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recombinant human ccl22  (R&D Systems)


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    R&D Systems recombinant human ccl22
    Fig. 3. Chemotaxis occurs at much higher rate in KK1 mutant (KK1m) compared to the KK1 revertant (KK1r). (A) Repre sentative timecourse of chemotaxis over 10 h, quantified as % confluence of cells in the top well, normalized to % confluence at t = 0 (left panel). Recombinant human <t>CCL22</t> (500 ng/mL) was added in the bottom well to induce migration. Statistical analysis of the chemotaxis timecourse replicates (n = 3) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the experiment (right panel). Significant differences are calculated and indicated by asterisks as in Fig. 2. (B) Representative images of KK1m and KK1r cell in the top well at t = 10 h.
    Recombinant Human Ccl22, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/recombinant+human+mdc/Recombinant+Human+CCL22%2FMDC+Protein/pm36442790-103-20-23
    Average 92 stars, based on 31 article reviews
    recombinant human ccl22 - by Bioz Stars, 2026-09
    92/100 stars

    Images

    1) Product Images from "A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells."

    Article Title: A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    doi: 10.1016/j.bbadis.2022.166601

    Fig. 3. Chemotaxis occurs at much higher rate in KK1 mutant (KK1m) compared to the KK1 revertant (KK1r). (A) Repre sentative timecourse of chemotaxis over 10 h, quantified as % confluence of cells in the top well, normalized to % confluence at t = 0 (left panel). Recombinant human CCL22 (500 ng/mL) was added in the bottom well to induce migration. Statistical analysis of the chemotaxis timecourse replicates (n = 3) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the experiment (right panel). Significant differences are calculated and indicated by asterisks as in Fig. 2. (B) Representative images of KK1m and KK1r cell in the top well at t = 10 h.
    Figure Legend Snippet: Fig. 3. Chemotaxis occurs at much higher rate in KK1 mutant (KK1m) compared to the KK1 revertant (KK1r). (A) Repre sentative timecourse of chemotaxis over 10 h, quantified as % confluence of cells in the top well, normalized to % confluence at t = 0 (left panel). Recombinant human CCL22 (500 ng/mL) was added in the bottom well to induce migration. Statistical analysis of the chemotaxis timecourse replicates (n = 3) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the experiment (right panel). Significant differences are calculated and indicated by asterisks as in Fig. 2. (B) Representative images of KK1m and KK1r cell in the top well at t = 10 h.

    Techniques Used: Chemotaxis Assay, Mutagenesis, Recombinant, Migration

    Fig. 5. Effect of ritonavir and ibrutinib on cell clumping and chemotaxis. (A) Mean rate of clumping (n = 4), in the presence of 100 U/mL IL-2, of KK1m and KK1r lines at the beginning (t = 0 h), middle (t = 20 h), and end (t = 40 h) of the timecourse, normalized to t = 0, with or without ritonavir (left panel) or ibrutinib (IB) (right panel). (B) As in (A) but measuring the average object area of the clumps with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel). (C) Mean rate of chemotaxis (n = 3), induced by 500 ng/mL CCL22, of KK1m cells with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the timecourse, normalized to t = 0. Significant differences are calculated and indicated by asterisks as in Fig. 2.
    Figure Legend Snippet: Fig. 5. Effect of ritonavir and ibrutinib on cell clumping and chemotaxis. (A) Mean rate of clumping (n = 4), in the presence of 100 U/mL IL-2, of KK1m and KK1r lines at the beginning (t = 0 h), middle (t = 20 h), and end (t = 40 h) of the timecourse, normalized to t = 0, with or without ritonavir (left panel) or ibrutinib (IB) (right panel). (B) As in (A) but measuring the average object area of the clumps with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel). (C) Mean rate of chemotaxis (n = 3), induced by 500 ng/mL CCL22, of KK1m cells with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the timecourse, normalized to t = 0. Significant differences are calculated and indicated by asterisks as in Fig. 2.

    Techniques Used: Chemotaxis Assay

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    Article Title: Macrophage-Derived Chemokine (CCL22) Is a Novel Mediator of Lung Inflammation Following Hemorrhage and Resuscitation
    Article Snippet: .. Confluent cells were treated with 100 ng/mL recombinant human MDC (R&D Systems) with and without 20 ng/mL recombinant human TNF-! (R&D Systems). .. After 24 h of treatment, culture media was collected, and cytokine levels analyzed by multiplex ELISA (Quansys Biosciences).

