mcp 1  (R&D Systems)

Journal: Brazilian Dental Journal

Article Title: Chemoattractive potential of stem cells from apical papilla in peripheral blood monocytes: an in vitro study

doi: 10.1590/0103-644020256694

Figure Lengend Snippet: Production of CCL2 quantified by ELISA assay in SCAP conditioned medium (CM-SCAP). Bars show the mean and standard deviation of the experiment (N=3) (A). Viability of monocytes stimulated with decreasing dilutions of SCAP supernatant (CM) compared with control (proliferation medium alone). Absorbance (570nm) data obtained from the MTT assay after 24 h of stimulus exposure (B). Fluorescence data obtained by Alamar Blue assay at experimental time of 24 h. Conditioned medium by SCAP (CM-SCAP) diluted ½ in the presence or not of a CCL2 neutralizer (CM-SCAP + α-CCL2) compared to control (proliferation medium alone) and Recombinant Human CCL2 (CCL2 rH) (C). The result showed the mean and standard deviation of the experiments performed in triplicate. Different letters represent statistical differences between groups. (One-Way ANOVA with Tukey's test, p<0.05) (B and C).

Article Snippet: 300 ng/ml of Recombinant Human CCL2 (279-MC-010 R&D Systems) was used for positive control, while α-MEM 10% was the negative control.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Control, MTT Assay, Fluorescence, Alamar Blue Assay, Recombinant

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Integrated analysis of chemokine/cytokine protein after 9-Gy myeloablative radiation, HSCs, and adoptive cellular therapy and in vitro migration assays and single-cell RNA sequencing analysis in KR158B glioma. (A) Cytokine and chemokine profiling of glioma protein lysates in the tumor secretome in adoptive cellular therapy–treated gliomas and untreated control. Statistical comparisons were made using the 1-way ANOVA test. (B) CCL12 protein levels in glioma lysates were quantified by ELISA. Statistical analysis was made using the unpaired Student t test. (C) Transcriptomic analysis of chemokine ligands and receptors using nanoString digital spatial profiling. Statistical analysis was performed using the 2-way ANOVA test. (D) In vitro trans-well migration assay of MDSCs migrating in response to glioma conditioned media from excised glioma tissue combined with neutralizing antibodies against CCL2 and/or CCL12. Statistical analysis was performed using the 1-way ANOVA test. (E) ELISA CCL12 protein quantification using immune cell–specific isolates from excised glioma tissue. Statistical analysis was performed using the 1-way ANOVA test. (F) Immune cell subset deconvolution from single-cell RNA sequencing analysis from excised glioma tissue. (G) mRNA expression of CCL12 overlayed on immune cell subset deconvolution using single-cell RNA sequencing analysis from excised glioma tissue. (H) In vitro trans-well migration assay of MDSCs migration in response to immune cell-specific isolates combined with either IgG or CCL12 neutralizing antibody. Statistical comparisons conducted using the 2-way ANOVA test. * P ≤ .05. ** P ≤ .01. *** P ≤ .001. **** P ≤ .0001; error bars: mean ± standard deviation. Abbreviations: ANOVA, analysis of variance; MDSCs, myeloid-derived suppressor cells.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: In Vitro, Migration, Single Cell, RNA Sequencing, Control, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Association of Ccl2 and macrophage-associated gene expression with clinical outcomes in glioblastoma patients. (A) Kaplan-Meier analysis of disease-free survival based on Ccl2 expression. (B) Kaplan-Meier analysis of overall survival of Cd14 expression. (C) Kaplan-Meier analysis of disease-free survival stratified by Fcgr3a expression. (D) Deconvolution of single-cell RNA sequencing cell subsets in the glioma microenvironment of GBM patients. (E) Single-cell RNA sequencing deconvolution of Ccl2 expression among cell clusters from GBM patients. (F) Deconvoluted quantification of Ccl2 expression from cell clusters identified from GBM patient samples. Statistical comparisons made using the log-rank test with P values presented on the survival graphs along with hazard ratios and P values for hazard ratios.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: Gene Expression, Expressing, Single Cell, RNA Sequencing

Journal: PLOS One

Article Title: Transport of CCL2 across an induced pluripotent stem cell-derived in vitro model of the human blood-brain barrier is heparan sulfate-dependent

doi: 10.1371/journal.pone.0338780

Figure Lengend Snippet: a. 125 I-CCL2 Pe in the presence of 10 µg/mL CCL2 compared to PBS vehicle. b. 131 I-Alb Pe in the presence of 10 µg/mL CCL2 compared to PBS vehicle. c. 125 I-CCL2 Pe corrected for non-specific leakage as quantified by 131 I-Alb Pe in the presence 10 µg/mL CCL2 compared to PBS vehicle. a-c. One differentiation was performed with n = 7-8 transwells per group. **p < 0.01, ***p < 0.001 (Unpaired two-tailed t-test). Means are displayed with their SD.

