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

Journal: Oncogene

Article Title: Enhanced expression of Duffy antigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential.

doi: 10.1038/sj.onc.1209703

Figure Lengend Snippet: Figure 3 The amount of CXCL8 (IL-8) (a) and CCL2 (MCP-1) (b) in the supernatant of the same number (1 107) of MDA-MB- 435HM-DARC, MDA-MB-435HM-vect, MDA-MB-435HM cells following 3, 6, 12 and 24 h incubation. The data represent the mean7s.e. of three independent experiments.

Article Snippet: And the amount of mouse CCL2 (JE) in DARC, mock-transfected and wild-type MDAMB-435HM xenografts in 4, 6 and 8 weeks were also determined by ELISA kit (Mouse CCL2/JE/MCP-1 DuoSet, R&D Systems Inc., USA).

Techniques: Incubation

Journal: Oncogene

Article Title: Enhanced expression of Duffy antigen receptor for chemokines by breast cancer cells attenuates growth and metastasis potential.

doi: 10.1038/sj.onc.1209703

Figure Lengend Snippet: Figure 4 (a): The growth curve of in vivo proliferation assay. The data represent the mean7s.e. of three independent experiments. Expression of MMP-9 was detected by RT–PCR (b) and Western blot (c). The relative expression of MMP-9 on mRNA and protein levels (d). For (b) and (c) lane 1, positive control (human spleen tissue); lane 2, MDA-MB-435HM-vect; lane 3, MDA-MB-435HM; lane 4, MDA-MB-435HM-DARC clone 1; lane 5, MDA-MB-435HM-DARC clone 2. lane 6: negative control (without mRNA template). (e) Representative the median level of mouse CCL2 (JE) protein in MDA-MB-435HM-DARC clones 1 and 2, MDA-MB- 435HM-vect, MDA-MB-435HM xenografts in 4, 6 and 8 weeks. Representative quantification of three independent experiments. The error bars represent the s.e. Forty-eight BALB/c nude mice were used in this study.

Article Snippet: And the amount of mouse CCL2 (JE) in DARC, mock-transfected and wild-type MDAMB-435HM xenografts in 4, 6 and 8 weeks were also determined by ELISA kit (Mouse CCL2/JE/MCP-1 DuoSet, R&D Systems Inc., USA).

Techniques: In Vivo, Proliferation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Positive Control, Negative Control, Clone Assay

Journal: EMBO Molecular Medicine

Article Title: Adaptively evolved human oral actinomyces‐sourced defensins show therapeutic potential

doi: 10.15252/emmm.202114499

Figure Lengend Snippet: An inhibition‐zone‐based assay for evaluating the stability of AMSIN in H 2 O or normal mouse serum. The bacterium used was BM and the peptide dose was 0.2 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann–Whitney U ‐test (* P < 0.05, ns: no significance; exact P values are listed in Table ). Hemolysis by AMSIN. Meucin‐18 was used as a positive control. Mean ± SD of three technical replicates is displayed. Comparison of cytotoxic effects of AMSIN and LL‐37 on HL‐60 cells. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk; * P < 0.05, *** P < 0.001, ns: no significance; exact P values are listed in Table ). An inhibition‐zone assay showing the inability of AMSIN to bind DNA. The bacterium used was MRSA P1374 and the peptide dose was 1.0 nmol/well. Mean ± SD of three biological replicates is displayed. P values were obtained by Mann–Whitney U ‐test (ns: no significance; exact P values are listed in Table ). The effect of AMSIN on human ion channels expressed in the CNS and heart. The asterisks mark steady‐state current traces after administering 100 μM AMSIN. Peptide‐induced IL‐8 release. A549 cells were treated with AMSIN or LL‐37 for 24 h. IL‐8 levels in culture supernatants from the FBS‐containing medium ( left ) or the serum‐free medium ( right ) were measured with an ELISA, as described in Materials and Methods. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk or ns; * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significance; exact P values are listed in Table ). Peptide‐induced MCP‐1 release. HL‐60 cells were treated with AMSIN or LL‐37 for 24 h. MCP‐1 levels in culture supernatants from the FBS‐containing medium ( left ) or the serum‐free medium ( right ) were measured with an ELISA. Mean ± SD of three biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by a red asterisk or ns; * P < 0.05, ** P < 0.01, ns: no significance; exact P values are listed in Table ). Surface plasmon resonance (SPR)‐based assay detecting anti‐AMSIN antibodies in mouse serum using Biacore T100. Sera were taken from three AMSIN‐treated mice (Samples 1–3) and one control mouse only injected with 0.9% NaCl. The sensorgrams present data of subtracting the background SPR signals from the control. Note: the abnormal peaks at 30 and 90 s yielded by the equipment due to sample injections were artificially removed. Insect, schematic diagram of SPR experiment in which 1:100 diluted serum was used as analyte to flow over the AMSIN‐immobilized CM5 chip. Treatment of pneumococcal pneumonia. Five mice per sampling point were inoculated with SP D39 through the nasopharynx and 24 h later the animals received AMSIN or penicillin (as a positive control) at different doses (AMSIN, at the doses of 8.35, 16.7, and 33 mg per kg; penicillin at 8.35, 16.7, and 30 mg per kg) via intramuscular injection (i.m.). One day after treatment, the animals were necropsied and their lungs were removed and homogenized for the determination of the level of SP , as described in the Materials and Methods section. Each point represents the determination from a single animal and the line shows the mean log value. Mean ± SD of five to ten biological replicates is displayed. P values were obtained by Student’s t ‐test or Mann–Whitney U ‐test (the latter indicated by red asterisks; ** P < 0.01, *** P < 0.001 denotes significant reduction in CFU compared with the saline group; exact P values are listed in Table ). Survival after peritoneal infection with MRSA P1374. The survival fraction of the vehicle‐treated control group was zero out of 11 mice and the treatment group contained six mice and all survived. Source data are available online for this figure.

Article Snippet: Medium was aspirated and replaced with fresh complete DMEM supplemented with 10% FBS or serum‐free medium, both containing different concentrations of polypeptides for 24 h. Then, the supernatants were harvested for quantification of the IL‐8 amount in A549 by the Human CXCL8/IL‐8 Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA) and the MCP‐1 amount in HL‐60 by the Human MCP‐1 ELISA Kit (Boster Biological Technology Co., Ltd, Wuhan, China) as per the manufactures’ instructions.

Techniques: Inhibition, MANN-WHITNEY, Positive Control, Comparison, Enzyme-linked Immunosorbent Assay, SPR Assay, Control, Injection, Sampling, Saline, Infection