    Article Title: Macrophage-Derived Chemokine (MDC/CCL22) is a Novel Mediator of Lung Inflammation Following Hemorrhage and Resuscitation
    Article Snippet: .. Confluent cells were treated with 100 ng/mL recombinant human MDC (R&D Systems) with and without 20 ng/mL recombinant human TNF-α (R&D Systems). .. After 24 h of treatment, culture media was collected and cytokine levels analyzed by multiplex ELISA (Quansys Biosciences).

    Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity
    Article Snippet: .. Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml. ..

    Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity
    Article Snippet: .. Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml. ..

    Control:

    Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity
    Article Snippet: .. Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml. ..

    Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity
    Article Snippet: .. Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml. ..

    Suspension:

    Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity
    Article Snippet: .. Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml. ..

    Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity
    Article Snippet: .. Where indicated, neutralizing anti-human MDC mAb or isotype-matched mouse IgG control Ab (R&D Systems Inc.) was added to the condition medium in the lower chamber at 10 μg/ml, or recombinant human MDC or recombinant human TARC (R&D Systems Inc.) was added to the cell suspension in the upper chamber at 100 ng/ml. ..



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    Fig. 3. Chemotaxis occurs at much higher rate in KK1 mutant (KK1m) compared to the KK1 revertant (KK1r). (A) Repre sentative timecourse of chemotaxis over 10 h, quantified as % confluence of cells in the top well, normalized to % confluence at t = 0 (left panel). Recombinant human <t>CCL22</t> (500 ng/mL) was added in the bottom well to induce migration. Statistical analysis of the chemotaxis timecourse replicates (n = 3) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the experiment (right panel). Significant differences are calculated and indicated by asterisks as in Fig. 2. (B) Representative images of KK1m and KK1r cell in the top well at t = 10 h.
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    Image Search Results


    Primer sequences used in the study

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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.

    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

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

    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

    Fig. 3. Chemotaxis occurs at much higher rate in KK1 mutant (KK1m) compared to the KK1 revertant (KK1r). (A) Repre sentative timecourse of chemotaxis over 10 h, quantified as % confluence of cells in the top well, normalized to % confluence at t = 0 (left panel). Recombinant human CCL22 (500 ng/mL) was added in the bottom well to induce migration. Statistical analysis of the chemotaxis timecourse replicates (n = 3) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the experiment (right panel). Significant differences are calculated and indicated by asterisks as in Fig. 2. (B) Representative images of KK1m and KK1r cell in the top well at t = 10 h.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

    doi: 10.1016/j.bbadis.2022.166601

    Figure Lengend Snippet: Fig. 3. Chemotaxis occurs at much higher rate in KK1 mutant (KK1m) compared to the KK1 revertant (KK1r). (A) Repre sentative timecourse of chemotaxis over 10 h, quantified as % confluence of cells in the top well, normalized to % confluence at t = 0 (left panel). Recombinant human CCL22 (500 ng/mL) was added in the bottom well to induce migration. Statistical analysis of the chemotaxis timecourse replicates (n = 3) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the experiment (right panel). Significant differences are calculated and indicated by asterisks as in Fig. 2. (B) Representative images of KK1m and KK1r cell in the top well at t = 10 h.

    Article Snippet: For chemotaxis experiments, growth medium was used without serum to minimize proliferation, with or without the addition of 500 ng/mL recombinant human CCL22 (R&D systems 336-MD-025) in the bottom well to induce migration.

    Techniques: Chemotaxis Assay, Mutagenesis, Recombinant, Migration

    Fig. 5. Effect of ritonavir and ibrutinib on cell clumping and chemotaxis. (A) Mean rate of clumping (n = 4), in the presence of 100 U/mL IL-2, of KK1m and KK1r lines at the beginning (t = 0 h), middle (t = 20 h), and end (t = 40 h) of the timecourse, normalized to t = 0, with or without ritonavir (left panel) or ibrutinib (IB) (right panel). (B) As in (A) but measuring the average object area of the clumps with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel). (C) Mean rate of chemotaxis (n = 3), induced by 500 ng/mL CCL22, of KK1m cells with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the timecourse, normalized to t = 0. Significant differences are calculated and indicated by asterisks as in Fig. 2.

