Journal: iScience

Article Title: Serglycin modulates inflammation and metabolism in macrophages

doi: 10.1016/j.isci.2026.115235

Figure Lengend Snippet: SRGN deficiency reshapes the inflammatory secretome and transcriptional programs in human macrophages (A) Volcano plot of differentially secreted proteins between SRGN −/− and wild-type THP-1 macrophages under M1 polarization. Among 1,507 quantified proteins, 53 were significantly altered (adjusted p < 0.05), with serglycin being the most downregulated protein in knockout cells. (B) Validation of selected targets by RT-qPCR. SRGN −/− M1 macrophages showed significantly increased expression of IL6 and TNF and reduced expression of CCL5 compared with wild-type cells (mean ± SEM; unpaired Student’s t test; p < 0.05, ∗ p < 0.01, and ∗∗∗ p < 0.0001; n = 4). (C) ELISA quantification of secreted cytokines in culture supernatants. SRGN −/− macrophages secreted significantly less TNF-α, CCL5, and IL-6 compared with wild-type macrophages ( n = 4), consistent with proteomics and RNA-seq data. (D) Transmission electron microscopy (TEM) images of THP-1 M0 and M1 macrophages. Scale bars, 5 μm. Vesicles were manually annotated and quantified in 10 cells per experimental group. The number of vesicles per cell and the percentage of cellular area occupied by vesicles were significantly reduced in both M0 and M1 SRGN −/− macrophages compared with wild-type macrophages. (E) Phagocytosis assay using fluorescently labeled bioparticles. SRGN −/− macrophages exhibited reduced phagocytic capacity under both M0 and M1 conditions (mean ± SEM; unpaired Student’s t test; p < 0.05 and ∗∗ p < 0.001; n = 6).

Article Snippet: THP-1: cytokine levels were measured using ELISA kits from R&D Systems: CCL5 (Cat. No. DY278), IL6 (Cat. No. DY206), IL-1β (Cat. No. DY201), TGF-β (Cat. No. DY240; activation kit DY010), and TNF-α (Cat. No. DY210), following the manufacturer’s instructions.

Techniques: Knock-Out, Biomarker Discovery, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Transmission Assay, Electron Microscopy, Phagocytosis Assay, Labeling

Journal: Toxicology and applied pharmacology

Article Title: MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure.

doi: 10.1016/j.taap.2015.05.017

Figure Lengend Snippet: Fig. 3. MUTZ-LC migration out of the epidermis after irritant but not allergen exposure is CCL5 dependent. SE-LC were exposed to water vehicle (V), 10 mM NiSO4 (N) or 2.5 mM Triton X-100 (T) for 16 h. Chemical exposure was performed in the presence of the neutralizing antibody to CCL5 (+) or IgG1 isotype control (−). Number of CD1a-PE positive MUTZ-LC in epidermal sheets was quantified using Image J software. Data represent the average of 4 individual experiments ± SEM. *p b 0.05, **p b 0.01 was calculated using the Mann–Whitney t-test.

Article Snippet: For blocking experiments 7 μg/mL goat anti-human CXCL12 (AF-310-NA, R&D systems, Minneopolis, MN, USA), 7 μg/mL goat anti-human CCL5 (AF-278-NA, R&D systems) or 2 μg/mL goat anti-human IL-10 (AF-217NA, R&D systems) was added to the culture medium prior to chemical exposure (optimal blocking concentration was previously determined) (Ouwehand et al., 2008, 2010a, 2011a).

