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

Journal: BMC Cancer

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility

doi: 10.1186/s12885-015-1201-5

Figure Lengend Snippet: Gene expression profiles of CXCR7, CXCR4, CXCL11, CXCL12 in human prostate cancer samples

Article Snippet: The following reagents were purchased from the indicated vendors: AR agonist R1881 (methyltrienolone) (Perkin Elmer Life Sciences, Waltham, MA); CXCL11 (672-IT) and CXCL12 (2716-SD) ligands (R&D Systems, Minneapolis, MN); double-stranded experimentally validated siRNAs for scrambled control (1027281), AR (SI02757258), CXCR4 (SI02664235), CXCR7 (SI02660644) (Qiagen, Valencia, CA), and CXCR7 (109229) (Life Technologies, Chicago, IL); RNeasy Mini kit, RT 2 qPCR primers for AR (PPH01016A), CXCR7 (PPH01182F), CXCR4 (PPH00621A), PSA (PPH01002B), FASN (PPH01012B), NKX3.1 (PPH02267C), TMPRSS2 (PPH02262C) (Qiagen); Oligofectamine Transfection Reagent, 4%-12% SDS-polyacrylamide gels, Superscript III enzyme, CyQUANT Cell Proliferation Assay Kit (Life Technologies); iQ SYBR-Green Supermix, Precision Plus Prestained Protein Standards, goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody (BioRad, Hercules, CA); mouse monoclonal AR antibody (AR441), rabbit polyclonal AR antibody (N-20), mouse monoclonal SBP antibody (SB19-C4) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal CXCR7 antibodies (ab38089 [a.a. 1–100], and ab72100 [a.a. 106–117, QHNQWPMGELTC]), rabbit polyclonal CXCR4 antibody (ab2074) (Abcam, Cambridge, MA); rabbit polyclonal CXCR4 antibody (PAB9849) (Abnova, Taipei, Taiwan); mouse monoclonal GM130 antibody (BD Transduction Laboratories, San Jose, CA); rabbit polyclonal PSA antibody (DAKO, Carpinteria, CA); mouse monoclonal PSMA antibody (Meridian Life Science Inc, Memphis, TN); rabbit polyclonal Histone H3 antibody, rabbit monoclonal GAPDH antibody (14C10) (Cell Signaling Technology, Beverly, MA); BCA Protein Assay Kit and ECL Western Blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA); Hyperfilm ECL film (GE Healthcare, Piscataway, NJ); fetal bovine serum, charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT); GeneRuler 1 kb DNA Ladder (MBI Fermentas, Hanover, MD); Protein Deglycosylation Mix (P6039S, New England BioLabs, Ipswich, MA); Synthetic peptides to CXCR7 (a.a. 348–362, RVSETEYSALEQSTK) and AR (a.a. 299–315, KSTEDTAEYSPFKGGY) were synthesized by Alpha Diagonistics (San Antonio, TX).

Techniques: Gene Expression

Journal: BMC Cancer

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility

doi: 10.1186/s12885-015-1201-5

Figure Lengend Snippet: CXCR7 expression and localization are modulated by CXCL11 and CXCL12. (A) Immunofluorescence staining of CXCR7 in AD -LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (100 nM), or CXCL12 (100 nM) for 30 min. Nuclei and F-actin are labeled with DAPI and Texas-red phalloidin, respectively. (B) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A) with pAbCXCR7 antibody. (C) Immunofluorescence staining of CXCR4 in AD -LNCaP cells as described in (A) . (D-E) Western blot of cytosolic, membrane, and nuclear protein fractions isolated from LNCaP cells, cultured as described in (A), with antibodies to (D) CXCR4, and (E) AR, GM130, and histone H3. Silver staining demonstrates equivalent loading across samples. The densitometry values were normalized to BSA-treated samples for each subcellular compartment and labeled below the blots.

