Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer

doi: 10.31557/APJCP.2024.25.3.725

Figure Lengend Snippet: ROR1 and ROR2 mRNA Expression in L132 Cells Treated with S1P. L132 cells were serum starved for 48 hrs and treated with vehicle or indicated S1P for 24 hrs. mRNA expression of ROR1 (A) and ROR2 (B) was quantified by qRT-PCR. GAPDH was used as a reference gene. Results represent an average of three experiments (N=3). Data is presented at mean ±SDEV. Means were compared by ANOVA followed by posthoc test. *p<0.01 compared to vehicle. C) ROR1 and ROR2 mRNA expression in A549 cells treated with PF543. Cells were treated with vehicle or 1µM concentration of PF543 for 24 hrs. mRNA expression of ROR1 and ROR2 was quantified by qRT-PCR.GAPDH was used as a housekeeping gene. Means were compared by t-test.*p<0.05 compared to vehicle;**p<0.01 compared to vehicle

Article Snippet: Establishment of cell lines and cell culture The lung cancer cell lines A549 and L132 were purchased from the National Center for Cell Science (NCCS), Pune India, while bronchial epithelial cells BEAS2B (ATCC# CRL-9609) was procured from the American Type Culture Collection (ATCC).

Techniques: Expressing, Quantitative RT-PCR, Concentration Assay

Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: A Crosstalk between the Receptor Tyrosine Kinase-Like Orphan Receptors ROR1/2 and S1P Signaling Pathways in Lung Cancer

doi: 10.31557/APJCP.2024.25.3.725

Figure Lengend Snippet: A-B) ROR1 and ROR2 mRNA Expression in SPHK1 knockdown BEAS2B. A) SPHK1 mRNA levels in HEK293T cells transfected with shSPHK1 when compared with shControl.B) Expression of ROR1 and ROR2 in SPHK1 knockdown BEAS2B. Fold Change expression was calculated. The level of significance was calculated using Two-way ANOVA (Sidak’s multiple comparison test). **P<0.01 compared to shControl. C-D) SPHK1 mRNA expression in ROR1 and ROR2 siRNA knocked down cells. L132 cells were transfected with ROR1 (C) and ROR2 (D), respectively. Forty-eight hours later, total RNA was isolated and mRNA expression of SPHK1 was quantified by qRT-PCR. GAPDH was used as a reference gene. Results represent an average of three experiments (N=3). Data is presented at the mean ±SDEV. **P<0.01 compared to untransfected

Article Snippet: Establishment of cell lines and cell culture The lung cancer cell lines A549 and L132 were purchased from the National Center for Cell Science (NCCS), Pune India, while bronchial epithelial cells BEAS2B (ATCC# CRL-9609) was procured from the American Type Culture Collection (ATCC).