Techniques: Migration, Control, Software, MANN-WHITNEY

Journal:

Article Title: Regulated on Activation, Normal T-Cell-Expressed and -Secreted mRNA Expression in Normal Endometrium and Endometriotic Implants

doi:

Figure Lengend Snippet: Isolated primary endometriotic stromal and epithelial cells were examined for RANTES mRNA expression by in situ hybridization. Subconfluent monolayers of endometriotic stromal cells were cultured for 48 hours. No RANTES mRNA was detectable under conditions in which the medium contained no added cytokine (A). After culturing the cells in the presence of TNF-α (100 ng/ml) for 48 hours (B), RANTES mRNA was expressed in 10 to 20% of the stromal cells. No specific autoradiographic signal was observed when TNF-α stimulated endometriotic stromal cells were hybridized with the RANTES sense RNA-control probe (C). Subconfluent monolayers of epithelial cells did not reveal evidence of RANTES expression, even after TNF-α stimulation (D). Original magnifications, ×600.

Article Snippet: Immunoperoxidase staining was performed overnight at 4°C, using mouse monoclonal IgG antibodies against human cytokeratin-18 (1:2000 dilution; Sigma, St. Louis, MO), human CD-68 (dilution 1:100; DAKO Corp., Carpinteria, CA), human RANTES (dilution 1:100; R&D Systems, Minneapolis, MN), or TNF-α (dilution 1:100, R&D Systems).

Techniques: Isolation, Expressing, In Situ Hybridization, Cell Culture, Control

Journal:

Article Title: Regulated on Activation, Normal T-Cell-Expressed and -Secreted mRNA Expression in Normal Endometrium and Endometriotic Implants

doi:

Figure Lengend Snippet: In situ hybridization of RANTES mRNA in normal mid-proliferative human endometrium (A). RANTES gene expression could be detected in the stromal compartment adjacent to epithelial glands, but the glands themselves did not contain RANTES message. No specific autoradiographic signal was observed when serial tissue sections were hybridized with the RANTES sense RNA-control probe (B). Endometriotic implants also showed RANTES message in the stromal compartment with several intense spots (black arrows) and other more diffuse hybridization (white arrows). The epithelial cell lining of the endometrioma and the unaffected ovarian stroma (top) did not contain RANTES message (C). No specific autoradiographic signal was observed when endometriotic tissue sections were hybridized with the RANTES sense RNA-control probe (D). Immunohistochemical localization of CD68-positive macrophages (E, black arrows) in an adjacent endometriotic tissue section. Monoclonal antibodies against human RANTES demonstrated this protein in the endometriotic stroma, with sparing of the cyst epithelium (F). Monoclonal antibodies against TNF-α showed the protein in the epithelial and stromal compartments of the endometriotic implant. Portions of the ovary uninvolved with endometriosis showed less TNF-α immunostaining (G). Control experiments were performed on serial endometriotic sections stained with TNF-α antibodies immunoabsorbed with excess TNF-α protein (H). Epithelial cells lining the endometriotic cyst were cytokeratin-positive (I). All sections were also counterstained with hematoxylin. H&E staining shows the morphology of the endometriotic implant (K). Original magnifications, ×200.

Article Snippet: Immunoperoxidase staining was performed overnight at 4°C, using mouse monoclonal IgG antibodies against human cytokeratin-18 (1:2000 dilution; Sigma, St. Louis, MO), human CD-68 (dilution 1:100; DAKO Corp., Carpinteria, CA), human RANTES (dilution 1:100; R&D Systems, Minneapolis, MN), or TNF-α (dilution 1:100, R&D Systems).

Techniques: In Situ Hybridization, Gene Expression, Control, Hybridization, Immunohistochemical staining, Bioprocessing, Immunostaining, Staining

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 1 – (a) Correlation between serum CCL5 level and tumor progression in patients with gastric cancer. Serum CCL5 levels in 91 patients with gastric cancer and 53 healthy volunteers were measured. Clinical stage was defined by the International Union Against Can- cer’s UICC TNM Classification of Malignant Tumors.22 All data are presented as the mean 6 SD. The number of patients in each group is displayed at the bottom of the graph. *p < 0.001 vs. healthy controls, p < 0.01 vs. T1–T2 group, p < 0.01 vs. N(2) group, §p < 0.01 vs. Stage I–II group. (b) Correlation between survival rate and serum CCL5 level in patients with gastric cancer. There was a significant dif- ference between the patients with high (>27 ng/ml) and low (27 ng/ ml) serum CCL5 levels as determined by the log-rank test (p 5 0.02). The 5-year survival rate in patients with high serum CCL5 levels was 60.4%, and in patients with low serum CCL5 levels was 86.1%. Dot- ted line: patients with high serum CCL5 levels (>27 ng/ml). Solid line: patients with low serum CCL5 levels (27 ng/ml).

