Journal: Molecular medicine reports

Article Title: miR‑137 decreases proliferation, migration and invasion in rheumatoid arthritis fibroblast‑like synoviocytes.

doi: 10.3892/mmr.2017.8225

Figure Lengend Snippet: Figure 4. CXCL12 is a direct target of miR‑137 in RA‑FLS. (A) Predicted binding site for miR‑137 in the 3'UTR of Wt‑CXCL12; mutations in the binding sites in the Mut‑CXCL12 sequence are also indicated. (B) Relative luciferase activity of in RA‑FLS co‑transfected with either Wt‑CXCL12 or Mut‑CXCL12 3'UTR reporter plasmid and either miR‑137 mimic or miR‑NC. **P<0.01 vs. miR‑NC. The expression levels of CXCL12 (C) mRNA and (D) protein were detected in FLS isolated the RA model rat group and the normal control group by RT‑PCR and western blotting, respectively. GAPDH was used as an internal control. **P<0.01 vs. normal FLS. (E and F) RA‑FLS were transfected with either miR‑137 mimic or miR‑NC, and CXCL12 (E) mRNA and (F) protein expression levels were determined. GAPDH was used as an internal control. **P<0.01 vs. miR‑NC. CXCL12, C‑X‑C motif chemokine ligand 12; FLS, fibroblast‑like synoviocytes; miR, microRNA; Mut, mutant; NC, negative control; RA, rheumatoid arthritis; UTR, untranslated region; Wt, wild‑type.

Article Snippet: Membranes were blocked with 5% non‐fat milk in Tris‐buffered saline (TBS; Sigma‐Aldrich; Merck KGaA) for 1 h at room temperature, and incubated overnight at 4 ̊C with the following primary antibodies: Mouse monoclonal anti-human CXCL12 (1:1,000; cat no. sc‐74271) and mouse monoclonal anti‐human GAPDH (1:5,000; cat no. sc-293335); both were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Binding Assay, Sequencing, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Isolation, Control, Western Blot, Transfection, Mutagenesis, Negative Control

Journal: Molecular Cancer Research

Article Title: Microenvironmental Regulation of Chemokine (C-X-C-Motif) Receptor 4 in Ovarian Carcinoma

doi: 10.1158/1541-7786.mcr-09-0463

Figure Lengend Snippet: FIGURE 1. CXCR4 is upregulated by 3DCI in EOC cells. A, ovarian carcinoma cell lines DOV13, OVCA433, SKOV-3, Caov-3, OVCA429, and OVCAR3 were cultured on 2DCI and 3DCI for 8 h. Cells were collected; RNA was extracted; cDNA was synthesized; and CXCR4 RNA was detected using real-time RT-PCR as described in Materials and Methods. CXCR4 RNA ratio was found using the 2−ΔΔCt method (23), was averaged (n = 4), and was plotted. *, P < 0.005 (Student's t test; comparisons: Ct values on 2DCI versus those on 3DCI). B, OVCA433, SKOV-3, Caov-3, and DOV13 were cultured on 2DCI and 3DCI for 24 h and subjected to Western blot thereafter. CXCR4-specific antibodies (Santa Cruz Biotechnology) were used at 1:200 dilution; anti–β-tubulin antibody was used at 1:1,000 dilution, as indicated in Materials and Methods. Histogram shows levels of CXCR4 expression relative to the loading control β-tubulin; *, P < 0.005 (Student's t test; comparisons included normalized band intensities on 2DCI versus those on 3DCI). C, DOV13 were cultured on 2DCI and 3DCI for 24 h and subjected to immunofluorescent staining. CXCR4-specific antibodies were used at 1:100 dilution. Confocal images were obtained using a Zeiss confocal microscope at ×60 magnification on the objective. Arrows, accumulation of CXCR4 on the tips of protruding pseudopodia in cells cultured in 3DCI, as indicated. Intensity of the CXCR4 staining was quantified from confocal images using ImageJ (NIH). Lines (white dotted) outlined with a circle were drawn across the tips of the protruding lamellipod (2DCI) and pseudopod (3DCI) to generate the intensity scan. Intensities of CXCR4 expression across the protrusions is plotted on the graph. Light gray line, CXCR4 in protrusions of cells cultured on 2DCI; black line, CXCR4 in protrusions of cells cultured on 3DCI, as indicated. Shown are representative images of three independent experiments. D, SDF-1 induces [Ca2+]i changes in DOV13. Cells were imaged with digital video microfluorimetry as described in Materials and Methods for 15 min as indicated. DOV13 responded to SDF-1 and ATP. A total of 46 cells (96%) from three independent experiments of 48 total (all cells in the field of view) showed [Ca2+]i increase when stimulated by SDF1. ATP-induced Ca responses were present in all cells tested. E, DOV13 was cultured on 3DCI, 2DCI, 3DPEG, RGD-coated plates, 3DMatrigel and thin-layer Matrigel for 8 h. Cells were collected; RNA was extracted; cDNA was synthesized; and CXCR4 RNA was detected using real-time RT-PCR as described in Materials and Methods. CXCR4 RNA ratio was found using the 2−ΔΔCt method (23), was averaged (n = 3), and was plotted. Black column, CXCR4 expression on 3DCI is from A and is shown here for comparison; *, P < 0.05 (comparisons: Ct values on two dimension versus those on three dimension for all matrices).

