Journal: American Journal of Cancer Research

Article Title: Lipopolysaccharide induces CCL2 through TLR4 signaling and promotes esophageal squamous cell carcinoma cell proliferation

doi: 10.62347/EIKE6128

Figure Lengend Snippet: LPS promotes ESCC cell proliferation and migration by activating CCL2 through NF-κB2. (A) Effect of LPS (10 μg/ml) on proliferation of cells of the indicated ESCC lines. Control cells grown in the absence of LPS were assigned a value of 100. (B, C) Comparison between CCL2 mRNA expression (B) and protein (C) in ESCC cell lines treated with 10 μg/ml LPS and untreated controls. (D) Levels of NF-κB2 mRNA expression in ESCC lines. (E) Wound healing assays showing the effect of LPS on migration of KYSE-150 and KYSE-450 cells. (F) Wound closure observed after 48 hours. *P<0.05; **P<0.001.

Article Snippet: The culture supernatants were then collected, and levels of expression of CCL2 were compared using an ELISA kit (Proteintech, Japan) [ 17 ].

Techniques: Migration, Control, Comparison, Expressing

Journal: American Journal of Cancer Research

Article Title: Lipopolysaccharide induces CCL2 through TLR4 signaling and promotes esophageal squamous cell carcinoma cell proliferation

doi: 10.62347/EIKE6128

Figure Lengend Snippet: Blocking TLR4 in ESCC cells reduces CCL2 production and cell proliferation. A. Effect of TLR4 blockade with TAK242 (10 μg/ml) on proliferation in ESCC lines treated with 10 μg/ml LPS. Untreated control cells were assigned a value of 100. B. Wound healing assays showing the effect of LPS and TAK242 on migration of KYSE-150 and KYSE-450 cells. C. Wound closure observed after 48 hours. D. Level of CCL2 protein in KYSE-150 and KYSE-450 cell supernatants after treatment with LPS and TAK242. *P<0.05.

Article Snippet: The culture supernatants were then collected, and levels of expression of CCL2 were compared using an ELISA kit (Proteintech, Japan) [ 17 ].

Techniques: Blocking Assay, Control, Migration

Journal: American Journal of Cancer Research

Article Title: Lipopolysaccharide induces CCL2 through TLR4 signaling and promotes esophageal squamous cell carcinoma cell proliferation

doi: 10.62347/EIKE6128

Figure Lengend Snippet: CCL2 expression assayed in a tissue microarray and corresponding survival data for 175 ESCC patients. (A) Whole images of CCL2 staining in the tissue microarray of samples from the 175 ESCC patients. (B) Representative images assigned IHC scores of 3+, 2+, and 1+. The triplicate cores are shown at 100× magnification (scale bar: 500 μm), along with high-magnification (400×) images on the right (scale bar: 100 μm). (C, D) Kaplan-Meier survival curves illustrating the association between CCL2 expression status (3+ or 2+ or 1+) and 5-year OS (C) and DSS (D) in ESCC patients after curative esophagectomy. The log-rank test was used to compare differences between the three groups (P<0.001). (E) CCL2 expression status and depth of invasion (pT). (F) CCL2 expression status and tumor differentiation. (G) CCL2 expression status and UICC 8th lymph node metastasis (pN). (H) CCL2 expression status and UICC 8th pathological stage. (I) CCL2 expression status and TLR4 expression.

Article Snippet: The culture supernatants were then collected, and levels of expression of CCL2 were compared using an ELISA kit (Proteintech, Japan) [ 17 ].

Techniques: Expressing, Microarray, Staining

Journal: American Journal of Cancer Research

Article Title: Lipopolysaccharide induces CCL2 through TLR4 signaling and promotes esophageal squamous cell carcinoma cell proliferation

doi: 10.62347/EIKE6128

Figure Lengend Snippet: The clinicopathological characteristics of 175 ESCC patients

Article Snippet: The culture supernatants were then collected, and levels of expression of CCL2 were compared using an ELISA kit (Proteintech, Japan) [ 17 ].

Techniques: Adjuvant, Expressing

Journal: American Journal of Cancer Research

Article Title: Lipopolysaccharide induces CCL2 through TLR4 signaling and promotes esophageal squamous cell carcinoma cell proliferation

doi: 10.62347/EIKE6128

Figure Lengend Snippet: Univariate (A) and Multivariate (B) analysis of the hazard ratios for 5-year OS in the tissue microarray cohort

Article Snippet: The culture supernatants were then collected, and levels of expression of CCL2 were compared using an ELISA kit (Proteintech, Japan) [ 17 ].

