Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a TIMP3 mRNA levels of oral cancer tissues and corresponding normal tissues. The TIMP3 mRNA levels of corresponding normal tissues were set as 1. Vertical bars, paired samples. N, corresponding normal tissue. T, oral cancer tissue. b TIMP3 protein levels of oral cancer tissues and corresponding normal tissues, β-actin was used as loading control. N, corresponding normal tissue. T, oral cancer tissue. c TIMP3 protein and mRNA levels of oral cell lines, β-actin and GAPDH was used as loading control. d TIMP3 mRNA levels of oral cell lines were detected by real-time PCR, GAPDH was used as internal control. e TIMP3 methylation levels of CpG island in HNSCC tissues and normal tissues from MethHC database. f The correlation between TIMP3 methylation levels of CpG island and TIMP3 mRNA levels from MethHC database. g TIMP3 mRNA levels of oral cancer cell lines after treatment of 5-aza. h Overview of the TIMP3 CpG locations. Three sequences a (CpG sites: 1–9), b (CpG sites: 10–13), and c (CpG sites: 14–26) were analyzed by pyrosequencing after amplifying P1 and P2 fragments. i The average methylation levels of a, b, and c sequences in oral cell lines. j The correlation between average methylation levels of three sequences and TIMP3 mRNA levels in oral cell lines

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Control, Real-time Polymerase Chain Reaction, Methylation

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Western blot results of Sp1 and TIMP3 after transfection of GFP vector and GFP-SP1 vector, β-actin was used as internal control. b Western blot results of Sp1 and TIMP3 after knockdown of Sp1, β-actin was used as internal control. c TIMP3 promoter activity after transfection of Sp1 overexpression vector, β-gal was used to normalize transfection efficiency. * p < 0.05 compared with GFP. d TIMP3 promoter activity after mutation of the Sp1 binding sites, β-gal was used to normalize transfection efficiency. * p < 0.05 compared with pGL3-TIMP3. e SCC9 and TW2.6 cells were transfected with the control siRNA or Sp1 siRNA. After 24 h, cells were treated with the vehicle control (DMSO) or 5-aza (10 μM) for 96 h before total RNA was subjected to qPCR analysis. * p < 0.05 compared with treatment of scrambled siRNA and DMSO. # p < 0.05 compared with treatment of Sp1 siRNA and DMSO. f SCC9 and TW2.6 cells were treated with the vehicle control (DMSO) or 5-aza (10 μM) for 96 h and were subjected to immunoprecipitation with an antibody against Sp1, DNMT1, and DNMT3B. The precipitates were subjected to PCR amplification using primers directed to Sp1 binding site of the TIMP3 promoter. g DNMT1 and DNMT3B levels in HNSCC tissues and normal tissues from TCGA database. h DNMT1 and DNMT3B levels in oral cancer cell lines and normal oral cell lines. i The mRNA expression of DNMT1 and DNMT3B after transfection of DNMT1 siRNA or DNMT3B siRNA. * p < 0.05 compared with scrambled siRNA. j TIMP3 levels after knockdown of DNMT1 or DNMT3B in SCC9 and TW2.6 cells. * p < 0.05 compared with scrambled siRNA

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Knockdown, Activity Assay, Over Expression, Mutagenesis, Binding Assay, Immunoprecipitation, Amplification, Expressing

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Western blot of SCC9 and TW2.6 stable clones, β-actin was used as internal control. b Cell proliferation was analyzed by MTT assay. The cell number in first day was set as 1 and used for normalization. c Clones were wounded for 0 h, 24 h, and 48 h (SCC9) or 0 h, 3 h, and 6 h (TW2.6). Phase-contrast pictures of the wounds at three different locations were taken. d Migration and invasion abilities were measured after 24 h and 48 h. * p < 0.05 compared with control group. e Migration and invasion abilities of SCC9 and TW2.6 cells exposed to their own stable conditioned medium (CM−: control cells; CM+: SCC9-T9 or TW2.6-T18) as chemoattractant, or to f recombinant TIMP3 protein (rTIMP3). g Migration and invasion abilities of SCC9-T9 and TW2.6-T18 after transfection with scrambled siRNA or TIMP3 siRNA. * p < 0.05 compared with scrambled siRNA

