Journal: Cancer Management and Research

Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

doi: 10.2147/CMAR.S171200

Figure Lengend Snippet: Cd treatment activates Wnt/β-catenin signaling and aberrant methylation of the CK1α promoter in NPC cell lines. Notes: ( A ) Western blot analysis of total β-catenin and CK1α in cCd-treated NPC cells and controls; β-actin was used as a loading control. ( B ) Immunofluorescence staining patterns of β-catenin and CK1α in NPC and CCT-NPC cells. Nuclei were stained with DAPI (magnification 400×). ( C ) Luciferase reporter assays using TOPflash/FOPflash reporter plasmids to assess the activity of Wnt/β-catenin. ( D ) RT-PCR analysis of relative transcript levels of the β-catenin target genes cyclin E, cyclin D1, c-Myc and c-Jun . * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; CK1α, casein kinase 1α; RT-PCR, reverse transcription-PCR.

Article Snippet: After blocking with goat serum for 30 minutes, the fixed and blocked cells were incubated with primary anti-β-catenin (rabbit antihuman, 1:100; Cell Signaling Technology), CK1α (rabbit anti human, 1:100; (Cell Signaling Technology) and actin filament antibodies overnight at 4°C.

Techniques: Methylation, Western Blot, Immunofluorescence, Staining, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

doi: 10.2147/CMAR.S171200

Figure Lengend Snippet: Hypermethylation of CK1α induces a switch in Wnt/β-catenin signaling and malignant progression in CCT-NPC cells. Notes: ( A ) Methylation status of the CK1α promoter analyzed by MS-PCR. ( B ) Western blot analyses of CK1α and β-catenin in CCT-CNE1 cells following treatment with 50 µM 5-aza-CdR for 48 hours. ( C ) RT-PCR analysis of relative transcript levels of β-catenin target genes following treatment of cells with 5-aza-CdR (50 µM). ( D ) Western blot and RT-PCR analyses of the effect of CK1α depletion and (or) 5-aza-CdR treatment on β-catenin expression and downstream gene transcription in CCT-CNE1 cells. ( E ) MTT assays for cell viability of CCT-CNE1 and CCT-CNE2 cells with increasing concentrations of 5-aza-CdR treatment. ( F ) Invasion and migration ability of 5-aza-CdR-treated CCT-CNE1 cells. * P <0.05; ** P <0.01; *** P <0.001, compared with parental cells. # P >0.05, compared with CK1α treated cells. Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; CK1α, casein kinase 1α; MS-PCR, methylation specific-PCR; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: After blocking with goat serum for 30 minutes, the fixed and blocked cells were incubated with primary anti-β-catenin (rabbit antihuman, 1:100; Cell Signaling Technology), CK1α (rabbit anti human, 1:100; (Cell Signaling Technology) and actin filament antibodies overnight at 4°C.

Techniques: Methylation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Migration

Journal: Cancer Management and Research

Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

doi: 10.2147/CMAR.S171200

Figure Lengend Snippet: Hypothesized model for the induction of Cd on Wnt/β-catenin signaling and malignant progression in NPC cells. Notes: Chronic low-dose Cd treatment of NPC cells induces CK1α promoter hypermethylation that downregulates CK1α expression, leading to accumulation and nuclear translocation of β-catenin thereby activating Wnt/β-catenin signaling to promote malignancy. Abbreviations: APC, adenomatous polyposis coli; CK1α, casein kinase α; EMT, epithelial–mesenchymal transition; GSK-3β, glycogen synthase kinase 3β; NPC, nasopharyngeal carcinoma; TCF/LEF, T-cell factor/lymphoid enhancer factor; β-Trcp, β-transducin repeats-containing proteins.

Article Snippet: After blocking with goat serum for 30 minutes, the fixed and blocked cells were incubated with primary anti-β-catenin (rabbit antihuman, 1:100; Cell Signaling Technology), CK1α (rabbit anti human, 1:100; (Cell Signaling Technology) and actin filament antibodies overnight at 4°C.