Article Snippet: The antibodies were used at the following dilutions: 1:200 for human anti-SDF1 antibodies (Santa Cruz www.aacrjournals.org on June 10, 2015. mcr.aacrjournals.org Downloaded from Biotechnology) in 3% bovine serum albumin (Sigma), 1:200 for human anti-CXCR4 polyclonal antibody (Santa Cruz Biotechnology) in 3% bovine serum in Tris-Buffered Saline Tween 20 (TBST), 1:1,000 for anti–β-tubulin monoclonal antibody (Sigma) in 5% skim milk in TBST, 1:2,000 for anti-p65 NFκB polyclonal antibodies (Rockland) in 3% bovine serum albumin, and 1:2,000 for antiactin monoclonal antibodies (Abcam) in 5% skim milk in TBST.

Techniques: Cell Culture, Synthesized, Quantitative RT-PCR, Western Blot, Expressing, Control, Staining, Microscopy, Comparison

Journal: Molecular Cancer Research

Article Title: Microenvironmental Regulation of Chemokine (C-X-C-Motif) Receptor 4 in Ovarian Carcinoma

doi: 10.1158/1541-7786.mcr-09-0463

Figure Lengend Snippet: FIGURE 5. The CXCR4-SDF1 axis in ovarian carcinoma cell invasion. A, a schematic representation of an invasion chamber setup used in the experiments. Dov13 cells were seeded into the inner well of a Boyden chamber overlaid with 3DCI in the presence or absence of additives (antibodies, small-molecule inhibitors). The well was placed in an outer chamber containing a layer of COS7 cells transfected with control or SDF-1 expression vector. The number of cells penetrating the 3DCI gel and adherent to the lower surface of the filter was enumerated and is shown in C. B, cells were transiently transfected with pBABE-SDF1 and a control vector containing no SDF1-specific sequence insert, were collected, and were lysed. Twenty micrograms of the total protein lysates was subjected to Western blot and probed with SDF1-specific antibodies and β-actin–specific antibodies (loading control). C, DOV13 cells were transiently transfected with control or CXCR4-specific siRNAs or preincubated in the presence or absence of CXCR4-blocking antibodies (0.2 μg/mL), nonspecific IgG (0.2 μg/mL), AMD3100 (10 μmol/L), or adhesion-activating CD29 clone TS2/16 antibodies (1:100 dilution) and allowed to invade for 18 h toward COS7-expressing SDF1, or control vector, as indicated. Experiments were done four times, each in triplicate, were averaged, and were plotted on a graph as a percent of invading cells; columns, mean of 10%; bars, SEM. DOV13 without additives, invading toward COS7 monolayer (white open columns) was arbitrarily set as 100% of invasion; *, P < 0.05; **, P > 0.05. D, expression of SDF1 in human ovarian carcinoma specimens was detected using real-time RT-PCR. Black columns, the values of the cycle numbers at which the accumulation of fluorescent signal from SYBR Green bound to the gene-specific double-stranded DNA PCR product was above the background. Absence of the black columns, no accumulation of the specific PCR product. A total of 48 samples were tested. Results for samples from 1 to 48 are plotted from left to right on the X axis and separated by small ticks. O, samples from normal ovary; I, II, III, and IV, those belonging to ovarian carcinoma FIGO stages I, II, III, and IV, and are separated by long ticks. E, malignant and nonmalignant ascites samples were obtained from women diagnosed with benign ovarian cysts (n = 6), stage I (n = 2), stage III (n = 21), and stage IV (n = 10) EOC. Patients ascites samples were analyzed using ELISA according to the manufacturer's specifications to determine the levels of CXCL12/SDF1 α; *, P < 0.05. Average for each group is shown with a vertical bar line across the individual values. F, cell proliferation assay. DOV13 were plated on 48-well plates, allowed to adhere followed by o/n serum starvation. Proliferation was initiated by addition of complete media [fetal bovine serum (FBS); open column], serum-free media (no fetal bovine serum; light gray column), 25 nmol/L SDF1 in serum-free media (black column), and 2.5 nmol/L SDF1 in serum-free media (dark gray column) and by the incubation for 24 h. Proliferation was measured by addition of WST-1 to the growing cells followed by 1 h of incubation and measurement of OD440. Experiments were repeated four times, each in triplicate, were averaged, and were plotted; columns, mean; bars, SEM; *, P < 0.05.