Techniques: Microarray, Expressing

Journal: PLoS Pathogens

Article Title: A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

doi: 10.1371/journal.ppat.1005410

Figure Lengend Snippet: Intratracheal administration of 0.5μg TNF (A and B) or 50μg poly(I:C) (C and D) to A20 AEC-KO or wild type littermates (A20 WT ). Absolute numbers of neutrophils or monocytes in bronchoalveolar lavages (BAL) as determined by flow cytometry at 6h and 24h post-treatment for TNF (A) or 24h post-treatment for poly(I:C) (C). IL-6, CXCL1 (KC), CCL2 (MCP-1) and TNF [only for poly(I:C)] protein levels in BAL fluid detected by Multiplex immunoassay (B and D). Data represent mean ± SEM of at least 4 mice per group (*p < 0.05; Student’s t -test).

Article Snippet: Recombinant mouse CCL2 (R&D Systems, endotoxin levels <0.01 EU per μg of protein as measured by the LAL method) was administered intranasally at a dose of 50 μg/kg at day 6 post infection.

Techniques: Flow Cytometry, Multiplex Assay

Journal: PLoS Pathogens

Article Title: A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

doi: 10.1371/journal.ppat.1005410

Figure Lengend Snippet: (A) Absolute numbers of monocytes, neutrophils and alveolar macrophages in bronchoalveolar lavages (BAL) of A20 AEC-KO or A20 WT mice at 2, 5, 8 and 12 days post-infection (days p.i.) with 0.05 X LD 50 X-47. (B) Absolute numbers of resident CD11b - or recruited CD11b + macrophages in the lungs of A20 WT and A20 AEC-KO mice. (C) CCL2 (MCP-1) protein levels in BAL fluid measured by Multiplex immunoassay at indicated time points post-infection. (D) Weight loss of A20 AEC-KO and A20 WT mice infected with 0.05 X LD 50 X-47. At day 6 p.i. (indicated by an arrow) mice received intranasal treatment with 50 μg/kg recombinant CCL2 (rCCL2) or PBS. Data were analysed using Student’s t -test (A, B and C *p < 0.05) and 2-way ANOVA (D, *p < 0.05 for A20 AEC-KO PBS vs. A20 WT PBS and # p < 0.05 for A20 AEC-KO PBS vs A20 AEC-Cre rCCL2). Data represent mean ± SEM of at least 3 mice per group. Data are representative of at least 2 independent experiments.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, endotoxin levels <0.01 EU per μg of protein as measured by the LAL method) was administered intranasally at a dose of 50 μg/kg at day 6 post infection.

Techniques: Infection, Multiplex Assay, Recombinant

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Inflammatory monocytes associate with survival in LUSC. a Kaplan–Meier plots of overall survival in lung squamous carcinoma (LUSC) patients split by median (left, p < 0.0001) and quartile (right, p = 0.0006) expression levels of CD14. P -values are obtained with the log-rank test; FDR were calculated according to Benjamini and Hochberg. b Proportion of patients by mRNA subtype that have CD14 expression levels above (red) or below (black) the median CD14 expression level. Binomial tests for proportions were performed. (Black asterisks: significant enrichment below the median, red asterisks: Significant enrichment above the median). c Kaplan–Meier plots of overall survival in LUSC by expression levels of CCL2 ( p = 0.001), CCL3 ( p = 0.018) and CSF1 ( p = 0.015) expression. d Pearson’s correlations of CCL2, CCL3, and CSF1 chemokines versus CD14 (gene expression). e Dynamic range of mRNA expression of CD14 and CCL2 for each LUSC mRNA subtype. P -values were obtained with analysis of variance. The purple shading for CD14 expression represents samples in the ‘IM-rich subset’ (above the median CD14 expression level). f Dynamic range of mRNA expression of CCL3 and CSF1 by LUSC mRNA subtype. g Representative multiplex IHC for CD14 (green), CCR2 (red) and pan-cytokeratin (light blue) in a LUSC tumor sample, and enumeration of CD14+/CCR2+ cells in CK+ and CK- regions. #1-3 represent CK- regions, #4 represents a CK + region. Scale bar 100 μm. h Representative dual CD14+/CCR2+ cells (white arrows) in CK- (#1-3) and CK+ (#4) regions. Note: CK + region shown in white channel to more easily appreciate green and red. Scale bar 25 μm. i Enumeration of CD14+/CCR2+ cells in CK- and CK+ regions by mRNA subtype. Classical ( n = 14), Basal ( n = 9), Primitive ( n = 6) and Secretory ( n = 12). p -values were obtained with Student’s t-test in comparison to the Classical subtype. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Multiplex Assay, Comparison