Techniques: Expressing, Translocation Assay

Journal: Cancer Management and Research

Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

doi: 10.2147/CMAR.S171200

Figure Lengend Snippet: Cd treatment activates Wnt/β-catenin signaling and aberrant methylation of the CK1α promoter in NPC cell lines. Notes: ( A ) Western blot analysis of total β-catenin and CK1α in cCd-treated NPC cells and controls; β-actin was used as a loading control. ( B ) Immunofluorescence staining patterns of β-catenin and CK1α in NPC and CCT-NPC cells. Nuclei were stained with DAPI (magnification 400×). ( C ) Luciferase reporter assays using TOPflash/FOPflash reporter plasmids to assess the activity of Wnt/β-catenin. ( D ) RT-PCR analysis of relative transcript levels of the β-catenin target genes cyclin E, cyclin D1, c-Myc and c-Jun . * P <0.05; ** P <0.01; *** P <0.001. Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; CK1α, casein kinase 1α; RT-PCR, reverse transcription-PCR.

Article Snippet: Western blot was performed as previously described using monoclonal antibodies against β-actin, β-catenin, (1:1,000; Cell Signaling Technology, Beverly, MA, USA), CK1α (1:1,000; Cell Signaling Technology) as well as E-cadherin, N-cadherin and Vimentin (1:1,000; Cell Signaling Technology), at 4°C overnight followed by rinsing and addition of an anti-rabbit/mouse secondary antibody (Gene Tech, Shanghai, People’s Republic of China) at 1:1,000 dilution for 1 hour.

Techniques: Methylation, Western Blot, Immunofluorescence, Staining, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Management and Research

Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

doi: 10.2147/CMAR.S171200

Figure Lengend Snippet: Hypermethylation of CK1α induces a switch in Wnt/β-catenin signaling and malignant progression in CCT-NPC cells. Notes: ( A ) Methylation status of the CK1α promoter analyzed by MS-PCR. ( B ) Western blot analyses of CK1α and β-catenin in CCT-CNE1 cells following treatment with 50 µM 5-aza-CdR for 48 hours. ( C ) RT-PCR analysis of relative transcript levels of β-catenin target genes following treatment of cells with 5-aza-CdR (50 µM). ( D ) Western blot and RT-PCR analyses of the effect of CK1α depletion and (or) 5-aza-CdR treatment on β-catenin expression and downstream gene transcription in CCT-CNE1 cells. ( E ) MTT assays for cell viability of CCT-CNE1 and CCT-CNE2 cells with increasing concentrations of 5-aza-CdR treatment. ( F ) Invasion and migration ability of 5-aza-CdR-treated CCT-CNE1 cells. * P <0.05; ** P <0.01; *** P <0.001, compared with parental cells. # P >0.05, compared with CK1α treated cells. Abbreviations: CCT-NPC, chronic cadmium-treated nasopharyngeal carcinoma; CK1α, casein kinase 1α; MS-PCR, methylation specific-PCR; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: Western blot was performed as previously described using monoclonal antibodies against β-actin, β-catenin, (1:1,000; Cell Signaling Technology, Beverly, MA, USA), CK1α (1:1,000; Cell Signaling Technology) as well as E-cadherin, N-cadherin and Vimentin (1:1,000; Cell Signaling Technology), at 4°C overnight followed by rinsing and addition of an anti-rabbit/mouse secondary antibody (Gene Tech, Shanghai, People’s Republic of China) at 1:1,000 dilution for 1 hour.

Techniques: Methylation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Migration

Journal: Cancer Management and Research

Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

doi: 10.2147/CMAR.S171200

Figure Lengend Snippet: Hypothesized model for the induction of Cd on Wnt/β-catenin signaling and malignant progression in NPC cells. Notes: Chronic low-dose Cd treatment of NPC cells induces CK1α promoter hypermethylation that downregulates CK1α expression, leading to accumulation and nuclear translocation of β-catenin thereby activating Wnt/β-catenin signaling to promote malignancy. Abbreviations: APC, adenomatous polyposis coli; CK1α, casein kinase α; EMT, epithelial–mesenchymal transition; GSK-3β, glycogen synthase kinase 3β; NPC, nasopharyngeal carcinoma; TCF/LEF, T-cell factor/lymphoid enhancer factor; β-Trcp, β-transducin repeats-containing proteins.