Article Snippet: The antibodies were used at the following dilutions: 1:200 for human anti-SDF1 antibodies (Santa Cruz www.aacrjournals.org on June 10, 2015. mcr.aacrjournals.org Downloaded from Biotechnology) in 3% bovine serum albumin (Sigma), 1:200 for human anti-CXCR4 polyclonal antibody (Santa Cruz Biotechnology) in 3% bovine serum in Tris-Buffered Saline Tween 20 (TBST), 1:1,000 for anti–β-tubulin monoclonal antibody (Sigma) in 5% skim milk in TBST, 1:2,000 for anti-p65 NFκB polyclonal antibodies (Rockland) in 3% bovine serum albumin, and 1:2,000 for antiactin monoclonal antibodies (Abcam) in 5% skim milk in TBST.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Sequencing, Western Blot, Blocking Assay, Quantitative RT-PCR, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Incubation

Journal: Journal of Translational Medicine

Article Title: CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells

doi: 10.1186/1479-5876-9-22

Figure Lengend Snippet: CXC12/CXCR4 expression in colorectal cancer (CRC) tissue specimens as determined by (A) Q-RT-PCR for CXCL12 and CXCR4, (B) ELISA analysis for CXCL12 and (C) immunohistochemistry for CXCR4 . (A) Q-RT-PCR data are expressed as mean +/- standard error of the mean (SEM), * P < 0.05, n = 50. Fold increase above 1 indicates gene overexpression in affected tissues related to unaffected neighbor tissues. (B) Detection of CXCL12 protein concentrations (pg/ml pro mg total protein) in total cell lysates of CRC and adjacent normal tissues from CRC patients ( n = 50). Protein data are expressed as mean +/- SEM, *P < 0.05. (C) Detection of CXCR4 protein expression in representative CRC specimens as assessed by immunohistochemical staining with CXCR4-specific antibodies showing positive cytoplasmic staining in CRC and in unaffected corresponding tissues (original magnification × 200 and × 400).

Article Snippet: CXCL12 levels were assayed using a validated commercial ELISA (Duo Set R&D Systems, DY350, Minneapolis, Minn., USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Over Expression, Immunohistochemical staining, Staining

Journal: Journal of Translational Medicine

Article Title: CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells

doi: 10.1186/1479-5876-9-22

Figure Lengend Snippet: CXCL12 protein expression in different human organs related to the liver as determined by ELISA . CXCL12 protein concentrations (pg/ml per mg total protein) were measured in total cell lysates of normal liver, pancreas, stomach, colon/rectum and esophagus tissues of 10 patients, respectively ( n = 10). Protein data are expressed as mean +/- SEM, *P < 0.05.