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: TNFα activation of NFκB promotes CCL2-mediated IM recruitment. a Microarray expression data (left) comparing murine bronchial epithelial cells (MBECs), parental KLN205 and the LN4K1 sub-clone. Top upstream pathways (right) from Ingenuity Pathway Analysis (IPA) are shown for the differentially regulated genes shown in brackets. b An upstream network visualization from IPA of all over-expressed genes (all nodes) in the upper portion of the heat map shown in Fig. with significant log-rank (survival analysis) p -value ( < 0.05). TNFα and NFκB (blue nodes) were amongst the top upstream regulators known to have direct roles (black lines) in promoting CCL2, CCL3 and CSF1 chemokines (the degree of redness corresponds with increasing statistical significance). c Relative expression of TNFα. d CCL2, CCL3 and CSF1 by qPCR. Data are averages ± s.e.m. P -values were obtained with Student’s t-test in comparison with KLN205. e Relative levels of CCL2 as measured by ELISA from secreted media of cells growing in vitro or, f , from plasma of tumor-bearing mice. Data are averages ± s.e.m. g Relative mRNA expression of CCL2 and p65 in LN4K1 cells following treatment with control or p65 siRNA with or without exogenous TNFα (100 ng/mL). h Relative expression of CCL2 mRNA (top) and phospho-p65 and p65 protein (bottom) following treatment with DMSO or an IKKβ inhibitor (Compound A, 5 μM) for 5 h. i Relative IM counts in the bone marrow, blood, spleen from healthy DBA2 mice versus those with LN4K1 tumors. j Relative IM, TAM and TReg counts from the lungs of healthy versus LN4K1-bearing mice. IMs were also assessed in age-matched DBA2 mice following HBSS ‘Mock’ injection. P -values obtained with one-sided Student’s t-test, n = 5 mice/group for f , i , and j . * P < 0.05, ** P ≤0.01, *** P ≤0.001

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Activation Assay, Microarray, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, In Vitro, Clinical Proteomics, Control, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: CCL2-mediated IM recruitment is critical for LUSC metastasis. a Relative expression of CCL2 for KLN205 and sub-clones. b Quantification of luciferase signal. c Representative images obtained 10 days after cell injection of (i) KLN205-Scr ORF, (ii) KLN205-CCL2 ORF, (iii) LN4K1-Cntrl shR, (iv) LN4K1-CCL2 shR#1 and (v) LN4K1-CCL2 shR#2. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. d Survival plots of mice following tail vein injection of KLN205 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. e Number of IMs per lung lobe, n = 12 lobes/group. f Survival plots of mice following tail vein injection of LN4K1 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. g Number of IMs per lung lobe, n = 12 lobes/group. h Schematic (left) and quantification of luciferase signal (right) of mice treated with vehicle or PF-04136309 to assess effects on established metastases. i FACS plots and ( j ) quantification of percent IMs in the blood and ( k ) right lung of LN4K1-bearing mice. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 5 mice/group. l , FACS analysis of percent immune infiltrates for TAMs (gated on F480), DCs (gated on SiglecF-/CD11c), CD4, CD8, Tregs (gated on TCRb + ) and NK cells (gated on SiglecF-/B220-/TCRb-). m Schematic (left) and quantification of luciferase signal (right) of mice treated at the time of cell injection to assess effects on preventing metastasis. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. n.s. = non-significant, * P ≤0.05, *** P < 0.001. For panel b , * FDR < 0.05, ** FDR < 0.01

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Expressing, Clone Assay, Luciferase, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Schematic of the ‘IM-rich subset’ of lung squamous carcinoma. TNFα activation of the canonical NFκB leads to LUSC cell secretion of chemo-attractant CCL2, which stimulates the bone marrow to release inflammatory monocytes (IMs) into circulation. The IMs bring large payloads of FXIIIA into the tumor microenvironment, leading to cross-linked fibrin, LUSC invadopodia formation and progression

