Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A . Differentially expressed genes in unstimulated or LPS or IL-4 stimulated LysM ΔZeb1 compared to LysM Ctrl BMDMs as measured by a customized RT2 array and depicted in log 2 fold change of expression. nd marks non-detectable mRNA levels. All transcripts were normalized to Gapdh (n=3). B . Relative mRNA expression of Ccl2 and Ccl22 in LysM Ctrl and LysM ΔZeb1 BMDMs (n=3; means ±SD; 2-way ANOVA). C . Comparison of transcript and secretome alterations of Ccl2 and Ccl22 in LysM ΔZeb1 compared to LysM Ctrl BMDMs (n≥5; means ±SD).

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A-B . Venn diagrams of differentially expressed genes (DEGs) of LysM Ctrl (blue) and LysM ΔZeb1 BMDMs (red) after stimulation with LPS (A) or IL-4 (B) compared to unstimulated in total (left) and divided in up-/downregulated DEGs (right). C . GO term enrichment analysis for DEGs (FDR<0.05) uniquely up- or downregulated by LysM ΔZeb1 BMDMs after LPS stimulation. D . Log 2 fold change of expression of selected trafficking genes after LPS stimulation. X marks no significant deregulation. E . Representative images and quantification of OPP incorporation of LysM Ctrl and LysM ΔZeb1 BMDMs with 0h, 4h and 16h LPS pre-stimulation (n=3; means ±SD; 2-way ANOVA). F-G . Representative arrays of intracellular cytokines of LysM Ctrl and LysM ΔZeb1 BMDMs with LPS stimulation or additional Brefeldin A and Monensin treatment (F) and quantification of intracellular CCL2 and CCL22 after LPS, Brefeldin A and Monensin treatment (G; n≥2).

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Expressing

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A . Confluence of KPC cells alone or co-cultured with LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). B . Representative images at t=28h and quantification over time of KPC cell invasion into a scratch wound without or with co-culture of LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). C . Confluence of KPC cells alone or with LysM Ctrl or LysM ΔZeb1 BMDM conditioned medium (CM) (n=2 KPC CM, n=3 LysM Ctrl and LysM ΔZeb1 CM). D . Transwell migration assay of CD8+ T cells alone or towards LysM Ctrl or LysM ΔZeb1 BMDMs in absence or presence of recombinant CCL2 and CCL22 (left panel, n>3) or absence or presence of anti-CCL2 and anti-CCL22 antibodies (right panel, n=3). Means ±SD; *:p<0.05; **:p<0.01; ns: not significant; 2-way ANOVA.

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Cell Culture, Co-Culture Assay, Transwell Migration Assay, Recombinant

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

Journal: Shock

Article Title: Macrophage-Derived Chemokine (CCL22) Is a Novel Mediator of Lung Inflammation Following Hemorrhage and Resuscitation

doi: 10.1097/shk.0000000000000253

Figure Lengend Snippet: FIG. 4. Recombinant mouse MDC (rmMDC) exacerbates lung inflam- mation following hemorrhage and resuscitation (H/R). Representative micrographs of lung tissue stained with hematoxylin-eosin (AYC) and antiYmouse Ly-6B.2 (DYF) for inflammatory cell identification. The lungs from mice injected with rmMDC prior to resuscitation (C, F) had substantial inter- stitial tissue edema and cell infiltration as compared with sham mice (A, D) and mice resuscitated with LR solution only (B, E). G, Quantification of posi- tively stained cells confirmed the effect of rmMDC on increased inflammatory cell recruitment. **P G 0.001 vs. all groups and *P G 0.001 vs. sham.

Article Snippet: In separate studies, mice were hemorrhaged and injected intravenously with 1 2g of recombinant mouse MDC (R&D Systems) prior to resuscitation.

Techniques: Recombinant, Staining, Injection

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

Article Title: Pivotal role of the CC chemokine, macrophage-derived chemokine, in the innate immune response.

doi: 10.4049/jimmunol.164.10.5362

Figure Lengend Snippet: FIGURE 1. MDC protects mice from the lethality induced by CLP. The CLP mice were treated with i.p. injection of either MDC (1 mg/mouse; F; 15 mice) or vehicle (E; 15 mice) immediately after CLP. The survival rates were monitored for 7 days after CLP. Two different experiments were conducted, and the data were pooled. The mortality rates were very similar in individual experiments.

Article Snippet: Immediately after CLP, the mice were treated with either i.p or i.v. injection of recombinant murine MDC (1 mg/mouse; R&D Systems, Minneapolis, MN; endotoxin content , 0.1 ng/mg).

Techniques: Injection

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

Article Title: Pivotal role of the CC chemokine, macrophage-derived chemokine, in the innate immune response.

doi: 10.4049/jimmunol.164.10.5362

Figure Lengend Snippet: FIGURE 5. MDC blockade reduces survival of mice after CLP. The CLP mice were treated with i.p. injection of either anti-MDC antiserum (F; 30 mice) or control serum (E; 30 mice) 2 h before CLP. The survival rates were monitored for 7 days after CLP. Three different experiments were conducted, and the data were pooled. The mortality rates were very similar in individual experiments.

Article Snippet: Immediately after CLP, the mice were treated with either i.p or i.v. injection of recombinant murine MDC (1 mg/mouse; R&D Systems, Minneapolis, MN; endotoxin content , 0.1 ng/mg).

Techniques: Injection, Control

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

Article Title: Pivotal role of the CC chemokine, macrophage-derived chemokine, in the innate immune response.

doi: 10.4049/jimmunol.164.10.5362

Figure Lengend Snippet: FIGURE 6. MDC induces respiratory burst in macrophages and lyso- zomal enzyme release from macrophages. Peritoneal cells were harvested from normal mice, the cells (1 3 106 cells) were incubated for 2 h at 37°C, and nonadherent cells were removed. A, The adherent macrophages were stimulated with murine MDC. After 30 min, the cultures were assayed for extracellular O2 2 release. B, The adherent macrophages were stimulated with murine MDC for 48 h at 37°C, and then the lysozyme enzyme ac- tivities in the culture supernatant were measured. The data are representa- tive of the three individual experiments (n 5 8 each). p, p , 0.05; ‡, p , 0.01; when compared with control (MDC 5 0 ng/ml).

Article Snippet: Immediately after CLP, the mice were treated with either i.p or i.v. injection of recombinant murine MDC (1 mg/mouse; R&D Systems, Minneapolis, MN; endotoxin content , 0.1 ng/mg).

Techniques: Incubation, Control