    Journal: Biochimica et biophysica acta. Molecular basis of disease

    Article Title: A frequent PLCγ1 mutation in adult T-cell leukemia/lymphoma determines functional properties of the malignant cells.

    doi: 10.1016/j.bbadis.2022.166601

    Figure Lengend Snippet: Fig. 5. Effect of ritonavir and ibrutinib on cell clumping and chemotaxis. (A) Mean rate of clumping (n = 4), in the presence of 100 U/mL IL-2, of KK1m and KK1r lines at the beginning (t = 0 h), middle (t = 20 h), and end (t = 40 h) of the timecourse, normalized to t = 0, with or without ritonavir (left panel) or ibrutinib (IB) (right panel). (B) As in (A) but measuring the average object area of the clumps with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel). (C) Mean rate of chemotaxis (n = 3), induced by 500 ng/mL CCL22, of KK1m cells with and without ritonavir (RITO) (left panel) or ibrutinib (IB) (right panel) at the beginning (t = 0 h), middle (t = 5 h), and end (t = 10 h) of the timecourse, normalized to t = 0. Significant differences are calculated and indicated by asterisks as in Fig. 2.

    Article Snippet: For chemotaxis experiments, growth medium was used without serum to minimize proliferation, with or without the addition of 500 ng/mL recombinant human CCL22 (R&D systems 336-MD-025) in the bottom well to induce migration.

    Techniques: Chemotaxis Assay

    FIGURE 1. CCL22 recruits iNKT cells, Tregs, and pDCs. (A) Chemotaxis assay showing migration of splenocytes toward 500 pg/ml recombinant CCL22. Migration index represents the ratio of cells that migrate toward CCL22 to cells that migrate to medium alone. Data are representative of four independent experiments, each performed in triplicate. **p , 0.01, ***p , 0.001 versus medium alone. (B) Chemotaxis assay showing migration of splenocytes toward untreated islets or islets transduced with Ad-CCL22. Migration index represents the ratio of cells that migrate toward untreated or CCL22-transduced islets to cells that migrate to medium only. Data are representative of three independent experiments, each performed in triplicate. *p , 0.05, **p , 0.01, ***p , 0.001. (C) Diabetic NOD mice were transplanted with 500 NOD.scid islets transduced with Ad-CCL22 (n = 6 at day 5 and n = 7 at day 10) or Ad-LacZ (n = 4 at day 5 and n = 5 at day 10). Immune infiltration in islet grafts was quantified by flow cytometry 5 and 10 d post- transplantation. *p , 0.05, **p , 0.01 versus LacZ at the same time point. lyDC, lymphoid DC; mDC, myeloid DC.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

    doi: 10.4049/jimmunol.1400567

    Figure Lengend Snippet: FIGURE 1. CCL22 recruits iNKT cells, Tregs, and pDCs. (A) Chemotaxis assay showing migration of splenocytes toward 500 pg/ml recombinant CCL22. Migration index represents the ratio of cells that migrate toward CCL22 to cells that migrate to medium alone. Data are representative of four independent experiments, each performed in triplicate. **p , 0.01, ***p , 0.001 versus medium alone. (B) Chemotaxis assay showing migration of splenocytes toward untreated islets or islets transduced with Ad-CCL22. Migration index represents the ratio of cells that migrate toward untreated or CCL22-transduced islets to cells that migrate to medium only. Data are representative of three independent experiments, each performed in triplicate. *p , 0.05, **p , 0.01, ***p , 0.001. (C) Diabetic NOD mice were transplanted with 500 NOD.scid islets transduced with Ad-CCL22 (n = 6 at day 5 and n = 7 at day 10) or Ad-LacZ (n = 4 at day 5 and n = 5 at day 10). Immune infiltration in islet grafts was quantified by flow cytometry 5 and 10 d post- transplantation. *p , 0.05, **p , 0.01 versus LacZ at the same time point. lyDC, lymphoid DC; mDC, myeloid DC.

