Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: Primer sequences used in the study

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Sequencing, Control

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: Hypomethylation states of CCL22 and CCR4 caused overexpression of CCL22 and CCR4 in CC . ( A ) An overview of mRNA levels of CCL22 and CCR4 in CC based on GEPIA database. ( B ) The mRNA levels of CCL22 and CCR4 in CC (Ca) (n=32) and normal cervical tissues (NC) (n=32) detected by RT-qPCR. ( C ) The correction between CCL22 and CCR4 in CC, analyzed by GEPIA database. ( D ) Predicted CpG islands in the promoter regions of CCL22 and CCR4 . Numbers indicate the positions in bp relative to the transcription start site. The blue region represents the CpG islands and the red vertical bars are the CpG loci in these input sequences. ( E and F ) DNA Methylation level of CCL22 and CCR4 promoter regions in CC (Ca) (n=9) and NC (n=9) detected by MS-PCR. MS-PCR images of 4 representative samples are shown from each group. ( G ) Detection of CCL22 and CCR4 promoter DNA methylation status by MS-PCR in SiHa, Hela and C33A cells; (M: methylated, U: unmethylated). ( H ) Relative mRNA expression of CCL22 and CCR4 in SiHa, HeLa and C33A cells after treatment with different concentrations of 5-Aza-CdR.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Over Expression, Quantitative RT-PCR, DNA Methylation Assay, Methylation, Expressing

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: DNMT3A reactivated the CCL22 and CCR4 expression through decreasing promoters' DNA methylation . ( A ) Detection of the expression of DNMT3A , CCL22 and CCR4 in DNMT3A specific siRNA transfected SiHa and HeLa cells by RT-qPCR. ( B ) Detection of the DNA methylation level of CCL22 and CCR4 in DNMT3A specific siRNA transfected SiHa and HeLa cells by MS-qPCR. ( C ) Schematic representation of the 4 regions of the CCL22 and CCR4 promoter regions amplified in the chromatin immunoprecipitation (ChIP)‑quantitative PCR (qPCR) experiment. ( D and E ) Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-DNMT3A ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for indicated four regions in the CCL22 and CCR4 promoter regions in qPCR. The enrichment of DNMT3A on CCL22 and CCR4 promoter regions relative to IgG in SiHa and HeLa cells, and H3 against RPL30 was used as positive control.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Expressing, DNA Methylation Assay, Transfection, Quantitative RT-PCR, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Positive Control

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: Inhibition of EZH2 promoted DNMT3A in cervical cancer cells with methylated the promoter regions of CCL22 and CCR4 . ( A ) Detection of the expression of EZH2, H3K27me3 and DNMT3A in EZH2 knocked-down or specific siRNA transfected or DZNep treated SiHa and HeLa cells by western blotting. ( B ) Schematic representation of the 4 regions of the DNMT3A promoter region amplified in the chromatin immunoprecipitation (ChIP)‑quantitative PCR (qPCR) experiment. ( C ) Chromatin was cross-linked, fragmented and immunoprecipitated with either IgG (mock) or anti-EZH2 and H3K27me3 ChIP-grade antibody and the purified DNA was used to amplify with respective primer pairs for indicated four regions in the DNMT3A promoter region in qPCR. The enrichment of EZH2 and H3K27me3 on DNMT3A promoter region relative to IgG in SiHa cell, and H3 against RPL30 was used as positive control. ( D , F and H ) The mRNA expression of EZH2 , DNMT3A and CCL22 - CCR4 in EZH2 knocked-down or specific siRNA transfected or DZNep treated SiHa and HeLa cells by RT-qPCR. ( E , G and I ) Detection of the methylation level of CCL22 and CCR4 in EZH2 knocked-down or specific siRNA transfected or DZNep treated SiHa and HeLa cells by MS-qPCR.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Inhibition, Methylation, Expressing, Transfection, Western Blot, Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Positive Control, Quantitative RT-PCR

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: Effect of CCL22-CCR4 on migration of CC cells . ( A ) The migratory potential of SiHa and HeLa cells which added recombinant human CCL22 protein or neutralization CCL22 antibody and the respective control cells was analyzed by the transwell cell migration assay. Number of migratory cells was shown as means ± standard error from three independent experiments using triplicate measurements and statistically analyzed with Student's t-test in each experiment. Magnification, ×200. ( B ) The expression of EMT-related proteins in SiHa or HeLa cells which added recombinant human CCL22 protein or neutralization CCL22 antibody and the respective control cells was determined by western blotting and the gray level analysis of the protein levels of EMT-related proteins.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Migration, Recombinant, Neutralization, Control, Cell Migration Assay, Expressing, Western Blot