Techniques: Expressing, Knockdown, Transfection, Comparison, Isolation, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Changes in CCL5 and HMGB1 protein levels changes at lesion sites following rat SCI. (A) ELISA measurement of CCL5 protein levels at lesion sites following SCI at 0 d, 1 d, 4 d and 7 d, respectively. (B) Western blot analysis of HMGB1 expression following SCI at 0 d, 1 d, 4 d and 7 d. (C) Quantification data are shown in ( B ). Quantities were normalized to endogenous β-actin. (D) ELISA analysis of CCL5 protein levels at lesion sites at 0 d, 1 d, 4 d and 7 d following with or without intrathecal injection of 10 µl of an HMGB1 neutralizing antibody (HMGB1 Ab, 50 µg/kg). (E) Immunofluorescence staining revealed the colocalization of CCL5 with GFAP-positive astrocytes before or after SCI at 4 d with or without the intrathecal injection of 10 µl of HMGB1 Ab (50 µg/kg). The rectangle indicates the region magnified. Arrowheads indicate positive signals. n = 6. Scale bars, 50 μm in (E) . The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Immunofluorescence, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Examination of CCL5 production in astrocytes following stimulation with rHMGB1. ( A , B ) Purified primary astrocytes were stained with GFAP and Hoechst 33,342, and the purity was greater than 90%. Scale bar, 50 μm. ( C , D ) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following stimulation with 0–2.5 µg/mL rat recombinant HMGB1 (rHMGB1) for 24 h, respectively. ( E , F ) ELISA assay was used to determine the production of CCL5 in the lysates and supernatants following primary astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of 2.5 µg/ml HMGB1 Ab, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of RAGE, TLR-2, or TLR-4 interference on HMGB1-induced CCL5 production in astrocytes. ( A , B ) Cell lysates and supernatants were tested by ELISA for the production of CCL5, following astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of the RAGE inhibitor FPS-ZM1 (2 µM) for 24 h. ( D , E ) ELISA was used to determine CCL5 protein levels in lysates and supernatants from astrocytes after stimulation with 0.5 µg/ml rHMGB1 in the presence of the TLR2 inhibitor C29 (10 µM) for 24 h. ( G , H ) CCL5 levels in lysates and supernatants of astrocytes were determined by ELISA after the astrocytes were treated with the TLR4 inhibitor TAK-242 (2 µM) in the presence of 0.5 µg/ml rHMGB1 for 24 h. ( C , F , I ) CCK8 assay of the effects of FPS-ZM1, C29, or TAK-242 on the viability of astrocytes. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Determination of CCL5 synthesis-related protein levels in astrocytes following stimulation with rHMGB1. (A) Western blot analysis of the phosphorylation of the ERK, JNK, P38 kinase and p65NF-κB proteins after astrocytes were treated with 0–2.5 µg/mL rHMGB1 for 24 h. (B-E) Quantification data are shown in (A). Quantities were normalized to endogenous β-actin. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of the inhibition of MAPK/NF-κB signaling on the HMGB1-induced production of CCL5 from astrocytes. ( A , B ) ELISA was used to determine the effects of 10 µM ERK inhibitor PD98059, 10 µM JNK inhibitor SP6001251 or 10 µM P38 inhibitor SB203580 in the presence of 0.5 µg/ml rHMGB1 for 24 h on CCL5 protein production levels in lysates and supernatants of astrocytes. (C) Western blot analysis of the activation of p65NFκB protein after astrocyte treatment with 10 µM ERK inhibitor PD98059 or 10 µM JNK inhibitor SP6001251 for 24 h in the presence of 0.5 µg/ml rHMGB1. (D) Quantification data are shown in (C). Quantities were normalized to endogenous β-actin. ( E , F ) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following challenge with 10 µM SN50 for 24 h in the presence of 0.5 µg/ml rHMGB1, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of HMGB1-mediated astrocytic CCL5 on the migration of BV2 microglia cells or RAW 264.7 macrophage cells in vitro. (A) Illustration of the BV2 or RAW 264.7 cells with rCCL5 coculture model. (B) Transwell assay analysis of migration the ability of BV2 or RAW 264.7 cells co-incubated with 0.1 µg/ml rCCL5 for 48 h. ( C ) and ( D ) Quantification data are shown in (B). (E) Illustration of the BV2 or RAW 264.7 cells and ACM coculture model. (F) Migration assay of BV2 or RAW 264.7 cells cocultured with ACM. ACM were prepared from astrocytes exposed to 0.5 µg/ml rHMGB1 for 24 h, followed by the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. (G) and (H) Quantification data are shown in (F). Scale bars, 100 μm in ( B ) and ( F ). The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Migration, In Vitro, Transwell Assay, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of HMGB1-induced astrocytic CCL5 on microglia/macrophage polarization. ( A ) and ( B ) qRT-PCR was used to determine the expression of M1 markers ( CD86 , iNOS and TNF-a ) and M2 markers ( CD206 , Arg1 and Ym1 ) after BV2 or RAW 264.7 cells incubated with 0.1 µg/ml rCCL5 for 24 h. ( C ) and ( D ) The expression levels of M1 and M2 markers were determined by qRT-PCR following BV2 or RAW 264.7 cells cocultured with ACM for 24 h. ACM was prepared by astrocytes challenge with 0.5 µg/ml rHMGB1 for 24 h, after which the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Immunofluorescence of microglia/macrophage and locomotor function assessment after the inhibition of HMGB1 or CCL5 by neutralizing antibodies in rat SCI. ( A ) Immunofluorescence of IBA1- or CD68-positive cells at 4 d following SCI after neutralizing antibody application by intrathecal injection of 10 µl of neutralizing antibodies (HMGB1 Ab, 50 µg/kg), CCL5 (CCL5 Ab, 50 µg/kg) or normal rabbit IgG (IgG, 50 µg/kg). n = 6. Scale bars, 50 μm. ( B , C ) Quantitative analysis of the intensity of IBA1- or CD68-labeled cells in ( A) , respectively. (D) HE staining of the injured spinal cord at 21 d after administration of 10 µl of HMGB1, CCL5 neutralizing antibodies or normal rabbit IgG. n = 6. Scale bars, 500 μm. (E) Quantification data are shown in ( D) . (F) Basso, Beattie, and Bresnahan (BBB) locomotor scale scores for hindlimb motor function in rats at 0 d, 7 d, 14 d, 21 d and 28 d following the intrathecal administration of the HMGB1 Ab, CCL5 Ab or normal rabbit IgG. n = 6. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Immunofluorescence, Inhibition, Injection, Labeling, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Mechanistic diagram of HMGB1-mediated astrocytic CCL5 contributes to microglia/macrophages activation and recruitment.