    Article Snippet: Recombinant CCL22 was purchased from R&D Systems and reconstituted in PBS with 0.1% BSA prior to addition to cells.

    Techniques: Chemotaxis Assay, Migration, Recombinant, Transduction, Cytometry, Transplantation Assay

    FIGURE 2. Effector T cells are suppressed in CCL22-expressing islet grafts. (A) Immunofluoresence of islet grafts harvested 10 d posttransplantation and stained for Ki67 (red), insulin (green), and DAPI (blue). Scale bar, 100 mm. (B) Flow cytometry analysis of conventional T cell proliferation in islet grafts and draining LN 10 d posttransplantation in CCL22 (n = 6) and LacZ (n = 4) recipients. **p , 0.01, ***p , 0.001 versus LacZ for the same cell population. (C) Flow cytometry analysis of conventional T cell expression of the cytotoxicity surface marker CD107a (n = 6 for CCL22 recipients and n = 4 for LacZ recipients). *p , 0.05, ***p , 0.001 versus LacZ for the same cell population. (D) IFN-g gene expression in CCL22-expressing islet grafts 5 and 10 d posttransplantation. n = 4 for both CCL22- and LacZ- recipients. Data shown as fold change gene expression compared with LacZ-transduced islet grafts. *p , 0.05 versus LacZ. (E and F) Cytokine gene expression in CD8conv and CD4conv cells, respectively, isolated from islet grafts 10 d post- transplantation (n = 3 for both CCL22 and LacZ recipients). Data are shown as fold change gene expression compared with LacZ-transduced islet grafts. *p , 0.05 versus LacZ.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

    doi: 10.4049/jimmunol.1400567

    Figure Lengend Snippet: FIGURE 2. Effector T cells are suppressed in CCL22-expressing islet grafts. (A) Immunofluoresence of islet grafts harvested 10 d posttransplantation and stained for Ki67 (red), insulin (green), and DAPI (blue). Scale bar, 100 mm. (B) Flow cytometry analysis of conventional T cell proliferation in islet grafts and draining LN 10 d posttransplantation in CCL22 (n = 6) and LacZ (n = 4) recipients. **p , 0.01, ***p , 0.001 versus LacZ for the same cell population. (C) Flow cytometry analysis of conventional T cell expression of the cytotoxicity surface marker CD107a (n = 6 for CCL22 recipients and n = 4 for LacZ recipients). *p , 0.05, ***p , 0.001 versus LacZ for the same cell population. (D) IFN-g gene expression in CCL22-expressing islet grafts 5 and 10 d posttransplantation. n = 4 for both CCL22- and LacZ- recipients. Data shown as fold change gene expression compared with LacZ-transduced islet grafts. *p , 0.05 versus LacZ. (E and F) Cytokine gene expression in CD8conv and CD4conv cells, respectively, isolated from islet grafts 10 d post- transplantation (n = 3 for both CCL22 and LacZ recipients). Data are shown as fold change gene expression compared with LacZ-transduced islet grafts. *p , 0.05 versus LacZ.

    Article Snippet: Recombinant CCL22 was purchased from R&D Systems and reconstituted in PBS with 0.1% BSA prior to addition to cells.

    Techniques: Expressing, Staining, Flow Cytometry, Marker, Gene Expression, Isolation, Transplantation Assay

    FIGURE 3. Tregs display an activated phenotype in CCL22-expressing islet grafts. Flow cytometry analysis of CD4 cell subsets in islet grafts and draining LNs of transplant recipients 10 d posttransplantation (n = 7 for CCL22 recipients and n = 5 for LacZ recipients). CD4 subsets were first gated as CD3+CD4+CD82 cells; Tregs and CD4conv cells were further identified as Foxp3+ and Foxp32 cells, respectively. (A) Cell surface expression of markers CTLA-4, ICOS, and CD62L. *p , 0.05, **p , 0.01 versus LacZ for same cell subset. (B) CCR4 expression levels on CD4conv cells and Tregs. **p , 0.01 versus LacZ for same cell subset. (C) Islet-specific CD4conv cells and Tregs were detected in the islet graft and LN using BDC2.5 MHC class II tetramers. p = NS. (D) Proliferation of Tregs in the islet graft and LN was measured by Ki67 staining. p = NS.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

