Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 1. Expression of CXCL12 (A) and CXCR4 (B) mRNA in the endometrium during

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 2. Localization of CXCL12 (A) and CXCR4 (B) proteins by

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques:

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 3. Immunoblot analysis of CXCL12 proteins in uterine flushings on Day 15 of

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Western Blot

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 4. Expression of CXCL12 and CXCR4 in conceptuses from Days 12 and 15 of

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Expressing

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 5. Effects of IFNG on CXCL12 and CXCR4 mRNA in endometrial explant

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques:

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 6. Effect of CXCL12 on pTr cell proliferation and migration. (A) RT-PCR

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction

Journal: Biology of reproduction

Article Title: Cysteine-X-cysteine motif chemokine ligand 12 and its receptor CXCR4: expression, regulation, and possible function at the maternal-conceptus interface during early pregnancy in pigs.

doi: 10.1093/biolre/ioy147

Figure Lengend Snippet: Figure 7. Effects of CXCL12 on migration of PBMCs and CD4+, CD8+, CD4+CD8+ T

Article Snippet: Blots were incubated overnight at 4°C with 1 μg/ml mouse monoclonal anti-CXCL12 antibody (R&D Systems), 1 μg/ml mouse monoclonal antiCXCL12 antibody neutralized with 100 ng recombinant human CXCL12 protein (rCXCL12; R&D Systems), or 1 μg/ml isotype-matched normal mouse IgG1 (Vector Laboratories) diluted in 2% (w/v) fat-free milk in TBST.

Techniques: Migration

Journal: Breast Cancer Research : BCR

Article Title: Loss of interferon regulatory factor 5 (IRF5) expression in human ductal carcinoma correlates with disease stage and contributes to metastasis

doi: 10.1186/bcr3053

Figure Lengend Snippet: Genes differentially regulated by IRF5 in MDA-MB-231 cells.

Article Snippet: Briefly, 100 ng/ml human recombinant CXCL12/SDF-1 (R&D Systems, Minneapolis, MN, USA) was added to 600 μl of phenol red-free DMEM medium supplemented with 10% FBS in the lower chamber.

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

doi: 10.1038/sj.bjc.6605968

Figure Lengend Snippet: CXCR4 and CXCL12 expression and growth response in pancreatic cancer cells. ( A ) Total protein was isolated from 12 pancreatic cancer cell lines and resolved on 10% SDS–polyacrylamide gels by electrophoresis. Subsequently, the gels were immunoblotted with anti-CXCR4 rabbit polyclonal antibodies and reprobed with anti- β -actin (internal control) mouse monoclonal antibody. CXCR4 was expressed (at varying levels) in all pancreatic cancer cell lines tested. ( B ) Enzyme-linked immunosorbant assay (ELISA) was performed on used culture media from pancreatic cancer cells grown under serum-free condition for 72 h using a commercial kit. Low level CXCL12 expression (13–230 pg per ml per 10 6 cells) was detected in all pancreatic cancer cell lines. ( C ) Growth response of pancreatic cancer cells (MiaPaCa and Panc1) on CXCL12 treatment (100 ng ml −1 ) indicating the functionality of CXCL12–CXCR4 signalling axis. CXCL12 stimulation (in serum-deprived and -supplemented media) led to the significant induction ( * P <0.01) of growth in pancreatic cancer cells. Responses were more pronounced under serum-free conditions than in serum-containing cultures likely due to the compensatory growth-promoting effects of other serum factors.

Article Snippet: Recombinant human CXCL12 and CXCL12 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Isolation, Electrophoresis, Control, Enzyme-linked Immunosorbent Assay

Journal: British Journal of Cancer

Article Title: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

doi: 10.1038/sj.bjc.6605968

Figure Lengend Snippet: Rescue of pancreatic cancer cells from gemcitabine-induced toxicity on CXCL12 treatment. Two pancreatic cancer cell lines, Panc1 ( A ) and MiaPaCa ( B ), were treated with various doses of gemcitabine (0–10 μ M ) under serum-supplemented condition in the presence and absence of CXCL12 (100 ng ml −1 ). Cancer cell viability was examined 72 h post-treatment by MTT assay. Significant protection of pancreatic cancer cells from gemcitabine toxicity (at 5 and 10 μ M ) by CXCL12 was observed. Data are presented as relative survival with respect to untreated or CXCL12 only-treated cells to control for the growth-promoting effect of CXCL12 ( * P <0.01).

Article Snippet: Recombinant human CXCL12 and CXCL12 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: MTT Assay, Control