Article Snippet: The following reagents were purchased from the indicated vendors: AR agonist R1881 (methyltrienolone) (Perkin Elmer Life Sciences, Waltham, MA); CXCL11 (672-IT) and CXCL12 (2716-SD) ligands (R&D Systems, Minneapolis, MN); double-stranded experimentally validated siRNAs for scrambled control (1027281), AR (SI02757258), CXCR4 (SI02664235), CXCR7 (SI02660644) (Qiagen, Valencia, CA), and CXCR7 (109229) (Life Technologies, Chicago, IL); RNeasy Mini kit, RT 2 qPCR primers for AR (PPH01016A), CXCR7 (PPH01182F), CXCR4 (PPH00621A), PSA (PPH01002B), FASN (PPH01012B), NKX3.1 (PPH02267C), TMPRSS2 (PPH02262C) (Qiagen); Oligofectamine Transfection Reagent, 4%-12% SDS-polyacrylamide gels, Superscript III enzyme, CyQUANT Cell Proliferation Assay Kit (Life Technologies); iQ SYBR-Green Supermix, Precision Plus Prestained Protein Standards, goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody (BioRad, Hercules, CA); mouse monoclonal AR antibody (AR441), rabbit polyclonal AR antibody (N-20), mouse monoclonal SBP antibody (SB19-C4) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal CXCR7 antibodies (ab38089 [a.a. 1–100], and ab72100 [a.a. 106–117, QHNQWPMGELTC]), rabbit polyclonal CXCR4 antibody (ab2074) (Abcam, Cambridge, MA); rabbit polyclonal CXCR4 antibody (PAB9849) (Abnova, Taipei, Taiwan); mouse monoclonal GM130 antibody (BD Transduction Laboratories, San Jose, CA); rabbit polyclonal PSA antibody (DAKO, Carpinteria, CA); mouse monoclonal PSMA antibody (Meridian Life Science Inc, Memphis, TN); rabbit polyclonal Histone H3 antibody, rabbit monoclonal GAPDH antibody (14C10) (Cell Signaling Technology, Beverly, MA); BCA Protein Assay Kit and ECL Western Blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA); Hyperfilm ECL film (GE Healthcare, Piscataway, NJ); fetal bovine serum, charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT); GeneRuler 1 kb DNA Ladder (MBI Fermentas, Hanover, MD); Protein Deglycosylation Mix (P6039S, New England BioLabs, Ipswich, MA); Synthetic peptides to CXCR7 (a.a. 348–362, RVSETEYSALEQSTK) and AR (a.a. 299–315, KSTEDTAEYSPFKGGY) were synthesized by Alpha Diagonistics (San Antonio, TX).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Western Blot, Membrane, Isolation, Cell Culture, Silver Staining

Journal: BMC Cancer

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility

doi: 10.1186/s12885-015-1201-5

Figure Lengend Snippet: CXCR7 modulates AR transcriptional activity. (A) Luciferase assay testing the effects of CXCR7 overexpression on the AR-target promoter probasin in LNCaP cells. LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40 Renilla vectors along with increasing amounts (30 ng experimental + 270 ng pcDNA3, 100 ng experimental + 200 ng pcDNA3, 300 ng experimental + 0 ng pcDNA3) of CXCR7 or ACTN4 cDNA mammalian expression vectors. The maximal amount (300 ng) of the pcDNA3 mammalian expression vector served as the positive control. Cells were subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and experimental cells within the androgen-treatment group. (B) Luciferase assay testing the effects of CXCR7 siRNA knockdown and treatment with CXCR7 ligand on the AR-target promoter probasin . LNCaP cells were co-transfected with the pGL4.10-Luc2- probasin and pRLSV40- renilla vectors, along with control or experimental siRNAs (50 nM). Next, cells were pre-treated with indicated ligands (BSA, CXCL11, or CXCL12) for 30 min. Cells were then subsequently treated with androgen (1 nM R1881) or vehicle (ethanol) for 18 hrs and tested for dual luciferase activity. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control cells and experimental cells within the androgen-treatment group. (C) RNA isolated from LNCaP cells treated with vehicle (0.1% BSA), CXCL11 (10 nM), or CXCL12 (10 nM) for 30 min and subsequently treated with vehicle or androgen (1 nM R1881) for 18 hrs were subjected to qPCR analysis for AR , FASN , NKX3.1 , PSA , and TMPRSS2 gene expressions. Student’s t -test was used to calculate significant differences (* p ≤ 0.05, n = 3) between control and chemokine ligand-treated cells.