    doi: 10.4049/jimmunol.1400567

    Figure Lengend Snippet: FIGURE 3. Tregs display an activated phenotype in CCL22-expressing islet grafts. Flow cytometry analysis of CD4 cell subsets in islet grafts and draining LNs of transplant recipients 10 d posttransplantation (n = 7 for CCL22 recipients and n = 5 for LacZ recipients). CD4 subsets were first gated as CD3+CD4+CD82 cells; Tregs and CD4conv cells were further identified as Foxp3+ and Foxp32 cells, respectively. (A) Cell surface expression of markers CTLA-4, ICOS, and CD62L. *p , 0.05, **p , 0.01 versus LacZ for same cell subset. (B) CCR4 expression levels on CD4conv cells and Tregs. **p , 0.01 versus LacZ for same cell subset. (C) Islet-specific CD4conv cells and Tregs were detected in the islet graft and LN using BDC2.5 MHC class II tetramers. p = NS. (D) Proliferation of Tregs in the islet graft and LN was measured by Ki67 staining. p = NS.

    Article Snippet: Recombinant CCL22 was purchased from R&D Systems and reconstituted in PBS with 0.1% BSA prior to addition to cells.

    Techniques: Expressing, Flow Cytometry, Staining

    FIGURE 4. CCL22 activates Tregs in vitro. (A and B) Phenotype of isolated CD4conv cells and Tregs cultured with or without 500 pg/ml recombinant CCL22 for 48 h. Data are representative of three independent experiments, each performed in triplicate. *p , 0.05, **p , 0.01 versus same subset untreated. (A) Cell surface expression of markers CTLA-4, ICOS, and CD62L after treatment with CCL22. (B) Mean fluorescence intensity of CCR4 expression on CD4 subsets after CCL22 treatment. (C and D) Expression of CTLA-4, ICOS, and CCR4 on isolated CD4conv cells and Tregs after 48 h culture with 500 pg/ml recombinant CCL22 in the presence or absence of TCR stimulation with CD3/CD28 beads. Data are representative of two in- dependent experiments performed in triplicate. *p , 0.05, **p , 0.01, ***p , 0.001 versus same subset untreated or as indicated by bar. (E) Viability assay of isolated Tregs cultured with CCL22 in the presence or absence of IL-2 as determined by staining with 7-aminoactinomycin D (7-AAD). Data are representative of two independent experiments performed in triplicate. *p , 0.05.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

    doi: 10.4049/jimmunol.1400567

    Figure Lengend Snippet: FIGURE 4. CCL22 activates Tregs in vitro. (A and B) Phenotype of isolated CD4conv cells and Tregs cultured with or without 500 pg/ml recombinant CCL22 for 48 h. Data are representative of three independent experiments, each performed in triplicate. *p , 0.05, **p , 0.01 versus same subset untreated. (A) Cell surface expression of markers CTLA-4, ICOS, and CD62L after treatment with CCL22. (B) Mean fluorescence intensity of CCR4 expression on CD4 subsets after CCL22 treatment. (C and D) Expression of CTLA-4, ICOS, and CCR4 on isolated CD4conv cells and Tregs after 48 h culture with 500 pg/ml recombinant CCL22 in the presence or absence of TCR stimulation with CD3/CD28 beads. Data are representative of two in- dependent experiments performed in triplicate. *p , 0.05, **p , 0.01, ***p , 0.001 versus same subset untreated or as indicated by bar. (E) Viability assay of isolated Tregs cultured with CCL22 in the presence or absence of IL-2 as determined by staining with 7-aminoactinomycin D (7-AAD). Data are representative of two independent experiments performed in triplicate. *p , 0.05.

    Article Snippet: Recombinant CCL22 was purchased from R&D Systems and reconstituted in PBS with 0.1% BSA prior to addition to cells.