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: HSF1 Attenuates LPS-Induced Acute Lung Injury in Mice by Suppressing Macrophage Infiltration

doi: 10.1155/2020/1936580

Figure Lengend Snippet: HSF1 attenuates the MCP-1 expression in serum, lung tissue, and BALF from LPS-induced ALI mice. MCP-1 levels were measured in (a) serum, (b) lung tissue, and (c) BALF at 12 h, 24 h, and 36 h after LPS treatment. (d) MCP-1 mRNA levels in the lung tissue from LPS-induced ALI mice using qRT-PCR. (e) HSF1 mRNA levels in the lung tissue from LPS-induced ALI mice using qRT-PCR. # p < 0.05, ## p < 0.01, versus HSF1 +/+ +NS group; ∗ p < 0.05, ∗∗ p < 0.01, versus HSF1 +/+ +LPS group; n = 6 mice per group. p values were determined using two-tailed Student's t -test for comparing two groups and one-way ANOVA for comparing multiple groups.

Article Snippet: Supernatants from lung tissue homogenate, BALF, and serum were collected to measure the levels of MCP-1 using an ELISA kit (MJE00B, RD, USA) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Oxidative Medicine and Cellular Longevity

Article Title: HSF1 Attenuates LPS-Induced Acute Lung Injury in Mice by Suppressing Macrophage Infiltration

doi: 10.1155/2020/1936580

Figure Lengend Snippet: HSF1 downregulated the transcription of MCP-1/CCR2 . (a, b, d) The binding of HSF1 to HSE in MCP-1 and CCR2 promoters was assessed using EMSA. (a, b) EMSA was used to detect the binding of the HSF1 protein with HSE1 (a) and HSE2 (b) in the MCP-1 promoter region in vitro. The biotin probe specific for HSE1 or HSE2 was bound by nuclear extracts of RAW 264.7 cells, which could be blocked by an HSF1 antibody. (d) EMSA was used to detect the binding of the HSF1 protein with HSE1 in the CCR2 promoter region in vitro. The biotin probe specific for HSE1 was bound by nuclear extracts, which could be blocked by an HSF1 antibody. Lane 1: negative control; lane 2: biotin-labeled HSE probe preincubated with nucleoproteins (HSF1-HSE complexes); lane 3: supershift analysis using anti-HSF1 antibodies; lane 4: competition using a 200-fold excess of the unlabeled competitive probe; lane 5: competition using a 200-fold excess of the unlabeled mutant probe. The HSF1/HSE complex is indicated by red arrowheads, the supershift band is indicated by blue arrowheads, and unbound HSE probe is indicated by black arrowheads. (c, e) Luciferase reporter assay analysis of HSF1 regulation on the promoter transcription activity of MCP-1 (c) and CCR2 (e) in RAW 264.7 cells transfected with the HSF1 overexpression vector (mHSF1) or empty expression vector pcDNA3.1. After 48 h of transfection, the cell lysate was extracted, and the firefly luciferase activity was measured and normalized to the Renilla luciferase activity. ∗∗ p < 0.01, pcDNA3.1 versus mHSF1 (intragroup comparison); # p < 0.05, versus mHSF1 + MCP-1-wt (or mHSF1 + CCR2-wt); ## p < 0.01, versus mHSF1 + MCP-1-wt (or mHSF1 + CCR2-wt). Data are representative of at least three independent experiments. p values were determined using two-tailed Student's t -test for comparing two groups and one-way ANOVA for comparing multiple groups.

Article Snippet: Supernatants from lung tissue homogenate, BALF, and serum were collected to measure the levels of MCP-1 using an ELISA kit (MJE00B, RD, USA) according to the manufacturer's instructions.

Techniques: Binding Assay, In Vitro, Negative Control, Labeling, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Transfection, Over Expression, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Cellular & Molecular Biology Letters

Article Title: PCSK9 inhibition ameliorates microplastic-induced endothelial redox imbalance via SIRT6 modulation

doi: 10.1186/s11658-025-00838-z

Figure Lengend Snippet: Effects of MPs on teloHAEC. Cell viability evaluated in teloHAEC exposed to different concentrations of A PE and B PVC (0–70 µg/mL) for 24, 48, and 72 h. C , D Cell viability assessed after 48 h of treatment with PE or PVC (30 µg/mL) and increasing concentration of PVC or PE (0–70 µg/mL), respectively. Evaluation of E MCP-1, F VCAM1, and G ICAM1 content in teloHAEC treated with PE (70 µg/mL), PVC (70 µg/mL), or PE + PVC (30 µg/mL + 30 µg/mL) for 48 h. H , I Representative cell cycle detection by FACS analysis in EC exposed to MPs. Data expressed as mean ± standard deviation (SD) of n = 4 independent experiments. * p < 0.05 versus 0 µg/mL or Ctr; ** p < 0.01 versus 0 µg/mL or Ctr; • p < 0.001 versus 0 µg/mL or Ctr; † p < 0.05 versus PE; ‡ p < 0.05 versus PVC; ns, not significant versus PE and PVC

Article Snippet: The levels of inflammatory mediators MCP-1 (RAF081R, BioVendor, Brno, Czech Republic), VCAM1 (EH0326, FineTest, Hubei, China), and ICAM1 (EH0161, FineTest, Hubei, China) were determined in cell culture supernatant, as previously reported [ ].