Article Snippet: The following reagents were purchased from the indicated vendors: AR agonist R1881 (methyltrienolone) (Perkin Elmer Life Sciences, Waltham, MA); CXCL11 (672-IT) and CXCL12 (2716-SD) ligands (R&D Systems, Minneapolis, MN); double-stranded experimentally validated siRNAs for scrambled control (1027281), AR (SI02757258), CXCR4 (SI02664235), CXCR7 (SI02660644) (Qiagen, Valencia, CA), and CXCR7 (109229) (Life Technologies, Chicago, IL); RNeasy Mini kit, RT 2 qPCR primers for AR (PPH01016A), CXCR7 (PPH01182F), CXCR4 (PPH00621A), PSA (PPH01002B), FASN (PPH01012B), NKX3.1 (PPH02267C), TMPRSS2 (PPH02262C) (Qiagen); Oligofectamine Transfection Reagent, 4%-12% SDS-polyacrylamide gels, Superscript III enzyme, CyQUANT Cell Proliferation Assay Kit (Life Technologies); iQ SYBR-Green Supermix, Precision Plus Prestained Protein Standards, goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody (BioRad, Hercules, CA); mouse monoclonal AR antibody (AR441), rabbit polyclonal AR antibody (N-20), mouse monoclonal SBP antibody (SB19-C4) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal CXCR7 antibodies (ab38089 [a.a. 1–100], and ab72100 [a.a. 106–117, QHNQWPMGELTC]), rabbit polyclonal CXCR4 antibody (ab2074) (Abcam, Cambridge, MA); rabbit polyclonal CXCR4 antibody (PAB9849) (Abnova, Taipei, Taiwan); mouse monoclonal GM130 antibody (BD Transduction Laboratories, San Jose, CA); rabbit polyclonal PSA antibody (DAKO, Carpinteria, CA); mouse monoclonal PSMA antibody (Meridian Life Science Inc, Memphis, TN); rabbit polyclonal Histone H3 antibody, rabbit monoclonal GAPDH antibody (14C10) (Cell Signaling Technology, Beverly, MA); BCA Protein Assay Kit and ECL Western Blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA); Hyperfilm ECL film (GE Healthcare, Piscataway, NJ); fetal bovine serum, charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT); GeneRuler 1 kb DNA Ladder (MBI Fermentas, Hanover, MD); Protein Deglycosylation Mix (P6039S, New England BioLabs, Ipswich, MA); Synthetic peptides to CXCR7 (a.a. 348–362, RVSETEYSALEQSTK) and AR (a.a. 299–315, KSTEDTAEYSPFKGGY) were synthesized by Alpha Diagonistics (San Antonio, TX).

Techniques: Activity Assay, Luciferase, Over Expression, Transfection, Expressing, Plasmid Preparation, Positive Control, Control, Knockdown, Isolation

Journal: BMC Cancer

Article Title: Androgen receptor and chemokine receptors 4 and 7 form a signaling axis to regulate CXCL12-dependent cellular motility

doi: 10.1186/s12885-015-1201-5

Figure Lengend Snippet: CXCR7 knockdown leads to a reduction in CXCR4 protein levels in LNCaP cells. (A) Transwell assay assessing the effects of CXCL12 on LNCaP migration. ANOVA was used to determine significant differences between vehicle (0.1% BSA) and CXCL12-treated cells (* p ≤ 0.05, n = 3). (B) Transwell assay assessing the effects of CXCR4 and CXCR7 knockdown on CXCL12-induced LNCaP cell migration. LNCaP cells transfected with 100 nM scrambled control, CXCR4, or CXCR7 siRNAs were seeded to the top chamber of the insert. The bottom chamber contained medium with 1% CS serum with 1 nM R1881 and CXCL12 at 0, 0.003, 0.03, or 0.3 nM concentrations. Data was normalized to control siRNA transfected, vehicle-treated cells. ANOVA was used to determine significant differences (*p ≤ 0.05, n = 3) between control and experimental cells. (C-E) Western blots to test the effects of CXCR7 and CXCR4 knockdown on AR signaling in LNCaP cells. Cells were transfected with control, CXCR7 or CXCR4 siRNA for 72 hrs and probed with antibodies against (C) CXCR7, (D) CXCR4, (E) AR, and PSA. The densitometry values were normalized to control siRNA transfected cells and labeled below the blots. (F) Silver staining demonstrates equivalent loading across the samples.