Article Snippet: The chloramine-T method was used to radioactively label recombinant carrier-free human CCL2 (R & D Systems, 279-MC-050/CF) with 125 I and bovine serum albumin (BSA, Sigma cat no. 17030-100G) with 131 I, as described previously [ ].

Techniques: Two Tailed Test

Journal: PLOS One

Article Title: Transport of CCL2 across an induced pluripotent stem cell-derived in vitro model of the human blood-brain barrier is heparan sulfate-dependent

doi: 10.1371/journal.pone.0338780

Figure Lengend Snippet: a. 125 I-CCL2 Pe in the presence of 20 U/mL heparin or 10 µM RS504393 (CCR2 antagonist) compared to control (“Ctrl”). b. 131 I-Alb Pe in the presence of 20 U/mL heparin or 10 µM RS504393 compared to an untreated control group. c. 125 I-CCL2 Pe corrected for non-specific leakage as quantified by 131 I-Alb Pe in the presence of 20 U/mL heparin or 10 µM RS504393 compared to an untreated control group. a-c. ***p < 0.001, ****p < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test). d. Comparison of 125 I-CCL2 and 131 I-Alb Pe in the untreated control group. ****p < 0.0001 (Unpaired two-tailed t-test). a-d. One differentiation was performed with n = 5-6 transwells per group. Means are displayed with their SD.

Article Snippet: The chloramine-T method was used to radioactively label recombinant carrier-free human CCL2 (R & D Systems, 279-MC-050/CF) with 125 I and bovine serum albumin (BSA, Sigma cat no. 17030-100G) with 131 I, as described previously [ ].

Techniques: Control, Comparison, Two Tailed Test

Journal: PLOS One

Article Title: Transport of CCL2 across an induced pluripotent stem cell-derived in vitro model of the human blood-brain barrier is heparan sulfate-dependent

doi: 10.1371/journal.pone.0338780

Figure Lengend Snippet: a. Representative immunocytochemistry of Heparan Sulfate clone F58-10E4 (HS(10E4)) (red) and DAPI (blue) in vehicle or HSase I, II, and III-treated iBECs. b-c. Immunofluorescence analysis of HS(10E4) mean fluorescence intensities (MFI) relative to DAPI area (b) or total HS(10E4) area relative to DAPI area (c). d-e. 125 I-CCL2 (d) or 131 I-Alb in the presence of 0.25 U/ml HSase I, II, and III compared to vehicle. f. 125 I-CCL2 Pe corrected for non-specific leakage as quantified by 131 I-Alb Pe in the presence of 0.25 U/ml HSase I, II, and III compared to vehicle. One differentiation was performed with n = 7-8 transwells per group. *p < 0.05, ***p < 0.001 (Unpaired two-tailed t-test). Means are displayed with their SD. Figure created with BioRender.

Article Snippet: The chloramine-T method was used to radioactively label recombinant carrier-free human CCL2 (R & D Systems, 279-MC-050/CF) with 125 I and bovine serum albumin (BSA, Sigma cat no. 17030-100G) with 131 I, as described previously [ ].

Techniques: Immunocytochemistry, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: PLOS One

Article Title: Transport of CCL2 across an induced pluripotent stem cell-derived in vitro model of the human blood-brain barrier is heparan sulfate-dependent

doi: 10.1371/journal.pone.0338780

Figure Lengend Snippet: a. Molecular structure of GalNAz. b. On day 3 post-subculture, transwells were organized into treatment groups such that TEER means were approximately equal and treated with GalNAz or DMSO (vehicle control).Transwells were re-treated on day 6 post-subculture, and the effects on TEER (c), 125 I-CCL2 Pe (d) and 131 I-Alb Pe (e) were measured on day 9 post-subculture. f. For each transwell, 125 I-CCL2 Pe was corrected for non-specific leakage as quantified by 131 I-Alb Pe. b-f. **p < 0.01, ***p < 0.001 (Unpaired two-tailed t-test). One differentiation was performed with n = 9 transwells treated per group. Means are displayed with their SD. Figure created with BioRender.

Article Snippet: The chloramine-T method was used to radioactively label recombinant carrier-free human CCL2 (R & D Systems, 279-MC-050/CF) with 125 I and bovine serum albumin (BSA, Sigma cat no. 17030-100G) with 131 I, as described previously [ ].