Techniques: Concentration Assay, Standard Deviation

Journal: Cellular & Molecular Biology Letters

Article Title: PCSK9 inhibition ameliorates microplastic-induced endothelial redox imbalance via SIRT6 modulation

doi: 10.1186/s11658-025-00838-z

Figure Lengend Snippet: iPCSK9 opposed the MP-related inflammation. A TeloHAEC viability evaluated after treatment with PE and PVC alone or combined PE + PVC, or pretreated with iPCSK9 (100 ng/mL) for 8 h and then exposed to PE, PVC, and PE + PVC. B Immunoblotting analysis of SIRT6 protein levels and ELISA assays of C MCP-1, D VCAM1, and E ICAM1. F Representative annexin V-FITC and PI-staining detected by FACS analysis and G cell cycle investigation. Data expressed as mean ± SD of n = 4 experiments. M, molecular weight markers; lane 1, Ctr; lane 2, PE; lane 3, PVC; lane 4, PE + PVC; lane 5, iPCSK9; lane 6, PE + iPCSK9; lane 7, PVC + iPCSK9; lane 8, PE + PVC + iPCSK9. * p < 0.05 versus Ctr; ** p < 0.01 versus Ctr; • p < 0.001 versus Ctr; † p < 0.05 versus PE; ‡ p < 0.05 versus PVC; + p < 0.05 versus PE + PVC; ¶ p < 0.01 versus PE; § p < 0.01 versus PVC; ▲ p < 0.01 versus PE + PVC; ns, not significant versus PE and PVC

Article Snippet: The levels of inflammatory mediators MCP-1 (RAF081R, BioVendor, Brno, Czech Republic), VCAM1 (EH0326, FineTest, Hubei, China), and ICAM1 (EH0161, FineTest, Hubei, China) were determined in cell culture supernatant, as previously reported [ ].

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Molecular Weight

Journal: Cellular & Molecular Biology Letters

Article Title: PCSK9 inhibition ameliorates microplastic-induced endothelial redox imbalance via SIRT6 modulation

doi: 10.1186/s11658-025-00838-z

Figure Lengend Snippet: SIRT6 silencing opposed the effects of iPCSK9 on inflammation and apoptosis. A Immunoblotting analysis of SIRT6 protein levels in teloHAEC treated with the empty transfection reagent (Vehicle) or transfected with negative control siRNA (NT) or with SIRT6 siRNA (siSIRT6). M, molecular weight markers; lane 1, Ctr; lane 2, Vehicle; lane 3, NT; lane 4, siSIRT6. B Immunoblotting analysis of PCSK9 protein levels and ELISA assays of C MCP-1, D VCAM1 and E ICAM1. F , G Representative dot plots and FACS analysis of annexin V-FITC and PI-staining of EC transfected with NT or siSIRT6 and exposed to PE + PVC alone or pretreated with iPCSK9 (100 µg/mL) before MP stimulation. M, molecular weight markers; lane 1, Ctr; lane 2, NT; lane 3, NT + PE + PVC; lane 4, NT + iPCSK9; lane 5, NT + PE + PVC + iPCSK9; lane 6, siSIRT6; lane 7, siSIRT6 + PE + PVC; lane 8, siSIRT6 + iPCSK9; lane 9, siSIRT6 + PE + PVC + iPCSK9. Q1: necrotic cells; Q2: late apoptotic cells; Q3: early apoptotic cells; Q4: viable cells. Data are expressed as mean ± SD of n = 3 experiments. ° p < 0.01 versus NT; # p < 0.001 versus NT; + p < 0.05 versus PE + PVC; ▲ p < 0.01 versus PE + PVC

Article Snippet: The levels of inflammatory mediators MCP-1 (RAF081R, BioVendor, Brno, Czech Republic), VCAM1 (EH0326, FineTest, Hubei, China), and ICAM1 (EH0161, FineTest, Hubei, China) were determined in cell culture supernatant, as previously reported [ ].