Article Snippet: The following reagents were purchased from the indicated vendors: AR agonist R1881 (methyltrienolone) (Perkin Elmer Life Sciences, Waltham, MA); CXCL11 (672-IT) and CXCL12 (2716-SD) ligands (R&D Systems, Minneapolis, MN); double-stranded experimentally validated siRNAs for scrambled control (1027281), AR (SI02757258), CXCR4 (SI02664235), CXCR7 (SI02660644) (Qiagen, Valencia, CA), and CXCR7 (109229) (Life Technologies, Chicago, IL); RNeasy Mini kit, RT 2 qPCR primers for AR (PPH01016A), CXCR7 (PPH01182F), CXCR4 (PPH00621A), PSA (PPH01002B), FASN (PPH01012B), NKX3.1 (PPH02267C), TMPRSS2 (PPH02262C) (Qiagen); Oligofectamine Transfection Reagent, 4%-12% SDS-polyacrylamide gels, Superscript III enzyme, CyQUANT Cell Proliferation Assay Kit (Life Technologies); iQ SYBR-Green Supermix, Precision Plus Prestained Protein Standards, goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody, goat anti-rabbit HRP-conjugated secondary antibody (BioRad, Hercules, CA); mouse monoclonal AR antibody (AR441), rabbit polyclonal AR antibody (N-20), mouse monoclonal SBP antibody (SB19-C4) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal CXCR7 antibodies (ab38089 [a.a. 1–100], and ab72100 [a.a. 106–117, QHNQWPMGELTC]), rabbit polyclonal CXCR4 antibody (ab2074) (Abcam, Cambridge, MA); rabbit polyclonal CXCR4 antibody (PAB9849) (Abnova, Taipei, Taiwan); mouse monoclonal GM130 antibody (BD Transduction Laboratories, San Jose, CA); rabbit polyclonal PSA antibody (DAKO, Carpinteria, CA); mouse monoclonal PSMA antibody (Meridian Life Science Inc, Memphis, TN); rabbit polyclonal Histone H3 antibody, rabbit monoclonal GAPDH antibody (14C10) (Cell Signaling Technology, Beverly, MA); BCA Protein Assay Kit and ECL Western Blotting Substrate Kit (ThermoFisher Scientific, Waltham, MA); Hyperfilm ECL film (GE Healthcare, Piscataway, NJ); fetal bovine serum, charcoal-stripped fetal bovine serum (Hyclone Laboratories, Logan, UT); GeneRuler 1 kb DNA Ladder (MBI Fermentas, Hanover, MD); Protein Deglycosylation Mix (P6039S, New England BioLabs, Ipswich, MA); Synthetic peptides to CXCR7 (a.a. 348–362, RVSETEYSALEQSTK) and AR (a.a. 299–315, KSTEDTAEYSPFKGGY) were synthesized by Alpha Diagonistics (San Antonio, TX).

Techniques: Knockdown, Transwell Assay, Migration, Transfection, Control, Western Blot, Labeling, Silver Staining

Journal:

Article Title: Stimulation of Chondrocyte Hypertrophy by Chemokine Stromal Cell-Derived Factor 1 in the Chondro-osseous Junction during Endochondral Bone Formation

doi: 10.1016/j.ydbio.2010.02.033

Figure Lengend Snippet: To determine whether SDF-1 can diffuse into cartilage, 17-day-chicken embryonic sternal cartilage was incubated with SDF-1 (100ng/mL) or without SDF-1 for 1h, 3h, and 24h. 10 µm frozen sections were used to detect SDF-1 by immuno-fluorescent staining with mAb against SDF-1. Fluorescence microscopy showed a progressive increase in SDF-1 staining (red color) surrounding chondrocytes during the 24 h time course (A, B,C) compared to control at 24 h (D). Scale bar = 20 µm.