Techniques: Control, Two Tailed Test

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 upregulates expression of PD-L1 and CCL2. (A) mRNA-seq analysis of ZEB2-overexpressing SW480 cells and analysis of KEGG pathways affected by ZEB2 expression. The size of each circle represents the number of genes involved in the corresponding pathway and the color scale denotes the P-value (upper). Changes in expression of cytokine-related genes in ZEB2-overexpressing SW480 cells are vs. those in control cells (lower). (B) RT-qPCR of CCL2 , CCL28 , CXCL2 , CXCL3 , CXCL6 and CXCL12 levels in ZEB2-overexpressing vs. control SW480 cells (upper) and in ZEB2-suppressed vs. control SNU-398 cells (lower; n=4). (C) RT-qPCR of CD274 mRNA levels in ZEB2-overexpressing vs. control SW480 cells (left) and in ZEB2-suppressed vs. control SNU-398 cells (right; n=4). (D) Analysis of CCL2 and PD-L1 protein levels in ZEB2-overexpressing vs. control SW480 cells (left), in ZEB2-suppressed vs. control SNU-398 cells (middle) and in ZEB2-overexpressing vs. control PC3 cells (right). Densitometric quantification of bands on the immunoblot was performed, with β-actin or GAPDH as a loading control. (E) Reporter analysis of CD274 and CCL2 promoter activity in ZEB2-overexpressing vs. control SW480 cells (upper) and in ZEB2-suppressed vs. control SNU-398 cells (lower; n=4). (F) Flow cytometry analysis of PD-L1 expression in SW480 cells transfected with ZEB2, TWIST1, or SNAIL expression vectors (n=3). Cells treated with IFN-γ or transfected with a PD-L1 expression vector were used as positive controls. Immunoblot analysis confirmed overexpression of ZEB2 (anti-myc), TWIST1 (anti-flag) and SNAIL (anti-SNAIL). (G) ELISA to measure secreted levels of CCL2 in conditioned medium from ZEB2-suppressed vs. control SNU-398 cells (n=3). (H, I) Scatter plots of ZEB2 mRNA expression vs. CD274 (H) and CCL2 (I) mRNA expression in colorectal adenocarcinoma (data from TCGA, Firehose Legacy and TCGA, Nature 2012). Correlations were statistically analyzed using the Spearman test. Spearman's correlation coefficients and equations were automatically generated using the cBioPortal webpage tool. (J) Kaplan-Meier analysis showing the relationship between overall survival of colon cancer (CPTAC-2, Prospective, Cell 2019; n=106) and pancreatic adenocarcinoma (TCGA, Firehose Legacy; n=178) patients and expression of ZEB2 and CD274 mRNA. P-values were calculated by the log-rank test. Values represent the mean ± standard deviation. * P<0.05; ** P<0.01; *** P<0.001; N.S, not significant. ZEB2, Zinc Finger E-Box Binding Homeobox 2; PD-L1, programmed cell death 1 ligand 1; CCL2, C-C motif chemokine ligand 2; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR; ELISA, enzyme-linked immunosorbent assay; TCGA, The Cancer Genome Atlas; sh, short hairpin.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Flow Cytometry, Transfection, Plasmid Preparation, Over Expression, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 cooperates with SP1 to promote transcription of CD274 and CCL2 by binding directly to their promoters. (A) SW480 cells were co-transfected with siRNA specific for SP1 (siSP1) and with a ZEB2 expression vector, for 48 h prior to immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed, with GAPDH as a loading control. (B) RT-qPCR of CD274 (upper) and CCL2 (lower) levels in SW480 cells co-transfected with siSP1 and the ZEB2 expression vector (n=4). (C) Mutation analysis of the SP1 site in the CD274 and CCL2 promoters. SW480 cells were transfected with reporter constructs containing SP1 site mutations and reporter activity was measured (n=4). Values represent mean ± SD. *** P<0.001, vs. vector + control siRNA; $$$ P<0.001, vs. ZEB2 + control siRNA. (D) ChIP analysis of the interaction between ZEB2 and SP1 and the CD274 and CCL2 promoters. Chromatin fragments from SNU-398 cells were immunoprecipitated by normal mouse IgG (lane 1), anti-ZEB2 (lane 2), or anti-SP1 (lane 3) and data were analyzed by semiquantitative PCR using CD274 (-181/-41) and CCL2 (-115/+25) promoter primers. The input control (1%) is shown in lane 4. Irrelevant regions (-807/-660 for CD274 and -1820/-1675 for CCL2 ) were also analyzed. ZEB2, Zinc Finger E-Box Binding Homeobox 2; si, small interfering; RT-qPCR, reverse transcription-quantitative PCR; PD-L1, programmed cell death 1 ligand 1; CCL2, C-C motif chemokine ligand 2; ChIP, chromatin immunoprecipitation.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR, Mutagenesis, Construct, Activity Assay, Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 promotes macrophage migration and polarization in a CCL2-dependent manner. (A) Migration of THP-1-derived macrophages by conditioned medium from and co-culturing with stable SNU-398 cells (control vs. ZEB2-suppressed cells) (n=4). CCL2 was used as a positive control. * P<0.05; ** P<0.01. (B) Migration of THP-1-derived macrophages toward conditioned medium from stable SNU-398 cells in the presence of an anti-CCL2 antibody (n=4) * P<0.05; ** P<0.01 vs. no antibody + no conditioned medium; $ P<0.05; $$ P<0.01 vs. no antibody + shSCR conditioned medium. (C) RT-qPCR of M1 ( TNF , CXCL8/IL8 , IL12B , NOS2/INOS , IL1B and IL6 ) and M2 ( TGFB1 , IL10 , MRC1 , CLEC10A , CCL17 and ARG1 ) markers secreted by THP-1-derived macrophages incubated with conditioned medium from stable SNU-398 cells in the absence and presence of an anti-CCL2 antibody (n=4). * P<0.05; ** P<0.01; *** P<0.001, vs. no antibody + no conditioned medium; $ P<0.05; $$$ P<0.001, vs. no antibody + shSCR conditioned medium. Values represent mean ± SD. ZEB2, Zinc Finger E-Box Binding Homeobox 2; CCL2, C-C motif chemokine ligand 2; sh, short hairpin.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Migration, Derivative Assay, Control, Positive Control, Quantitative RT-PCR, Incubation, Binding Assay