Article Snippet: The lysates were centrifuged at 12,000×g for 15 min. CCL5 levels (R&D systems, cat. no. MMR00) were assessed via ELISA kits according to the manufacturer’s directions.

Techniques: Activation Assay

Journal: EBioMedicine

Article Title: Batf3-dependent CD8α + Dendritic Cells Aggravates Atherosclerosis via Th1 Cell Induction and Enhanced CCL5 Expression in Plaque Macrophages

doi: 10.1016/j.ebiom.2017.04.008

Figure Lengend Snippet: Lack of CD8α + DCs led to decreased IFN-γ and CCL5 expression in the aorta. The Apoe −/− mice (n = 8) and the Batf3 −/− Apoe −/− mice (n = 8) were fed either a chow diet or Western diet for 12 weeks. (a) Analysis of aortic Ifng , Foxp3 , and Tbx21 mRNA expression by qPCR. (b) An aortic tissue ELISA analysis of IFN-γ. Data represent pg/mg aortic protein. (c) qPCR analysis of Il12a , Tnf , Il6 , Tgfb1 , and Il10 mRNA expression. (d) qPCR analysis of Ccl5 expression. (e) ELISA analysis of the CCL5 concentration in the aortic tissue. Data represent pg per mg aortic protein. Data are presented as mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Data are representative of three independent experiments. See also Fig. S6.

Article Snippet: For the immunohistofluorescence analysis, the cryosections were stained with an antibody against CD45 (104; eBioscience Cat# 47-0451-82, RRID: AB_1548781 ), Mac3 (M3/84; BD Biosciences Cat# 550292, RRID: AB_393587 ), CCL5 (Bioss Inc. Cat# bs-1324R-Biotin, RRID: AB_11099534 ).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: EBioMedicine

Article Title: Batf3-dependent CD8α + Dendritic Cells Aggravates Atherosclerosis via Th1 Cell Induction and Enhanced CCL5 Expression in Plaque Macrophages

doi: 10.1016/j.ebiom.2017.04.008

Figure Lengend Snippet: IFN-γ promoted CCL5 expression on macrophages, promoting leukocytes infiltration into the plaque area. (a) Female Apoe −/− mice (n = 15) were put on a Western diet for eight weeks, then the aortic cells were pooled to prepared as described in the methods and stained with an antibody against CD45. Then, CD45 + leukocytes and CD45 − non-leukocytes were sorted by FACS, and qPCR was used to analyze Ccl5 mRNA expression. (b and c) Immunofluorescence of the aortic root. The Apoe −/− mice (n = 8) and the Batf3 −/− Apoe −/− mice (n = 8) were fed a Western diet for 12 weeks. The cryosections of the aortic root were stained with an antibody against CD45 (red), MAC3 (red), and CCL5 (green). The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images were viewed and captured with a Laser Scanning Confocal Microscope. Scale bars: 100 μm, dashed lines indicate the internal elastic lamina, arrows pointing to representative colocalized cells. (d) Primary splenic macrophages were sorted as described in supplementary data, and then treated with100 ng/mL IFN-γ for 6 h. Ccl5 mRNA expression was analyzed by qPCR. The Apoe −/− mice (n = 8) and the Batf3 −/− Apoe −/− mice (n = 8) were fed a Western diet for 6 weeks, and cryosections of the aortic root were performed. (e) H&E staining. Scale bars: 100 μm. (f) Immunohistochemistry. Representative images of leukocytes (CD45), T cells (CD3), DCs (CD11c), and macrophages (Mac3) in the aortic are shown. Scale bars: 200 μm. Data are presented as mean ± SD. Differences of a P < 0.05 were considered to be statistically significant. ** P < 0.01; *** P < 0.001; ns, not significant. Data are representative of three independent experiments.

Article Snippet: For the immunohistofluorescence analysis, the cryosections were stained with an antibody against CD45 (104; eBioscience Cat# 47-0451-82, RRID: AB_1548781 ), Mac3 (M3/84; BD Biosciences Cat# 550292, RRID: AB_393587 ), CCL5 (Bioss Inc. Cat# bs-1324R-Biotin, RRID: AB_11099534 ).

Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Microscopy, Immunohistochemistry

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