Article Snippet: Before collecting samples for experiment, the cells were stimulated with SDF-1 (100ng/mL; Cat# 351-FS, R&D Systems, Inc. Minneapolis, MN) for 24h or pretreated with AMD3100 for 2 h (5ug/mL; Cat# 155148–31–5, Sigma-Aldrich, St. Louis, MO), a specific inhibitor for CXCR4, before stimulation with SDF-1.

Techniques: Incubation, Staining, Fluorescence, Microscopy, Control

Journal:

Article Title: Stimulation of Chondrocyte Hypertrophy by Chemokine Stromal Cell-Derived Factor 1 in the Chondro-osseous Junction during Endochondral Bone Formation

doi: 10.1016/j.ydbio.2010.02.033

Figure Lengend Snippet: To confirm that SDF-1 induces chondrocyte hypertrophy in growth plates, 12-day-old chicken tibia growth plates were cultured in the presence of SDF-1 (100ng/mL) or in the absence of SDF-1 for 2, 4, and 6 days. 10 µm frozen sections were used to detect Type X collagen expression by immuno-fluorescent staining with mAb against type X collagen. A progressive increase in the size of the hypertrohic growth plate based on Type X collagen staining was seen. The ratio of the length of the hypertrophic zone to that of the total growth plate was calculated at the different time points (B). (* p<0.05). Scale bar = 100 µm.

Article Snippet: Before collecting samples for experiment, the cells were stimulated with SDF-1 (100ng/mL; Cat# 351-FS, R&D Systems, Inc. Minneapolis, MN) for 24h or pretreated with AMD3100 for 2 h (5ug/mL; Cat# 155148–31–5, Sigma-Aldrich, St. Louis, MO), a specific inhibitor for CXCR4, before stimulation with SDF-1.

Techniques: Cell Culture, Expressing, Staining

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: CXCL12 compromises neuronal survival in a concentration-dependent fashion. Mixed neuronal-glial cerebrocortical cell cultures were incubated for 24 h with CXCL12 at concentrations of 2, 20, and 50 nM. BSA (0.001 % final concentration) served as vehicle control in the absence of CXCL12. Assessment of neuronal survival was performed as described in the text using cell counting after fluorescence staining for neuronal MAP-2 and nuclear DNA. Values are mean ± SEM; n ≥ 3 with duplicate or triplicate samples per condition; ** P ≤ 0.01; *** P ≤ 0.001 by ANOVA followed by Fisher’s PLSD post hoc test

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Incubation, Control, Cell Counting, Fluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Ca 2+ channel blockers MK-801 and nimodipine abrogate neurotoxicity of CXCL12 in cerebrocortical cell cultures. Incubation with CXCL12 and Ca 2+ channel blockers and assessment of neuronal survival was performed as described in the text using staining for neurons (MAP-2) and nuclear DNA. Representative images are shown for six experimental conditions (MK-801 at 10 μM). Scale bar , 20 μm. Values in the graph are mean ± SEM; n ≥ 3 with duplicate or triplicate samples per condition; * P ≤ 0.05, *** P ≤ 0.001 by ANOVA followed by Fisher’s PLSD post hoc test

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Staining

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Neurotoxic CXCL12 transiently activates p38 MAPK and JNK in rat cerebrocortical cultures. a Complete neuro-glial cell cultures and neuron-depleted cerebrocortical cell cultures were analyzed by Western blotting for the presence of active p38 MAPK and JNK. To obtain glial cells, neurons were depleted by treating cerebrocortical cultures with 300 μM NMDA for 20 min 2 days prior to the preparation of cell lysates. Equal amounts of cellular protein (30 μg) were separated by SDS-PAGE and analyzed by immunoblotting for the indicated proteins. A representative Western blot from one of the three independent neuron depletion experiments is shown. b Cerebrocortical cultures were incubated with recombinant CXCL12 (20 nM) for the indicated time periods, prior to cell lysis on ice. Equal amounts of cellular protein (100 μg) were used for the performance of immunocomplex kinase assays as described in the “ ” section. The Western blots show representative samples of phosphorylated indicator substrates observed with samples of the 12 and 24 h time points. Kinase activity in CXCL12-exposed samples and vehicle-treated controls was measured for each time point. Vehicle controls were defined as the 100 % baseline value. Note the difference in the dimension of time on the split X -axis. Each time point represents four to seven assessments in duplicate or triplicate in independent experiments. * P ≤ 0.05 compared to control by student’s t test. ‘pp38’/‘p-p38’ and ‘p-JNK’ indicate phosphorylated p38 MAPK and JNK, respectively