Article Snippet: CXCL12 levels were assayed using a validated commercial ELISA (Duo Set R&D Systems, DY350, Minneapolis, Minn., USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells

doi: 10.1186/1479-5876-9-22

Figure Lengend Snippet: CXC12/CXCR4 expression in Caco-2, HT-29 and SW480 cells as determined by (A) Q-RT-PCR for CXCL12 and CXCR4, (B) ELISA analysis for CXCL12 and (C) immunocytochemistry for CXCR4 . (A) Q-RT-PCR data are expressed as mean +/- standard error of the mean (SEM), * P < 0.05. Fold increase above 1 indicates gene overexpression related to housekeeping gene B2M. (B) Detection of CXCL12 protein concentrations in pg/ml in cell culture supernatant of Caco-2, HT-29 and SW480 cells. Protein data are expressed as mean +/- SEM. (C) Detection of CXCR4 protein expression in representative cell culture slides of Caco-2, HT-29 and SW480 cells as assessed by immunocytochemical staining with CXCR4-specific antibodies.

Article Snippet: CXCL12 levels were assayed using a validated commercial ELISA (Duo Set R&D Systems, DY350, Minneapolis, Minn., USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunocytochemistry, Over Expression, Cell Culture, Staining

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Sequences of primers used in this study

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques:

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modifies the expression of the CXCL12 signaling axis in MCF-7 cells. (A) Characterization of the control and COUP clones. An immunofluorescence cytochemistry assay was used to detect the relative expression of HA/COUP-TFI or COUP-TFI proteins in the control (Cont.) and COUP clones. The cells were fixed and processed for immunofluorescence as described in Methods; the nuclei were stained with DAPI. (B) CXCL12 , CXCR4 , and CXCR7 mRNAs were quantified by a real-time PCR analysis from two independent MCF-7 control and COUP clones. The results were normalized to GAPDH mRNA used as an internal control. The results were expressed as the relative mRNA expression level of CXCL12 , CXCR4 , or CXCR7 . Data are the mean values ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (C) The amount of intracellular HA/COUP-TFI, COUP-TFI, CXCL12, CXCR4, and CXCR7 protein was determined from whole-cell extracts of the different MCF-7 clones and compared to total ERK. A representative western blot is shown. (D) The control and COUP clones were fixed, and an immunofluorescence cytochemistry assay was used to detect the relative expression of CXCL12, CXCR4, and CXCR7 proteins. Staining with DAPI is also presented to visualize the nucleus of the cells.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Control, Clone Assay, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI modulates the chromatin structure of the CXCL12 and CXCR4 gene promoters. The FAIRE assay was performed as described in Methods. Real-time PCR was performed to monitor the enrichment of DNA corresponding to the proximal promoter of the CXCL12 (A) , the CXCR4 (B) and the CXCR7 (C) genes relative to the input chromatin from the control (Cont.) and COUP clones. The data are from triplicate samples and are representative of three separate experiments. The asterisk indicates significant differences ( p < 0.05) between the control and COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Real-time Polymerase Chain Reaction, Control, Clone Assay