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: Inhibition EZH2 represses migration in CC cells through downregulating CCL22-CCR4 . ( A ) The migratory potential of EZH2 knocked-down SiHa or HeLa cells and the respective control cells was analyzed by the transwell cell migration assay. Number of migratory cells was shown as means ± standard error from three independent experiments using triplicate measurements and statistically analyzed with Student's t-test in each experiment. Magnification, ×200. ( B ) The migratory potential of DNMT3A specific siRNA transfected SiHa and HeLa cells and the respective control cells was analyzed by the transwell cell migration assay. Number of migratory cells was shown as means ± standard error from three independent experiments using triplicate measurements and statistically analyzed with Student's t-test in each experiment. Magnification, ×200. ( C ) The expression of EMT-related proteins in EZH2 knocked-down or DZNep treated SiHa and HeLa cells were determined by western blotting and the gray level analysis of the protein levels of EMT-related proteins. ( D ) The expression of EMT-related proteins in DNMT3A specific siRNA transfected SiHa and HeLa cells were determined by western blotting and the gray level analysis of the protein levels of EMT-related proteins and DNMT3A.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Inhibition, Migration, Control, Cell Migration Assay, Transfection, Expressing, Western Blot

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: CCL22-CCCR4 promotes migration in CC cells . ( A ) The migratory potential of EZH2 knocked-down SiHa or HeLa cells with or without CCL22 and the respective control cells was analyzed by the transwell cell migration assay. Number of migratory cells was shown as means ± standard error from three independent experiments using triplicate measurements and statistically analyzed with Student's t-test in each experiment. Magnification, ×200. ( B ) The expression of EMT-related proteins in EZH2 knocked-down SiHa and HeLa cells with or without CCL22 were determined by western blotting. ( C ) The migratory potential of DNMT3A specific siRNA transfected SiHa and HeLa cells with or without anti-CCL22 and the respective control cells was analyzed by the transwell cell migration assay. Number of migratory cells was shown as means ± standard error from three independent experiments using triplicate measurements and statistically analyzed with Student's t-test in each experiment. Magnification, ×200. ( D ) The expression of EMT-related proteins in DNMT3A specific siRNA transfected SiHa and HeLa cells with or without anti-CCL22 determined by western blotting.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Migration, Control, Cell Migration Assay, Expressing, Western Blot, Transfection

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: Epigenetic modulation of CCL22-CCR4 mediated by EZH2 in vivo . ( A ) SiHa-shEZH2 and HeLa-shEZH2 tumor xenografts in nude mice. ( B ) The tumors weight formed from SiHa-shEZH2 and HeLa-shEZH2. ( C ) Tumors formed from SiHa-shEZH2 and HeLa-shEZH2 cells as well as tumor growth curves. ( D , F and G ) Western blotting and RT-qPCR results of EZH2, H3K27me3, DNMT3A, CCL22-CCR4 and EMT-related proteins in SiHa-shEZH2 and HeLa-shEZH2 cells formed tumors. ( E ) The methylation level of CCL22 and CCR4 promoter regions were monitored by MS-qPCR in tumor tissues. ( H - K ) Chromatin was cross‑linked, fragmented and immunoprecipitated with either IgG (mock) or anti‑EZH2, H3K27me3 and DNMT3A ChIP‑grade antibody and the purified DNA was used to amplify with respective primer pairs for the indicated 4 regions in the DNMT3A , CCL22 - CCR4 promoters in qPCR. The enrichment of EZH2 and H3K27me3 on DNMT3A , CCL22 - CCR4 promoters and the enrichment of DNMT3A CCL22 - CCR4 promoters on relative to IgG in tumor tissues.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: In Vivo, Western Blot, Quantitative RT-PCR, Methylation, Immunoprecipitation, Purification

Journal: Journal of Cancer

Article Title: Activation of the CCL22/CCR4 causing EMT process remodeling under EZH2-mediated epigenetic regulation in cervical carcinoma

doi: 10.7150/jca.101881

Figure Lengend Snippet: The pathway of EZH2 regulated CCL22-CCR4 expression through epigenetic modification causing EMT remodeling.

Article Snippet: Recombinant human CCL22 (NBP2-34977, NOVUS, USA) was reconstituted according to instructions provided by the manufacturer.

Techniques: Expressing, Modification