Journal: British Journal of Cancer

Article Title: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

doi: 10.1038/sj.bjc.6605968

Figure Lengend Snippet: Antiapoptotic effects of CXCL12 treatment on gemcitabine-induced cell death. ( A ) DNA fragmentation assay. Cells were seeded in 6-cm Petri dishes and treated with 5 and 10 μ M gemcitabine in the absence or presence of CXCL12 (100 ng ml −1 ) for 48 h. Subsequently, genomic DNA was isolated and resolved (2 μ g per lane) on 1% agarose gel. Lane 1: untreated, lanes 2 and 3: gemcitabine-treated (5 and 10 μ M ), respectively, and lanes 4 and 5: gemcitabine-treated (5 and 10 μ M , respectively) in the presence of CXCL12. CXCL12-treated pancreatic cancer cells exhibit reduced DNA laddering compared with cells treated with gemcitabine only. ( B ) In situ determination of apoptosis. Panc1 and MiaPaCa cells were cultured on chamber slides and treated with gemcitabine (5 μ M ) in the absence and presence of CXCL12 (100 ng ml -1 ). Apoptosis was detected by staining the cells with CaspACE FITC-VAD-FMK solution in PBS for 2 h at 37°C. Following fixation, bound marker was visualised by fluorescent detection under a confocal microscope. Representative pictures (overlay of FITC and DAPI) are from one of the random fields of untreated, gemcitabine only, and gemcitabine+CXCL12-treated Panc1 and MiaPaCa cells. Apoptotic cells that stained positively with FITC-labelled marker were counted in 10 random fields and presented in a bar diagram (mean±s.d.). * Significant difference as compared with gemcitabine only-treated cells. CXCL12 co-treated cells exhibited 53 and 55% reduced apoptosis by gemcitabine in Panc1 and MiaPaCa cells, respectively.

Article Snippet: Recombinant human CXCL12 and CXCL12 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: DNA Fragmentation Assay, Isolation, Agarose Gel Electrophoresis, DNA Laddering, In Situ, Cell Culture, Staining, Marker, Microscopy

Journal: British Journal of Cancer

Article Title: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

doi: 10.1038/sj.bjc.6605968

Figure Lengend Snippet: CXCL12-induced activation of FAK, Akt, and ERK pathways. Sub-confluent Panc1 and MiaPaCa cell cultures were treated with CXCL12 (100 ng ml −1 ) for 5, 15, and 30 min durations. Protein was extracted and resolved on SDS–polyacrylamide gels by electrophoresis. Activation of FAK, Akt, and ERK pathways was assessed by immunoblotting using total and phospho-form-specific antibodies as indicated. β -Actin served as an internal control. CXCL12 treatment induced the phosphorylation of all three effector proteins with a concomitant inactivating phosphorylation of proapoptotic BAD protein in both Panc 1 and MiaPaCa cell lines.

Article Snippet: Recombinant human CXCL12 and CXCL12 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Electrophoresis, Western Blot, Control, Phospho-proteomics

Journal: British Journal of Cancer

Article Title: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

doi: 10.1038/sj.bjc.6605968

Figure Lengend Snippet: Induction of β -catenin/TCF and NF- κ B transcriptional activities and expression of survival proteins by CXCL12 in pancreatic cancer cells. ( A ) Pancreatic cancer cells were transfected with TOPflash or FOPflash or NF- κ B luciferase reporter constructs along with Renilla luciferase construct to control for the transfection efficiency. Cells were treated with CXCL12 24 h post-transfection and protein isolated in passive lysis buffer. Luciferase activity was assessed using a dual-luciferase assay system and data presented as fold change in luciferase activity after normalisation. Bars represent the average of triplicates±s.d.; * statistically significant difference ( P <0.01). ( B ) Change in the expression of Bcl-2, Bcl-xL, Notch 1, and survivin was examined in CXCL12-treated cells at different time durations by immunoblotting. An increased expression of all the four survival proteins was detected in CXCL12-treated pancreatic cancer cells.

Article Snippet: Recombinant human CXCL12 and CXCL12 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Transfection, Luciferase, Construct, Control, Isolation, Lysis, Activity Assay, Western Blot

Journal: British Journal of Cancer

Article Title: CXCL12–CXCR4 signalling axis confers gemcitabine resistance to pancreatic cancer cells: a novel target for therapy

doi: 10.1038/sj.bjc.6605968

Figure Lengend Snippet: Effect of CXCR4 targeting and blockade of PI3K or Erk pathways on the cytoprotective effect of CXCL12 in pancreatic cancer cells from gemcitabine-induced toxicity. ( A ) Pancreatic cancer cells (Panc1 and MiaPaCa) were treated with AMD3100 (5 μ g ml −1 ) or LY294002 (20 μ M ) or PD98059 (25 μ M ) for 1 h before induction with CXCL12. Total protein was isolated 15 min after CXCL12 treatment, and activation of Akt and ERK was examined by immunoblotting for their total and phospho-forms. AMD3100 inhibited the activation of both Akt and ERK pathways, whereas LY294002 and PD98059 specifically inhibited Akt and ERK pathways, respectively. ( B ) Cells were pretreated with AMD3100 or LY294002 or PD98059 or PBS for 1 h. Subsequently, cells were treated with CXCL12 or gemcitabine either alone or in combination. Cell viability was assessed by MTT assay. Bars represent the average of triplicates±s.d.; * statistically significant difference ( P <0.01) with respect to gemcitabine+CXCL12-treated cells. Bars 1: untreated, 2: CXCL12 treated, 3: AMD3100 treated, 4: AMD3100 pretreated+CXCL12 treated, 5: gemcitabine treated, 6: gemcitabine+CXCL12 treated, 7: AMD3100 pretreated+gemcitabine+CXCL12 treated, 8: LY294002 pretreated+gemcitabine+CXCL12 treated, and 9: PD98059 pretreated+gemcitabine+CXCL12 treated.

Article Snippet: Recombinant human CXCL12 and CXCL12 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Activation Assay, Western Blot, MTT Assay