Article Snippet: Tumor and PBMC bearing mice were injected i.p. with 25 lg of anti-human CCL5 monoclo- nal antibody (MAB678; R&D Systems) or mouse IgG1 (MAB002; R&D Systems) in 100 lL PBS on day 0, day 1, day 2 and day 7 to monitor survival.

Techniques:

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 3 – CCL5 production by cocultures of whole PBMCs, CD41 cells, CD42 cells and CD81 cells with gastric cancer cell lines. Whole PBMCs, CD41 cells, CD42 cells and CD81 cells were all obtained from healthy volunteers. Cells (1 3 106) were cocultured with viable (a) or irradiated (b) gastric cancer cell lines (5 3 105), MKN45 or KATO III, for 48 h. Viable (a) or irradiated (b) MKN45 and KATO III cells were cultured for 48 h without any lymphocytes. PBMCs were also cultured alone for 48 h (a). CCL5 levels in the supernatants were measured. All data are presented as the mean 6 SE of 4 individual experiments. *p < 0.01, p < 0.05 vs. other groups.

Article Snippet: Tumor and PBMC bearing mice were injected i.p. with 25 lg of anti-human CCL5 monoclo- nal antibody (MAB678; R&D Systems) or mouse IgG1 (MAB002; R&D Systems) in 100 lL PBS on day 0, day 1, day 2 and day 7 to monitor survival.

Techniques: Irradiation, Cell Culture

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 2 – (a) Representative immunohistochemical staining for CCL5 (3400) and CCL5 receptors (3200), CCR1, CCR3 and CCR5, on resected gastric cancer tissue. (b) Representative fluorescent double staining for CCL5/CD4 or CCL5/CD8 on resected gastric cancer tis- sue. Left column: CCL5-red fluorescence and CD4-green fluores- cence. Right column: CCL5-red fluorescence and CD8-green fluores- cence. Upper panels (3200), Lower panels (31,000).

Article Snippet: Tumor and PBMC bearing mice were injected i.p. with 25 lg of anti-human CCL5 monoclo- nal antibody (MAB678; R&D Systems) or mouse IgG1 (MAB002; R&D Systems) in 100 lL PBS on day 0, day 1, day 2 and day 7 to monitor survival.

Techniques: Immunohistochemical staining, Staining, Double Staining

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 4 – (a) CCR1, CCR3 and CCR5 expression on the gastric cancer cell lines, MKN28 and KATO III by flowcytometric analyses. Representative data are shown from 5 individual experiments. Gray shadow indicates CCR1, 3 or 5. Solid line indicates isotype-matched controls. (b) The effect of CCL5 stimulation on cell proliferation of the gastric cancer cell lines MKN28 and KATO III. Tumor cells (1 3 104/well) were seeded in 96-well flat-bottomed plates. After 12 h, tu- mor cells were stimulated with or without indicated concentrations of rh CCL5. Incorporation of [3H] thymidine into the DNA of proliferat- ing tumor cells was measured by a liquid scintillation counter. All data are presented as the mean 6 SE of 4 individual experiments. *p < 0.05 vs. the value without CCL5.

Article Snippet: Tumor and PBMC bearing mice were injected i.p. with 25 lg of anti-human CCL5 monoclo- nal antibody (MAB678; R&D Systems) or mouse IgG1 (MAB002; R&D Systems) in 100 lL PBS on day 0, day 1, day 2 and day 7 to monitor survival.