Techniques: Western Blot, Transfection, Negative Control, Molecular Weight, Enzyme-linked Immunosorbent Assay, Staining

Journal:

Article Title: Inflammatory cytokines stimulate the chemokines CCL2/MCP-1 and CCL7/MCP-7 through NF?B and MAPK dependent pathways in rat astrocytes

doi: 10.1016/j.brainres.2009.06.081

Figure Lengend Snippet: Rat astrocytes were stimulated with serum-free media (Control) (□), 2.5 ng/ml IL-1β (●) or 25 ng/ml TNF-α (▼) for increasing amounts of time over 48 hrs (A,B), or were stimulated with increasing concentrations of IL-1β (●) or TNF-α (▼) for 6 hr (C) or 24 hr (D). Conditioned media was analyzed by ELISA for CCL2 (A,C) or CCL7 (B,D) as described in Methods. Data are mean ± SEM from triplicate determinations and show a representative 1 of 3 independent experiments. Data were analyzed with two way ANOVA followed by Bonferroni post test (A,B) or with one way ANOVA followed by Dunnett’s multiple comparison test (C,D). *p<0.05, **p<0.01, ***p<0.001, vs. controls.

Article Snippet: The levels of CCL2 and CCL7 released into the conditioned media were measured by ELISA (CCL2 BD OptEIA, BD Biosciences, San Jose, CA and CCL7 Construction Kit, Antigenix, Huntington Station, NY) according to the manufacturer’s instructions.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal:

Article Title: Inflammatory cytokines stimulate the chemokines CCL2/MCP-1 and CCL7/MCP-7 through NF?B and MAPK dependent pathways in rat astrocytes

doi: 10.1016/j.brainres.2009.06.081

Figure Lengend Snippet: Rat astrocytes were pre-treated with 10 µM of selective inhibitors of MAPK pathways (SB203580 and SB202190 for p38, U0126 for ERK1/2, or SP600125 for JNK1/2) or control compounds (SB202474, U0124, or JNK ctr- B-F only) for 20 min before addition of 2.5 ng/ml IL-1β or 25 ng/ml TNF-α for 30 min (A), 24 hr (B,E,F) or 6 hr (C,D). Inhibition of specific MAPK pathways was analyzed by Western blots of cell lysates with antibodies against the activated (phosphorylated) form of a substrate downstream of the inhibitor target: phospho MK2 (for p38 inhibitor SB203580), phospho ERK1/2 (for MEK1/2 inhibitor U0126), phospho c-jun (for JNK1/2 inhibitor SP600125), and β-Actin loading control (A). Cell viability was measured by MTS assay (B). After stimulation of cells with IL-1β or TNF-α for 24 hr, MTS reagent was added into each well, cultures were incubated for an additional hr, and the absorbance at 490 nm was measured as described in Methods. Levels of CCL2 (C,D) or CCL7 (E,F) in conditioned media from astrocytes stimulated with IL-1β (C,E) and TNF-α (D,F) were determined by ELISA as described in Methods. Data are expressed as % IL-1β alone or % TNF-α alone. Data are mean ± SEM from triplicate determinations and show a representative 1 of 3 independent experiments (A,B), or are the mean ± SEM of 9–11 independent experiments of triplicate determinations (C–F). Data were analyzed by one way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05, **p<0.01, ***p<0.001 vs. IL-1β alone or vs. TNF-α alone.

Article Snippet: The levels of CCL2 and CCL7 released into the conditioned media were measured by ELISA (CCL2 BD OptEIA, BD Biosciences, San Jose, CA and CCL7 Construction Kit, Antigenix, Huntington Station, NY) according to the manufacturer’s instructions.

Techniques: Control, Inhibition, Western Blot, MTS Assay, Incubation, Enzyme-linked Immunosorbent Assay, Comparison

Journal:

Article Title: Inflammatory cytokines stimulate the chemokines CCL2/MCP-1 and CCL7/MCP-7 through NF?B and MAPK dependent pathways in rat astrocytes

doi: 10.1016/j.brainres.2009.06.081

Figure Lengend Snippet: Rat astrocytes were pre-treated with 10 µM of either SB203580 (SB580) + U0126 (U6), SB203580 + SP600125 (SP125), U0126 + SP600125, SB202474 (SB474) + U0124 (U4), SB202474 + JNK ctr, or U0124 + JNK ctr for 20 min before addition of 2.5 ng/ml IL-1β or 25 ng/ml TNF-α for 6 hr (A,B) or 24 hr (C,D), and conditioned media was analyzed by ELISA for CCL2 (A,B) or CCL7 (C,D). Data are expressed as % IL-1β alone or % TNF-α alone. Data are means ± SEM of 3 independent experiments of triplicate determinations. Data were analyzed by one way ANOVA followed by Dunnett’s multiple comparison test for treatments versus stimulus alone, *p<0.05, **p<0.01, ***p<0.001, or by student’s t-test for treatments versus inhibitors alone (Fig 3), ϕ =versus SB203580, ≠ =versus U0126, and ┴=versus SP600125, p<0.05.