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Estrogenic regulation of CXCL12 and CXCR4 in control and COUP clones. Control (Cont.) and COUP clones were treated with ethanol (EtOH) as the vehicle or E2 10 −8 M and ICI 10 −6 M alone or both together for 48 h. The CXCL12 (A) and CXCR4 (B) relative mRNA levels were monitored by a real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Control, Clone Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: The effect of COUP-TFI on the CXCL12/CXCR4 axis is mediated by EGF/EGFR activation. (A) The relative expression of EGF and EGFR mRNA was monitored by a real-time PCR analysis using MCF-7 control (Cont.) and COUP clones. The results were normalized against GAPDH as the internal control and are expressed as the mean EGF or EGFR mRNA/GAPDH mRNA ratio ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the control and COUP clones. (B) ERK activation was examined in the MCF-7 control (Cont.) and COUP clones after a 5- or 10-min stimulation with EGF (10 −9 M) or CXCL12 (200 ng/mL). Western blots were performed using antibodies against phospho-ERK (P-ERK) and total ERK (ERK1/2); a representative western blot is presented. The importance of EGFR-specific signaling and general ERK signaling on CXCL12 (C) and CXCR4 (D) regulation was assayed by treating the cells with EGF (10 −9 M), AG1478 (25 μM), or U0126 (25 μM) for 48 h. The CXCL12 and CXCR4 relative mRNA levels were monitored by the real-time PCR analysis, normalized to GAPDH mRNA as the internal control, and were expressed as the relative mRNA expression of CXCL12 or CXCR4 . Data are the mean ± SEM of at least three independent experiments. The asterisks indicate significant differences ( p < 0.05) between the untreated and treated control clones. The pound sign indicates significant differences ( p < 0.05) between the untreated and treated COUP clones.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Clone Assay, Western Blot

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: COUP-TFI overexpression influences cellular responses to CXCL12. (A) The relative growth of the control (Cont.) and COUP clones was assayed with or without CXCL12 treatment for 7 days. The basal and CXCL12-induced cell growth were evaluated by MTT assays (n = 6) and determined in three independent experiments. The results are expressed as the relative cell number obtained when the control cells were treated with the vehicle control. Significant differences between the unstimulated control cells and the other conditions ( p < 0.05) are indicated with an asterisk. Significant differences between stimulated control cells and stimulated COUP clones ( p < 0.05 ) are indicated with a pound sign. (B) The migratory capacity of control (Cont.) and COUP clones was analyzed. The cells were maintained in phenol red-free DMEM/2.5% dsFBS for 48 h and then seeded in phenol red-free DMEM/0.5% dsFBS in the upper chamber of a PET 8-μm pore insert. The cells were allowed to migrate for 24 h toward the phenol red-free DMEM/2.5% dsFBS medium complemented or not with CXCL12 (200 ng/mL) and AMD3100 (50 μM). (C) CXCL12 was also added to the culture medium in the upper chamber prior to migration. The results are expressed as the mean ± SEM of the relative number of migratory cells compared to the basal migration of the control cells measured in three independent experiments. The asterisks indicate significant differences ( p < 0.05) from the basal migration of the control clones. The pound sign indicates significant differences ( p < 0.05) between two conditions linked by black lines.

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Over Expression, Control, Clone Assay, Migration

Journal: BMC Cancer

Article Title: COUP-TFI modifies CXCL12 and CXCR4 expression by activating EGF signaling and stimulates breast cancer cell migration

doi: 10.1186/1471-2407-14-407

Figure Lengend Snippet: Box plots of CXCR4, CXCR7, CXCL12, and COUP-TFI mRNA expression in breast cancer and normal tissue. CXCR4 (A) , CXCR7 (B) , CXCL12 (C) , and COUP-TFI (D) mRNA expression was measured by real-time PCR in 23 normal breast tissue samples (NT), in 20 SBR grades 1 and 2, and in 19 SBR grades 3. The expression level was normalized by 18S RNA expression and analyzed with IQ5 software (Bio-Rad). The data are presented as whisker plots in which the horizontal bar represents the median, the grey boxes are the 25 th and 75 th percentiles, the vertical bar is the standard deviation, and the plus signs are the extreme points. All the Mann-Whitney tests were performed with Minitab 16 software, and the p value is indicated on the different graphs (ns denotes non-significant).

Article Snippet: A goat polyclonal antibody against human CXCL12 (R&D Systems AF-310-NA), rabbit polyclonal antibody against CXCR4 (Abcam Inc. ab2074), mouse monoclonal antibody against human CXCR7/RDC1 (R&D Systems clone 11G8; MAB42273), a rabbit polyclonal antibody against COUP-TFI (Abcam Inc. ab11954) and a rabbit polyclonal antibody against HA epitope (Santa Cruz sc-805) were used for the immunofluorescence and western blot assays.

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Software, Whisker Assay, Standard Deviation, MANN-WHITNEY