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 SUMOylation through PC2 is required for ZEB2 acting as a transcriptional activator and playing subsequent cellular functions. (A) SW480 cells were transfected with ZEB2WT and ZEB2_K391/866R for 48 h prior to lysis and immunoblot analysis. (B) Reporter assay of ITGA5 (integrin α5), VIM (vimentin), VEGFA , CDH1 , CD274 and CCL2 promoter activity in SW480 cells transfected with ZEB2WT and ZEB2_K391/866R (n=4). (C) Invasion (representative fields at magnification, ×100), (D) survival and (E) anchorage-independent growth of SW480 cells transfected with ZEB2WT and ZEB2_K391/866R (n=3). (F) SW480 cells were co-transfected with shRNA specific for CBX4 (shPC2) and with a ZEB2-expression vector, for 48 h prior to lysis and immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed, with GAPDH as a loading control. Values represent mean ± SD. * P<0.05; ** P<0.01; *** P<0.001; N.S, not significant. ZEB2, Zinc Finger E-Box Binding Homeobox 2; SUMO, small ubiquitin-like modifier; CCL2, C-C motif chemokine ligand 2; PD-L1, programmed cell death 1 ligand 1; VEGF, vascular endothelial growth factor; sh, short hairpin; WT, wild type; Mut, mutant.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Transfection, Lysis, Western Blot, Reporter Assay, Activity Assay, shRNA, Expressing, Plasmid Preparation, Control, Binding Assay, Ubiquitin Proteomics, Mutagenesis

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: SUMOylation of ZEB2 is required for cooperation between ZEB2 and SP1. Reporter assay to determine transcriptional activity of SP1 in SW480 cells (n=4). (A) Cells were transfected with ZEB2WT and ZEB2_K391/866R expression vectors for 48 h. (B) Cells were co-transfected with a ZEB2 expression vector and siRNA specific for CBX4 (siPC2) for 48 h. Values represent mean ± SD. * P<0.05; ** P<0.01; *** P<0.001. (C) SW480 cells transfected with ZEB2WT and ZEB2_K391/866R expression vectors were treated with cycloheximide for the indicated times prior to lysis and immunoblot analysis. (D) A cytosolic fraction and a nuclear fraction were prepared from 293E cells transfected for 48 h with ZEB2WT and ZEB2_K391/866R expression vectors. GAPDH and PARP were used as internal controls for the cytosolic and nuclear fractions, respectively. (E) Co-immunoprecipitation analysis of the interaction between ZEB2 and SP1 in 293E cells co-transfected with ZEB2 (WT vs. K391/866R) and SP1 expression vectors. (F) Kaplan-Meier analysis showing the probability of progression-free survival of patients with colorectal adenocarcinoma (TCGA, PanCancer Atlas; n=588) in relation to CBX4 mRNA expression. (G) Overall survival of patients with colorectal adenocarcinoma (TCGA, PanCancer Atlas; n=568) in relation to expression of ZEB2 and CBX4 mRNA. P-values were calculated using the log-rank test. SUMO, small ubiquitin-like modifier; ZEB2, Zinc Finger E-Box Binding Homeobox 2; CCL2, C-C motif chemokine ligand 2; PD-L1, programmed cell death 1 ligand 1; VEGF, vascular endothelial growth factor; TCGA, The Cancer Genome Atlas; si, small interfering; WT, wild type; Mut, mutant.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Reporter Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Lysis, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Mutagenesis