at 37°C, with 5% CO 2 and 95% humidity.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Kras-driven intratumoral heterogeneity triggers infiltration of M2 polarized macrophages via the circHIPK3/PTK2 immunosuppressive circuit

doi: 10.1038/s41598-021-94671-x

Figure Lengend Snippet: Monocytic MDSC subsets Gr1 − /CD11b − , Gr1 − /CD11b + elicit M2 macrophage enrichment. ( A ) Relative infiltration rate of MDSC subsets in M1/M2 macrophage regions. MDSC infiltration was evaluated by the presence of gMDSC (Gr − /CD11b − ) and mMDSC (Gr − /CD11b + ) cells in primary and metastatic tissues. ( B ) Induction and infiltration of mMDSCs (CD11bLy6C+) and gMDSCs (CD11b + Ly6G+) in, lung and lymph node tumor. A549 cells (2 × 106) were orthotopically implanted in C57BL/6 mice. Monitoring of MDSCs infiltration started three days post impantation. ( C ) Graph showing positive correlation between the M2/MDSC ratio and M2 macrophage cell numbers. ( D ) Immunofluorescence analysis of Ly6C/Ly6G positive cells in mice bearing A549 tumors (magnification × 200). Confocal microscopy images of Ly6C (green), Ly6G labeled cells (red) and DAPI (blue). Images were captured using Carl Zeiss fluorescence confocal microscope. ( E , F ) Data represent the mean ± SD of three independent experiments (*p < 0.05; **p < 0.01). Differences were considered statistically significant at p < 0.05. Statistically significant data are indicated by asterisks (*p < 0.05, **p < 0.01).

Article Snippet: Human lung adenocarcinoma cells A549 were grown in DMEM, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biochrom AG, Berlin, Germany).

Techniques: Immunofluorescence, Confocal Microscopy, Labeling, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Kras-driven intratumoral heterogeneity triggers infiltration of M2 polarized macrophages via the circHIPK3/PTK2 immunosuppressive circuit

doi: 10.1038/s41598-021-94671-x

Figure Lengend Snippet: Co-inhibition of circPTK2 and M2 polarization reduces tumor volume and metastatic foci development. ( A ) Characteristic images of excised tumors from mice following si-circPTK2/PLX-3397 treatment. ( B ) Tumor volume analysis of mice treated at various groups. ( C ) In vivo metastatic analysis of lymph node metastasis from si-circPTK2/PLX-3397-treated groups. Images show representative lymph node metastatic foci highlighted in yellow from different groups. ( D ) Statistical analysis of the number of metastatic foci of each group. ( E ) Representative photographs of excised tumors from C57BL/6 mice after intravenous treatment with si-circPTK2/PLX-3397. ( F ) Relative tumor volumes (V/V0) of A549 tumor bearing mice following intravenous administration. ( G ) Survival rates of tumor-bearing mice after a 60-day tumor challenge in each group. Data were given as the mean ± SD (n = 6) (*p < 0.05, **p < 0.01). Data represent the mean ± SD of three independent experiments.

Article Snippet: Human lung adenocarcinoma cells A549 were grown in DMEM, supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biochrom AG, Berlin, Germany).

Techniques: Inhibition, In Vivo

Journal: The EMBO Journal

Article Title: The EGFR phosphatase RPTPγ is a redox‐regulated suppressor of promigratory signaling

doi: 10.15252/embj.2022111806

Figure Lengend Snippet: A Left: Western blot showing endogenous p22 phox (top row) and GAPDH (loading control; bottom row) in cell lysates obtained from different clones of MCF7 cells subjected to CRISPR‐Cas9‐mediated p22 phox ‐KO (lanes 1–9) and MCF7 WT cells (last lane). Middle: Western blot showing Akt and phospho‐Akt (pS473) in lysates of MCF7 WT cells (lane 1) and selected p22 phox ‐KO cell lines (lanes 2–5) treated for 5′ with 160 ng/ml EGF. Right: Same as middle for Erk and phospho‐Erk (pT202 and pY204). B Western blot showing endogenous RPTPγ (top row) and Na + /K + ATPase (loading control; bottom row) in membrane protein extracts of MCF7 WT cells (first lane) and different clones of MCF7 cells subjected to CRISPR‐Cas9‐mediated RPTPγ‐KO (lanes 2–6). C, D (C) Representative western blots showing EGFR (left), Akt (middle) and Erk (right) in the top rows with corresponding phosphorylation (middle row: EGFR: pY1068, Akt: pS473, Erk: pT202 and pY204) in WT (lanes 1–4) and p22 phox ‐KO (lanes 5–8) MCF7 cells, without EGF and upon 5′ stimulation with EGF‐Alexa647 (20, 80 and 160 ng/ml). Bottom row: GAPDH (loading control). (D) Same arrangement as (C) for WT and RPTPγ‐KO MCF7 cells. E Top panel: Representative western blot showing phosphorylated EGFR at tyrosine 1068 (pY1068) in MCF7 WT and HT29 cells, without EGF and upon 5′ stimulation with EGF‐Alexa647 (20, 80 and 160 ng/ml). Bottom graph: Quantification with mean ± SD, N = 3 biological replicates, P : unpaired two‐tailed t ‐test. F Top panel: Representative confocal micrographs of immunostained endogenous EGFR (left image), phosphorylated EGFR at tyrosine 1068 (middle image: pY1068) and ectopically expressed RPTPγ‐mTFP (right image) in HT29 cells in absence of EGF‐stimulus. Scale bar: 10 μm. Bottom panel: Quantification of phosphorylated (pY1068) over total EGFR staining in cells without (blue) and with (yellow) RPTPγ‐mTFP expression. Individual cells with mean ± SD, N = 3, n > 75 cells per condition, P : unpaired two‐tailed t ‐test. G Representative western blots showing EGFR (left), Akt (middle) and Erk (right) in the top rows with corresponding phosphorylation (lower row: EGFR: pY1068, Akt: pS473, Erk: pT202 and pY204) in WT (lanes 2–5) and RPTPγ‐KO (lanes 6–9) MCF7 cells treated with 10 μM of EGFR‐inhibitor gefitinib for 1 h, without EGF and upon 5′ stimulation with EGF‐Alexa647 (20, 80 and 160 ng/ml). Lane 1: WT MCF7 cells treated with 80 ng/ml EGF in the absence of gefitinib. H Representative fluorescence micrographs of EGF‐Alexa647 (green) bound to EGFR in WT MCF7 cells at the corresponding, indicated concentrations applied for 5′. Blue: Hoechst33342, scale bar 10 μm. Insets: Individually contrast‐stretched fluorescence micrographs.

Article Snippet: HT29 cells (HTB‐38, LGC Genomics GmbH) were cultured in Ham's F12 culture medium (PAN‐Biotech GmbH) supplemented with 2 mM L‐Glutamine, 1% (NEAA) and 10% FBS.

Techniques: Western Blot, Control, Clone Assay, CRISPR, Membrane, Phospho-proteomics, Two Tailed Test, Staining, Expressing, Fluorescence