    Techniques: In Vitro, Isolation, Cell Culture, Recombinant, Expressing, Viability Assay, Staining

    FIGURE 5. CCL22 enhances the ability of Tregs to induce IDO expression in DCs. (A) DCs were cultured with or without Tregs at a ratio 1:2 (DC/Treg) in the presence or absence of CCL22. To test the role of CTLA-4, a blocking Ab or isotype control (ctr IgG) was added to the media. IDO gene expression was analyzed after 48 h and expressed as fold change compared with untreated DCs. Data are representative of two independent experiments, each performed in triplicate. *p , 0.05, **p , 0.01 as indicated by bars. (B) Immunohistochemistry of islet grafts 10 d posttransplantation showing the presence of IDO+ cells (arrows). IDO was stained with New Fuschin as chromogen and hematoxylin counterstain. Scale bar, 100 mm. IDO+ cells were quantified and normalized to the graft area on two graft sections per mouse, with three animals in each group (p = NS). (C) IDO gene expression in islet grafts and draining LN harvested 10 d posttransplantation. Data are expressed as fold change compared with LacZ recipients (n = 4 at day 5 and n = 3 at day 10 for both CCL22 and LacZ recipients). (D) Expression of IFN-g, TNF-a, and IDO genes in CD11c+ cells isolated from islet grafts 10 d posttransplantation. *p , 0.01 CCL22 (n = 3) versus LacZ (n = 3) recipients. Data are expressed as fold change compared with LacZ recipients.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

    doi: 10.4049/jimmunol.1400567

    Figure Lengend Snippet: FIGURE 5. CCL22 enhances the ability of Tregs to induce IDO expression in DCs. (A) DCs were cultured with or without Tregs at a ratio 1:2 (DC/Treg) in the presence or absence of CCL22. To test the role of CTLA-4, a blocking Ab or isotype control (ctr IgG) was added to the media. IDO gene expression was analyzed after 48 h and expressed as fold change compared with untreated DCs. Data are representative of two independent experiments, each performed in triplicate. *p , 0.05, **p , 0.01 as indicated by bars. (B) Immunohistochemistry of islet grafts 10 d posttransplantation showing the presence of IDO+ cells (arrows). IDO was stained with New Fuschin as chromogen and hematoxylin counterstain. Scale bar, 100 mm. IDO+ cells were quantified and normalized to the graft area on two graft sections per mouse, with three animals in each group (p = NS). (C) IDO gene expression in islet grafts and draining LN harvested 10 d posttransplantation. Data are expressed as fold change compared with LacZ recipients (n = 4 at day 5 and n = 3 at day 10 for both CCL22 and LacZ recipients). (D) Expression of IFN-g, TNF-a, and IDO genes in CD11c+ cells isolated from islet grafts 10 d posttransplantation. *p , 0.01 CCL22 (n = 3) versus LacZ (n = 3) recipients. Data are expressed as fold change compared with LacZ recipients.

    Article Snippet: Recombinant CCL22 was purchased from R&D Systems and reconstituted in PBS with 0.1% BSA prior to addition to cells.

    Techniques: Expressing, Cell Culture, Blocking Assay, Control, Gene Expression, Immunohistochemistry, Staining, Isolation

    FIGURE 6. NKT cells contribute to CCL22-mediated protection against recurrent autoimmune diabetes. (A) Syngeneic islet transplantation in diabetic wild-type (WT) or CD1d- deficient NOD recipients. Islets were transduced with an MOI of 10 of Ad- CCL22 or Ad-LacZ. Survival curves were compared using the log-rank test. Compared to WT CCL22, p , 0.05 for CD1d2/2 CCL22, p , 0.01 for CD1d2/2 LacZ, and p , 0.001 for WT LacZ. p = NS for CD1d2/2

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Cellular mechanisms of CCL22-mediated attenuation of autoimmune diabetes.

    doi: 10.4049/jimmunol.1400567

    Figure Lengend Snippet: FIGURE 6. NKT cells contribute to CCL22-mediated protection against recurrent autoimmune diabetes. (A) Syngeneic islet transplantation in diabetic wild-type (WT) or CD1d- deficient NOD recipients. Islets were transduced with an MOI of 10 of Ad- CCL22 or Ad-LacZ. Survival curves were compared using the log-rank test. Compared to WT CCL22, p , 0.05 for CD1d2/2 CCL22, p , 0.01 for CD1d2/2 LacZ, and p , 0.001 for WT LacZ. p = NS for CD1d2/2

    Article Snippet: Recombinant CCL22 was purchased from R&D Systems and reconstituted in PBS with 0.1% BSA prior to addition to cells.

    Techniques: Transplantation Assay, Transduction