Article Snippet: Western blot was performed as previously described using monoclonal antibodies against β-actin, β-catenin, (1:1,000; Cell Signaling Technology, Beverly, MA, USA), CK1α (1:1,000; Cell Signaling Technology) as well as E-cadherin, N-cadherin and Vimentin (1:1,000; Cell Signaling Technology), at 4°C overnight followed by rinsing and addition of an anti-rabbit/mouse secondary antibody (Gene Tech, Shanghai, People’s Republic of China) at 1:1,000 dilution for 1 hour.

Techniques: Expressing, Translocation Assay

Journal: Stem Cells International

Article Title: Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

doi: 10.1155/2017/3508907

Figure Lengend Snippet: Effect of ASP on β -catenin expression of Sca-1 + HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) The protein expression analyses of β -catenin by Western blot. Proteins were extracted from cellular cytoplasm and nucleus, respectively; β -actin was used as the internal control. (b) The cellular localization of β -catenin by immunofluorescence, PI (red) to visualize nucleus. (c) β - catenin mRNA expression by qRT-PCR, β -actin was used as the internal control. Different letters: P < 0.05.

Article Snippet: Slides were then incubated with antibodies against β -catenin (1 : 100) (Cell Signaling Technology, USA) overnight at 4°C.

Techniques: Expressing, Western Blot, Immunofluorescence, Quantitative RT-PCR

Journal: Stem Cells International

Article Title: Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

doi: 10.1155/2017/3508907

Figure Lengend Snippet: Effect of ASP on Wnt/ β -catenin signaling mediators of Sca-1 + HSC/HPCs in D-gal- induced aging mice. The Sca-1+ HSC/HPCs were collected after the treatment. (a) GSK-3 β , Ser9-phosphorylated GSK-3 β and TCF-4 protein expressions by Western blot. β -actin was used as the internal control. (b) The relative protein expression of GSK-3 β . (c) The relative protein expression of Ser9-phosphorylated GSK-3 β . (d) The relative protein expression of TCF-4. Different letters: P < 0.05.

Article Snippet: Slides were then incubated with antibodies against β -catenin (1 : 100) (Cell Signaling Technology, USA) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Journal of Cancer

Article Title: MUC 15 Promotes Osteosarcoma Cell Proliferation, Migration and Invasion through Livin, MMP-2/MMP-9 and Wnt/β-Catenin Signal Pathway

doi: 10.7150/jca.49641

Figure Lengend Snippet: Mechanisms of MUC15 on osteosarcoma proliferation, migration and invasion. Western blot detected the apoptosis-inhibiting protein Livin ( A ), the migration-related proteins MMP-9 /MMP-2 ( B ), EMT related proteins ( C ) and the Wnt/b-Catenin signaling pathway of OS cell ( D ).

Article Snippet: The primary antibodies included anti-MUC15 (Abcam, ab224468), anti-Livin (Abcam, ab97350), anti-MMP2 (Invitrogen, 35-1300Z), anti-MMP9 (Invitrogen, MA5-15886), anti-E-cadherin (GeneTex, GTX124198), anti-Vimentin (Abcam, ab92547), anti-b-Catenin (CST, #9562) and anti-c-Myc (CST, #5605).

Techniques: Migration, Western Blot

Journal: BMC Cancer

Article Title: Properties of resistant cells generated from lung cancer cell lines treated with EGFR inhibitors

doi: 10.1186/1471-2407-12-95

Figure Lengend Snippet: Characterization of H1650-ER1 cells . (A) mRNA expression of E-cadherin, vimentin, occludin and fibronectin in H1650 and H1650-ER1 cells was measured by quantitative RT-PCR. Fold change expression was normalized with respect to H1650 cells. (B) mRNA expression of Snail, Twist and Zeb1 in H1650 and H1650-ER1 cells was measured by quantitative RT-PCR. Fold change expression was normalized with respect to H1650 cells. Error bars represent s.e.m. (n = 3). (C) Immunofluorescence of H1650 and H1650-ER1 cells stained with DAPI (blue) and antiβ-catenin antibody (green). (D) H1650 and H1650-ER1 cells were seeded on 6 well plates. After 48 hr, a scratch was induced in the confluent cell monolayer. Images were obtained at time t = 0 and after 12 hr (t = 12) to monitor cell migration. Percent cell migration was calculated based on migration of H1650 cells. The error bars represent s.e.m. (n = 3).

Article Snippet: Then the cells were incubated overnight with anti-β-catenin antibody (Cell Signaling Technology, Danvers, MA) at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Migration