Techniques: Expressing

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 5 – (a) The effect of CCL5-treated cancer cells on the CD41/CD81 proportion and apoptosis of CD81 cells with cocul- tured PBMCs. Gastric cancer cells (KATO III, 5 3 105) were incu- bated with 50 ng/ml of CCL5 or medium alone for 24 h. After wash- ing cells to remove CCL5, 1 3 106 PBMCs were cocultured with CCL5-pretreated gastric cancer cells for 48 h. The CD41/CD81 pro- portion of PBMCs was examined using a flow cytometer (left column) and are shown as mean 6 SE (middle column). Upper-left quadrants indicate the percentage of CD41 cells and lower-right quadrants indi- cate the percentage of CD81 cells. Annexin V expression on CD81 cells in the cocultured PBMCs is shown (right column). Bars indicate Annexin V positive expression as determined by isotype controls. Inserted data indicate the mean percentage 6 SE of Annexin V posi- tive cells. Four individual experiments were done and the representa- tive data are depicted. Upper panel: coincubation with PBMCs and CCL5 pretreated gastric cancer cells. Lower panel: coincubation with PBMCs and untreated gastric cancer cells. (b) The effect of CCL5- treated cancer cells on the Fas/Fas Ligand expression of CD41/ CD81 cells with cocultured PBMCs. Gastric cancer cells (KATO III, 5 3 105) were incubated with 50 ng/ml of CCL5 or medium alone for 24 h. After washing cells to remove CCL5, 1 3 106 PBMCs were cocultured with CCL5-pretreated gastric cancer cells for 48 h. The CD41/CD81 proportion of PBMC was examined using a flow cy- tometer and the expression of Fas (upper) and Fas Ligand (lower) was examined. Four individual experiments were done and the representa- tive data are depicted. Solid line indicates CCL5-pretreated cells; dot- ted line indicates no treatment; dashed line indicates isotype-matched controls.

Article Snippet: Tumor and PBMC bearing mice were injected i.p. with 25 lg of anti-human CCL5 monoclo- nal antibody (MAB678; R&D Systems) or mouse IgG1 (MAB002; R&D Systems) in 100 lL PBS on day 0, day 1, day 2 and day 7 to monitor survival.

Techniques: Cytometry, Expressing, Incubation

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 6 – The effect of neutralization of CCL5 on tumor survival rate in PBMC-bearing SCID mice. Six-week-old SCID mice were irra- diated (2 Gy, 150 kV, 5.0 mA) and 1 h later were injected i.p. with 1 3 107 KATO III cells and 1 3 107 human PBMCs. SCID mice were also injected i.p. with 1 3 107 KATO III cells alone. Tumor- and PBMC-bearing mice were given i.p. injections of 25lg/body anti- human CCL5 neutralizing antibody (n 5 10) or mouse IgG1 (n 5 10) on day 0, day 1, day 2 and day 7. The survival rates were generated using the Kaplan-Meier method, and the significance of the difference in the survival rates was determined by the log-rank test (p < 0.05).

Article Snippet: Tumor and PBMC bearing mice were injected i.p. with 25 lg of anti-human CCL5 monoclo- nal antibody (MAB678; R&D Systems) or mouse IgG1 (MAB002; R&D Systems) in 100 lL PBS on day 0, day 1, day 2 and day 7 to monitor survival.