Article Snippet: The levels of CCL2 and CCL7 released into the conditioned media were measured by ELISA (CCL2 BD OptEIA, BD Biosciences, San Jose, CA and CCL7 Construction Kit, Antigenix, Huntington Station, NY) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal:

Article Title: Inflammatory cytokines stimulate the chemokines CCL2/MCP-1 and CCL7/MCP-7 through NF?B and MAPK dependent pathways in rat astrocytes

doi: 10.1016/j.brainres.2009.06.081

Figure Lengend Snippet: Rat astrocytes were pre-treated with 100 µM of SC-514 or 1 µM of MG-132 (NFκB inhibitors) for 20 min before addition of serum-free media (Control), 2.5 ng/ml IL-1β or 25 ng/ml TNF-α for 15 min (A). Inhibition of the NFκB pathway was analyzed by Western blots of cell lysates with antibodies against IκB-α and β-Actin loading control (A). Rat astrocytes were pre-treated with increasing concentrations of SC-514 or MG-132 for 20 min before addition of IL-1β or TNF-α, and after 24 hr MTS reagent was added into each well and cultures were incubated for an additional hr. The absorbance at 490 nm in each well was then measured as described in Methods (B). Rat astrocytes were pre-treated with increasing concentrations of SC-514 or MG-132 for 20 min before addition of IL-1β or TNF-α, and media was collected at 6 hr (for CCL2) and 24 hr (for CCL7) and analyzed by ELISA (C–F). Data are expressed as % IL-1β alone or % TNF-α alone (C–F). Data are mean ± SEM of triplicate determinations of 1 of 3 independent experiments (A,B) or are the mean ± SEM of 6 independent experiments of triplicate determinations (C–F). Data were analyzed by one way ANOVA followed by Dunnett’s multiple comparison test, *p<0.05, **p<0.01, vs. IL-1β or vs. TNF-α.

Article Snippet: The levels of CCL2 and CCL7 released into the conditioned media were measured by ELISA (CCL2 BD OptEIA, BD Biosciences, San Jose, CA and CCL7 Construction Kit, Antigenix, Huntington Station, NY) according to the manufacturer’s instructions.

Techniques: Control, Inhibition, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Inflammation induced by incomplete radiofrequency ablation accelerates tumor progression and hinders PD-1 immunotherapy

doi: 10.1038/s41467-019-13204-3

Figure Lengend Snippet: Tumor cell-derived CCL2 is critical for TAM accumulation in residual tumors. a CCL2 mRNA and protein expression in the untreated and iRFA-treated CT26 and MC38 tumors on days 3, 6, 9, and 12 by real-time PCR and Western blot ( n = 5). b Representative microphotographs are showing CCL2, TNFα, and F4/80 staining of the untreated and residual CT26 and MC38 tumor (the necrotic ablation area is below the red dotted line). Scale bar = 100 μm ( n = 3). c Flow cytometric analysis and quantification of CCR2 expression on total myeloid cells and monocytes (gate on CD11b + cells) in the untreated and iRFA-treated CT26 and MC38 tumors on day 9 after iRFA ( n = 5). d Flow cytometric analysis and quantification of CD11b + , F4/80 + , Ly6G + and Ly6C + cells in residual wild-type and CCL2 -/- CT26 and MC38 tumors on day 9 after iRFA (gate on CD45 + liver cells and CD11b + cells, respectively) ( n = 5). e mRNA expression of IL-10 , IL-6 , TGFβ , Arg1 , IDO , and TNFα in residual wild-type and CCL2 −/− CT26 and MC38 tumors on day 9 after iRFA ( n = 5). f The growth curve of residual wild-type and CCL2 −/− CT26 and MC38 tumors treated with iRFA (one-sided ANOVA test, *** P < 0.001, n = 5). g The number of distant metastases in wild-type and CCL2 −/− CT26 and MC38 tumor-bearing mice on day 14 after iRFA (yellow arrow indexes metastasis) ( n = 6). Data represent cumulative results from 1/2 independent experiments. The data are represented as mean ± SEM. Statistical differences between pairs of groups were determined by a two-tailed Student’s t -test (** P < 0.01, *** P < 0.001).

