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

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) HBEpC were cultured with cGAMP (14nM), H 2 O 2 , (100μM) or cGAMP+H 2 O 2 for 4 hours. (B-F) qPCR analysis of human IFNα (B) and IFNβ (C) and epithelial cytokines, IL-33 (D), TSLP (E), and RANTES (F). RANTES was stimulated by cGAMP and cGAMP+ H 2 O 2 . IFNβ was also stimulated by cGAMP+H 2 O 2 . Statistical analysis is performed by one-way ANOVA (means ± SEM, n=4). * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) 8 weeks mice were treated with 20 ng of RANTES with 1 μg of HDM or 1 μg of HDM intra nasally on Day 1. Mice were challenged with 1 μg of HDM intranasally on Day 7, then lungs were extracted and analyzed on Day 8. (B) Pictures of lung sections of control and RANTES-adjuvanted, HDM-sensitized mice stained with H-E. Scale bar: 200 μm. Scale bar in picture of high magnification view: 40 μm. (C) Number of cells between bronchus and alveoli analyzed by ImageJ. Statistical analysis is performed by ordinary Mann Whitney’s U test (means ± SEM, 3 points/ section, n=5). (D) Pictures of lung sections in PBS and RANES-adjuvanted, HDM-sensitized mouse stained with PAS/Alcian blue. Scale bar: 80 μm. (E) Ratio of PAS/Alcianble area per epithelial cells (%). **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Control, 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: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 3 ADSCs may be affected by endogenous CCL5 signaling. (A) Expression of CCR1 ligands CCL3 and CCL5 in peripheral blood mononuclear cells (BCs), 6 samples of ADSCs (ADSCs 1–6) and skin primary fibroblasts (FBs 1–2) was examined by RT-PCR (CCL3/CCL5 and GAPDH targets were amplified for 40 and 20 cycles, respectively). (B) The level of CCL5 mRNA expression in different samples of ADSCs (I, IV, V, VII, VIII) cultivated for different periods of time (passages p1–p12) was measured using qRT-PCR, normalized with GAPDH expression level and set as 1 in the sample Ip9. The other samples were calculated accordingly. The data are presented as an average mean of one measurement performed in triplicates±SD. Concurrently, concentrations of secreted CCL5 protein (pg/ml) in the conditioning media of the samples I p2, I p9, VIII p2, VII p1, VII p3 and VII p4 were measured by ELISA; NA — not analyzed. (C) CCL5 protein in permeabilized naive or treated with 50 ng/ml of recombinant CCL5 ADSCs (left and right panels, respectively) was examined using CCL5 antibody (red). Nuclear staining was performed using DAPI (blue) (40× magnification). (D) Proliferation and viability of ADSCs cultured in the presence of 25 and 50 ng/ml of CCL5 for 24 and 48 h were tested using ViaLight™plus kit. (E) Three independent lineages of ADSCs were treated with 50 ng/ml of CCL5 for 8, 24, 48 and 72 h, and the level of CCL5 mRNA expression was measured in triplicates, normalized with GAPDH expression level and set as 1 in untreated samples. The data are presented as fold of induction of CCL5 expression in CCL5 treated samples over untreated controls±SD; **pb0.01.

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Staining, Cell Culture

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 4 ADSCs respond to CCL5. (A) ADSCs were stimulated with 50 ng/ml of CCL5 for 3 h (right panel) and subjected to immunostaining using CCR1 antibody (red) and DAPI (blue). Non-stimulated cells are shown in the left panel. (B) ADSCs were treated with different concentrations of CCL5 for 20 min. The levels of phospho-AKT, phospho-ERK, AKT, ERK and GAPDH proteins were detected by WB. (C) CCR1, CCR3 and CCR5 expression levels in two ADSC samples were measured in triplicates by qRT-PCR, normalized with GAPDH mRNA expression levels and set as 1 in sample V (left panel). ADSCs were stimulated with 50 ng/ml of CCL5 for 10 min or 1 h, the cells were lysed and subjected to WB analysis using phospho-ERK, phospho-AKT, ERK, AKT and GAPDH antibodies. (D) Migration capacity of ADSCs towards 50 ng/ml of CCL5 (left panel) was examined using a modified Boyden chamber. As a control, CCL5-free media were used (right panel). The migratory cells were stained and observed under a light microscope using 20× objective.

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Immunostaining, Expressing, Quantitative RT-PCR, Migration, Modification, Control, Staining, Light Microscopy

Journal: Stem cell research

Article Title: CCL5/CCR1 axis regulates multipotency of human adipose tissue derived stromal cells.

doi: 10.1016/j.scr.2012.11.004

Figure Lengend Snippet: Fig. 5 CCL5 induces NFκB transcriptional activity. (A) ADSCs were co-transfected with plasmids encoding firefly luciferase reporter under the control of NFκB dependent promoter and Renilla luciferase reporter under SV40 promoter. The cells were stimulated with CCL5 at indicated concentrations for 24 h. Average firefly luciferase activity obtained from three indepen- dent experiments measured in three replicates were normalized to Renilla luciferase values and presented as a fold of induction±SD over luciferase activity in a non-stimulated ADSC sample (set as 1). (B) ADSCs were stimulated with 50 ng/ml of CCL5 for indicated periods of time and several NFκB target gene mRNA expression levels were analyzed by qRT-PCR. Target gene mRNA levels were measured in triplicates and normalized with GAPDH mRNA expression level. The level of the particular gene expression in the untreated control cells at the same time point was set as 1 (indicated by line). Data are represented as an average mean of fold of induction of the indicated gene expression over non- stimulated control±SD obtained from three independent experi- ments. ((A, B) *pb0.05, **pb0.01.)

Article Snippet: Cells were exposed to CCR1 antibody CKR-1 (C-20), mouse monoclonal CCL5 capture antibody from CCL5/RANTES DuoSet ELISA Development System (R&D System, Wiesbaden, Germany), or BrdU antibody mAbG3G4 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA) at dilutions of 1:300, 1:500 and 1:200, respectively, for 2 h at RT.

Techniques: Activity Assay, Transfection, Luciferase, Control, Expressing, Quantitative RT-PCR, Gene Expression