Techniques: Neutralization, Injection, Generated

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×10 5 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 10 6 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 10 6 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 10 6 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 10 6 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Real-time Polymerase Chain Reaction, Control, Sterility, Incubation, Gene Expression, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 48 h, and the expression of key regulators in Notch, NF-κB, STAT3 and HIF, were detected by western blot. (B) 3×10 5 THP-1 macrophages were cultured with 15 mM lactate for 24 h, and the mRNA levels of Notch ligands and receptors were measured by quantitative PCR. (C) Western blot for Notch ligands and receptors in THP-1 (10 6 ) macrophages after 48 h lactate treatment. (D) Lactate stimulated the expression of NICD in a time and dose-dependent manner. 10 6 THP-1 macrophages were treated with 15 mM lactic acid for 48 h. Data presented were representatives of at least three independent experiments. (E) 10 6 THP-1 macrophages were transfected with 50nM siNotch1, or pretreated with 50μM DAPT for 2 h, and then cultured with 15 mM lactate for 48 h. The secretion of CCL5 was measured by ELISA. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 72 h, and then cells were washed twice and fresh media were added. Macrophages were cultured for another 24 h and the conditional media (lactate CM) was collected. The effect of CM on breast cancer cell migration was measured by double chamber transwell assay. 5μg/ml anti-CCL5 neutralizing antibody significantly decreased lactate CM-induced cell migration. (B) 10 6 MCF-7 cells were co-cultured with 15 mM lactate-activated macrophages in the presence of 5μg/ml anti-CCL5 antibody or not, and protein levels of EMT markers were tested by western blot. (C) 10 6 breast cancer cells were co-cultured with 10 6 lactate-activated THP-1 macrophages (or 10 6 lactate-activated primary macrophages) for different time points, and the expression of CCR5 was monitored by western blot. (D) MDA-MB-231 and MCF-7 cells were transfected with shCCR5 plasmids, or pre-treated with 5μM Maraviroc for 2 h, then cell migration induced by lactate CM was detected by double chamber transwell assay. Lactate CM was described in (A). (E) MCF-7 cells (10 6 ) were transfected with pcDNA3.1-CCR5, and then cultured with 10ng/ml CCL5 for 24 h. The expression of E-cadherin, N-cadherin and vimentin was investigated by western blot. (F) 10 6 Human primary macrophages (No. 4 and No. 9) were treated with 15 mM lactate for 72 h and CM was collected as described in (A). The migration of MDA-MB-231 cells was measured in the presence of primary macrophage CM. 5μg/ml anti-CCL5 neutralizing antibody, shRNAs designed against CCR5, or 5μM Maraviroc, significantly reduced primary macrophage CM-induced cell migration. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Migration, Transwell Assay, Western Blot, Expressing, Transfection

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 MDA-MB-231 and MCF-7 cells were stimulated with 1-5ng/ml TGF-β1 for 24 h, and total RNA was isolated and tested for CCR5 mRNA by quantitative PCR. (B) Western blot for CCR5 protein in breast cancer cells (10 6 ) under TGF-β1 stimulation for 48 h. Data presented were representatives of at least three independent experiments. (C) MDA-MB-231 and MCF-7 cells (3×10 5 ) were co-transfected with pGL3-CCR5 and pRL-TK and exposed to different concentrations of TGF-β1 for 24 h, and luciferase activities were determined. (D) MDA-MB-231 and MCF-7 cells were pre-treated with 5μM SIS3 for 2 h, and cells were subjected to luciferase assay. (E) 10 6 MCF-7 cells were transfected with TGFβRI/ALK5 siRNA, and were then co-cultured with lactate-activated THP-1 macrophages (ratio 1:1) for 24 h. The protein levels of CCR5 were assayed by western blot. (F) The expression of TGF-β1, CCL5 and CCR5 in clinical samples obtained from breast cancer patients. The mRNA levels were measured by quantitative PCR, and the correlation between TGF-β1 and CCL5-CCR5 axis was shown. (G) Representative IHC staining for TGF-β1, CCL5 and CCR5 in breast cancer samples. The sample used was derived from 28 breast cancer cases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Cell Culture, Expressing, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure . (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure . (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Transfection, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Western blot for AMPK, c-Myc, HIF-1α and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPKα1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 10 6 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Incubation, Inhibition, Co-Culture Assay, Recombinant, Phospho-proteomics

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) MDA-MB-231 cells were co-cultured with 15 mM lactate-activated THP-1 macrophages for 7 days, in the presence of 5μg/ml anti-CCL5 neutralizing antibody or not. MDA-MB-231 cells were then collected and injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted. (B) CCR5, HK2 and p-AMPK were immunostained in MDA-MB-231 metastases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Injection