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Western Blot, Clone Assay, Control, MTT Assay, Migration, Recombinant, Transfection

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Morphology and cell size of SCC9 and TW2.6 stable clones. * p < 0.05 compared with control cells. b Adhesion assays of oral stable cells were performed by seeding cells for 30 min on plates coated with collagen. * p < 0.05 com p ared with control cells. c Heat map including 84 EMT-related genes in SCC9-control and SCC9-T9 cells was assessed by Human OneArray ® . Red arrows indicate the downregulation of fibronectin (FN1) and upregulation of E-cadherin (CDH1) in TIMP3 overexpression SCC9-T9 cells. d EMT markers of stable clones were analyzed by real-time PCR. The relative mRNA expression was normalized to GAPDH. * p < 0.05 compared with the control cells. e Western blot of EMT-related protein expression. β-actin was used as loading control. f Western blot of EMT-related protein expression after transfection of scrambled siRNA or TIMP3 siRNA. β-actin was used as loading control. g Knockdown of E-cadherin by siRNA. h Adhesion ability of TIMP3 stable cells after E-cadherin knockdown for 2 days. i Migration ability of TIMP3 stable cells after E-cadherin knockdown for 2 days

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Clone Assay, Control, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Knockdown, Migration

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a The E-cadherin promoter contains three E-box sites was cloned into the luciferase reporter vector. b The E-cadherin promoter activity of SCC9 and TW stable cells after transfection of scrambled siRNA or TIMP3 siRNA. * p < 0.05 compared to control stable cell treated with scrambled siRNA. # p < 0.05 com p ared to TIMP3 stable cell treated with scrambled siRNA. c The mRNA levels of EMT-related transcription factors in TIMP3 stable clones. The relative mRNA expression was normalized to GAPDH. * p < 0.05 compared with the control cells. d The mRNA levels of EMT-related transcription factors of TIMP3 stable cells after transfection with scrambled siRNA or TIMP3 siRNA. The relative mRNA expression was normalized to GAPDH. * p < 0.05 compared with the scrambled siRNA. e The protein expression of EMT-related transcription factors. β-actin was used as loading control. f The protein expression of EMT-related transcription factors after transfection with scrambled siRNA or TIMP3 siRNA. β-actin was used as loading control

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Control, Stable Transfection, Expressing

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a The protein expression of EMT-related signaling pathway in oral stable cells. β-actin was used as loading control. b The protein expression of EMT-related signaling pathway in TIMP3 stable cells after transfection of scrambled siRNA or TIMP3 siRNA. β-actin was used as loading control. c EMT-related protein expression after treatment of PD98059 for 48 h. β-actin was used as loading control. d Migration and invasion abilities after treatment of PD98059 for 48 h. * p < 0.05 compared with DMSO. e EMT-related protein expression after transfection of TIMP3 siRNA for 24 h and treatment of PD98059 for another 24 h. β-actin was used as loading control. f Migration and invasion abilities after transfection of TIMP3 siRNA for 24 h and treatment of PD98059 for another 24 h. * p < 0.05 compared to treatment with TIMP3 siRNA and DMSO

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Expressing, Control, Transfection, Migration

Journal: Cell Death & Disease

Article Title: Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

doi: 10.1038/s41419-019-2016-0

Figure Lengend Snippet: a Luciferase activity image of mice after injecting with luciferase-tagged TW2.6/pcDNA3 or TW2.6/TIMP3 cells. b After 35 days of tumor cell injection, tumors from six mice injected with TW2.6/pcDNA3 or TW2.6/TIMP3 were quantified by measuring the photon influx. c , d Lymph node metastasis was imaged at the end of the study with the mean signal for each group indicated ( n = 6). * p < 0.05 compared with the TW2.6/pcDNA3 groups. e , f Macroscopic analysis of neck lymph nodes. The appearance, number, and volume of neck lymph nodes were photographed, enumerated, and measured after removal. * p < 0.05 compared with the TW2.6/pcDNA3 groups. g Proposed model for the role of TIMP3 methylation contributes to oral cancer metastasis

Article Snippet: TIMP3 (MAB3318) antibodies were purchased from Millipore (Billerica, MA, USA).

Techniques: Luciferase, Activity Assay, Injection, Methylation