Article Snippet: The level of CCL2 secreted protein was measured in the cell supernatants using an ELISA kit specific for mouse CCL2 (Cat SMZE00B, R&D Systems), following the manufacturer’s instructions.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Inflammation induced by incomplete radiofrequency ablation accelerates tumor progression and hinders PD-1 immunotherapy

doi: 10.1038/s41467-019-13204-3

Figure Lengend Snippet: TAMs promote tumor cells to produce CCL2 through TNFα. a , b Mice were administered etanercept intraperitoneally before iRFA treatment. a CCL2 mRNA expression was quantified by real-time PCR on day 3 ( n = 5). b CCL2 protein expressions were analyzed by Western blot on day 3 ( n = 5). c , d CT26 or MC38 cancer cells were cocultured with TAMs (CD11b + F4/80 + ) isolated from residual tumors. To block TNFα and TNFR1, anti-mouse TNFα IgG (R&D Systems) or anti-TNFR1 mAb were added. c Total RNA was extracted, and CCL2 mRNA was quantified by real-time PCR. Relative mRNA expression was expressed as fold-change ( n = 5). d Concentration of secreted CCL2 protein in culture media was determined by ELISA ( n = 5). e , f CT26 or MC38 cells were stimulated with recombinant mouse TNFα. e Relative mRNA expression was expressed as fold-change ( n = 3). f CCL2 protein was detected by immunofluorescence analyses ( n = 3). g Schematic depiction of the crosstalk between residual tumor cells and TAMs promotes CCL2 production by tumor cells, sustaining the infiltration of myeloid cells and inhibiting T cell activity in residual tumors. Data represent cumulative results from 1/2 independent experiments. The data are represented as mean ± SEM. Statistical differences between pairs of groups were determined by a two-tailed Student’s t -test (*** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: The level of CCL2 secreted protein was measured in the cell supernatants using an ELISA kit specific for mouse CCL2 (Cat SMZE00B, R&D Systems), following the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Isolation, Blocking Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Immunofluorescence, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Inflammation induced by incomplete radiofrequency ablation accelerates tumor progression and hinders PD-1 immunotherapy

doi: 10.1038/s41467-019-13204-3

Figure Lengend Snippet: CCL2/CCR2 blockade inhibits tumor progression and overcomes resistance to anti-PD-1 therapy. a – f iRFA treatment was performed in CT26 and MC38 colon cancer models as shown in Fig. . Anti-PD-1 mAb (200 μg, clone: J43) was administered through intraperitoneal injection to mice every 3 days for a total of four times. The CCR2 antagonist (CCR2a) (RS504393, Tocris) was given subcutaneously at a dose of 5 mg/kg twice per day for 9 days. a Growth curve of the CT26 and MC38 residual tumor (one-sided ANOVA test, n = 8). b The weight of the residual CT26 and MC38 tumor examined on day 14 after iRFA by dissection of the mice ( n = 6). c The number of metastases examined on day 14 after iRFA by dissection the mice ( n = 6). d Kaplan–Meier survival curves are shown, and the log-rank test was performed ( n = 8). e Flow cytometric analysis and quantification of CD3 + and CD8 + infiltration (gate on single live cells) in residual CT26 tumors. f Granzyme B and IFNγ expression on CD8 + cells in residual CT26 tumors. (gate on CD8 + cells) ( n = 5). g , h iRFA treatment was performed in mice bearing wild type and CCL2 −/− CT26 or MC38 tumor. g Growth curve of the CT26 and MC38 residual tumor (one-sided ANOVA test, n = 5). h Kaplan–Meier survival curves are shown, and the log-rank test was performed ( n = 8). Data represent results from 1/2 independent experiments. The data are represented as mean ± SEM. Statistical differences between pairs of groups were determined by a two-tailed Student’s t-test (ns present not significant, * P < 0.05, ** P < 0.01, *** P < 0.001). Source data are provided as a Source Data file.

Article Snippet: The level of CCL2 secreted protein was measured in the cell supernatants using an ELISA kit specific for mouse CCL2 (Cat SMZE00B, R&D Systems), following the manufacturer’s instructions.

Techniques: Injection, Dissection, Expressing, Two Tailed Test