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

Journal: Scientific Reports

Article Title: Biomimetic post-capillary venule expansions for leukocyte adhesion studies

doi: 10.1038/s41598-018-27566-z

Figure Lengend Snippet: Spatial statistics of leukocyte adhesion as a function of coatings. ( a ) Leukocyte adhesion distribution heatmap for all ICAM-1 containing conditions in the straight channel device (Supplemental Fig. ). Flow direction is left to right. These consist of a length 4 expansion and an extended straight section to capture relaxation of the expansion effect. Wall associated adhesion peaks immediately after the initial expansion, and again at the collision-inducing point where the expansions end. Channel center adhesion subtly increases over the length of the channel. ( b ) Mean number of adherent cells per ROI in response to factorial combinations of CXCL12, SDC4, and ICAM1 in the coating, demonstrating additive effects. Each point represents mean numbers for a single participant, N = 6 donors. Boxes indicate mean and standard error. ( c ) Sub-ROI quantification using a mask for wall-associated vs. center of channel leukocytes as a function of flow distance along the ROI, demonstrating relaxation of the expansion effect for wall-associated but not channel center leukocytes. Wall associated adhesion probability peaks immediately after the expansion, then declines across the ROI until the junction with the constriction induces further collisions between erythrocytes and leukocytes, generating a second peak of adhesion. In contrast, channel center adhesion gradually increases across the length of the channel. ( d ) Validation of the sub-ROI quantification method by quantifying across the cross section of the ROI: Wall associated adhesion is restricted to the sidewalls, whereas channel center adhesion peaks in the channel center. ( e ) Linear model fits across the ROI cross section for the relative contributions of CXCL12, SDC4, and ICAM1 to adhesion. As expected, the contribution of adhesion molecule ICAM1 is most significant in the channel center, where shear stress is highest. ( f ) Linear model fits along the ROI for coating contributions to leukocyte adhesion on the sidewall. The initial spike of adhesion at the expansion is strongly ICAM-1 dependent. ( g ) Linear model fits along the ROI for leukocyte adhesion in the center of the channel. Adhesion immediately after the expansion is dependent on all three coatings, whereas ICAM-1 becomes more dominant as the expansion effect relaxes.

Article Snippet: Recombinant human CXCL12 and recombinant human Fc-ICAM1 chimeric fusion protein were obtained from R&D Systems (Minneapolis, MN).

Techniques: Biomarker Discovery, Shear