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, SDS Page, Incubation, Recombinant, Lysis, Activity Assay, Control

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Phosphorylated p38 MAPK in cerebrocortical cell cultures localizes to neurons with and without CXCL12 exposure. Cerebrocortical cell cultures from rats were incubated with CXCL12 (20 nM). After 12 h of treatment, cells were fixed, permeabilized, and stained for MAP-2 ( red ), as a neuronal marker, activated-phospho p38 MAPK ( green ), and nuclear DNA (DRAQ5; shown in blue pseudocolor for better visualization). Samples were analyzed using immunofluorescence microscopy as described in the “ ” section. The overlap of red and green signals appears yellow in the merged images. Scale bars , 20 μm

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Staining, Marker, Immunofluorescence, Microscopy

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Ca 2+ channel blockers limit activity of p38 MAPK in cerebrocortical cells upon exposure to CXCL12. Rat cerebrocortical cells were incubated for the indicated time periods with CXCL12 (20 nM) in the presence or absence of MK-801 (10 μM) or nimodipine (10 nM), prior to cell lysis on ice. 30 μg of protein per lane were analyzed by Western blotting using the specific antibodies against the indicated molecules. A representative immunoblot is shown for each time point. Quantification by densitometry; n = 3 to 4 per time point; * P ≤ 0.05, ** P ≤ 0.01 by ANOVA followed by Fisher’s PLSD post hoc test

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activity Assay, Incubation, Lysis, Western Blot

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Phosphorylated p38 MAPK localization in neurons in the presence of CXCL12 and Ca 2+ channel blockers. Cerebrocortical cell cultures were exposed to CXCL12 (20 nM), nimodipine (10 nM), or MK801 (10 μM) or combinations thereof for 12 h. Afterwards, cells were fixed, permeabilized, and stained for MAP-2 ( red ), activated/phospho p38 MAPK ( green ) and nuclear DNA (DRAQ5; shown in blue pseudocolor for better visualization). Images were analyzed using fluorescence microscopy as described in the “ ” section. The overlap of red and green signals appears yellow in the merged images. Scale bars , 20 μm

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Staining, Fluorescence, Microscopy

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: Pharmacological inhibition of p38 MAPK protects cerebrocortical neurons from toxicity of CXCL12. Cerebrocortical cell cultures were incubated with CXCL12 (20 nM) in the presence and absence of p38 MAPK inhibitor SB203580 (SB, 10 μM) for 24 h. Analysis of neuronal survival was performed as described in the “ ” section using staining for neurons (MAP-2) and nuclear DNA (H33342). Values are mean ± SEM; n ≥ 3 with duplicate or triplicate samples per condition; ** P ≤ 0.01, *** P ≤ 0.001 by ANOVA followed by Fisher’s PLSD post hoc test

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Incubation, Staining

Journal: Journal of Neuroinflammation

Article Title: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l -type Ca 2+ channels upstream of p38 MAPK

doi: 10.1186/s12974-016-0724-2

Figure Lengend Snippet: NMDA receptors and l -type Ca 2+ channels can regulate CXCL12 induced neuronal death upstream of p38 MAPK activation. Activity of p38 MAPK (phosphorylation indicated by p ) increases above baseline levels ( upward arrow ) as a critical mediator of CXCR4-mediated neurotoxicity of CXCL12. The present study implicates besides NMDAR-gated ion channels for the first-time l -type Ca 2+ channels ( l -type CC) in CXCL12 neurotoxicity as the blockade of both independently can prevent CXCL12 neurotoxicity while concomitantly preventing an increase (nimodipine; equal sign ) or reducing (MK-801; downward arrow) the activity of p38 MAPK compared to baseline

Article Snippet: Recombinant CXCL12 (SDF-1β) was purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Activity Assay, Phospho-proteomics