Journal: PLoS ONE

Article Title: Glutathione S-Transferase P Influences Redox and Migration Pathways in Bone Marrow

doi: 10.1371/journal.pone.0107478

Figure Lengend Snippet: (A) Migration of BMDDCs to CXCL12. Wide type and Gstp1/p2 −/− BMDDCs were either left untreated (control) or stimulated with 200 ng/ml CXCL12 for 16 h. Values are average percentages of migration (± SD ) from six independent experiments with asterisks (*) indicating statistical significant differences between WT and Gstp1/p2 −/− BMDDCs ( p <0.05). ( B, C ) Intracellular calcium (B); plasma membrane potential dynamics (C) in WT and Gstp1/p2 −/− BMDDCs in response to CXCL12. Arrows indicate the addition of CXCL12. Data are representative traces of three independent experiments. ( D ) Kinetics of Akt and ERK phosphorylation in BMDDCs with response to CXCL12. Wild type and Gstp1/p2 −/− BMDDCs were serum starved for 2 h. 200 ng/ml CXCL12 was then added and cells were collected at various time points. Representative immunoblots show phosphorylation of Akt and ERK in WT and Gstp1/p2 −/− BMDDCs after stimulation with CXCL12 for the indicated periods. p-Akt or p-ERK levels were quantified and normalized relative to total Akt or ERK protein for each time point. The ratio (p-Akt:total Akt or p-ERK:total ERK) at 0 min CXCL12 exposure was assigned a value of one and all other ratios are shown relative to this value. Bars represent the means (± SD ) from three independent experiments. ( E, F ) Impact of GSTP ablation on CXCR4, IP3R and SHP-2 levels in BMDDCs. Total protein levels were evaluated by immunoblots. Actin served as a loading control. Surface expression of CXCR4 was further analyzed by flow cytometry. Representative histograms are shown: CXCR4 expression in WT and Gstp1/p2 −/− BMDDCs compared to isotype control showing the mean fluorescence intensity (MFI) and statistical summary of three independent measurements presented as means (± SD ) with asterisks (*) indicating statistically significant differences between WT and Gstp1/p2 −/− BMDDCs (p<0.05). ( E ). Relative gene expression levels were quantified by Real-Time RT-PCR. Bars represent the means (±SD ) from three independent experiments ( F ). ( G ) Effect of SHP-2 specific inhibitor PHPS1 and CXCR4 specific antagonist AMD3100 on CXCL12-stimulated chemotaxis of BMDDCs. WT and Gstp1/p2 −/− BMDDCs were either untreated (control) or pretreated with 10 µM PHPS1 or 1 µM AMD3100 for 2 h, and then cells were either left untreated or stimulated with 200 ng/ml CXCL12 for 6 h. Values are average percentages of specific chemotaxis (percentage of cells migrating to medium alone was subtracted from the percentage of cells migrating to medium with CXCL12) (± SD ) from three independent experiments, with asterisks (*) indicating statistically significant differences between control and drug treatments ( p <0.05). Chemotaxis of WT cells in the absence of inhibitor is considered 100% migration.

Article Snippet: Cell surface CXCR4 expression was determined by fluorescence-activated cell sorter (FACS) analysis using FITC-conjugated rat anti-mouse CXCR4 mAb or FITC-conjugated rat IgG2b (both from BD Pharmingen) as isotype control.

Techniques: Migration, Western Blot, Expressing, Flow Cytometry, Fluorescence, Quantitative RT-PCR, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Close Interactions between Mesenchymal Stem Cells and Neuroblastoma Cell Lines Lead to Tumor Growth Inhibition

doi: 10.1371/journal.pone.0048654

Figure Lengend Snippet: SH-SY5Y, Htla-230 and GI-LI-N NB cells incubated or not with hMSCs in a transwell system for 24 hours, were placed in the upper wells on Matrigel®-coated invasion chambers and separated by an 8 μm membrane from CXCL12 or bone marrow sample. After 24 hours, NB cells migrated into the lower chamber were stained and counted after May Grunwald-Giemsa. Panels A–C show the invasiveness of SH-SY5Y (Panel A), GI-LI-N (Panel B) and Htla-230 (Panel C) to CXCL12. Panels D shows the invasiveness of SH-SY5Y to CXCL12 in presence or absence of CXCR4 antagonist AMD3100 that was added to some wells. Panel E shows the invasiveness of SH-SY5Y to the bone marrow of an healthy donor. The data are expressed as mean values obtained from the count of 10 fields/well. Bars are SD. Statistical analysis was performed by Unpaired t test with Welch's correction.

Article Snippet: CXCR4 surface expression was evaluated on NB cells incubated with CXCR4-FITC mAb as well as isotype-matched control mAbs (BD Bioscience) for 30 min at 4°C, and then analyzed by flow cytometry with a FACScan instrument (Beckmann Coulter Gallius Cytometry).

Techniques: Incubation, Staining

Journal: PLoS ONE

Article Title: Silibinin Inhibits HIV-1 Infection by Reducing Cellular Activation and Proliferation

doi: 10.1371/journal.pone.0041832

Figure Lengend Snippet: A , Cytotoxicity profile of SIL in TZM-bl cells. Cells were infected with LAI, a CXCR4-using virus, or BAL, a CCR5-using virus, at an MOI of 0.05 in the presence of the indicated concentrations of SIL and ATP was measured using the ATPlite kit 48 hours later. The data are representative of 2 (BAL) and 3 (LAI) independent technical repeats. B , Antiviral profile of SIL in TZM-bl cells. Serial dilutions of SIL were tested for inhibition of infection in TZM cells. Following addition of compounds and virus, cells were incubated for 48 hours before luciferase activity was measured. Percent inhibition refers to percent reduction in luciferase activity of SIL versus untreated cultures. Error bars represent standard deviation of 3 independent technical repeats. C , SIL inhibits pseudovirus replication in TZM-bl cells. TZM-bl cells were infected with the indicated viruses in the presence of the indicated concentrations of SIL and luciferase activity was measured 48 hours post-infection. The D013M12 psuedovirus contains a subtype D envelope sequence, while the D769 psuedovirus contains a subtype A envelope sequence. Error bars represent standard deviations of triplicate wells per condition.

Article Snippet: Cells were washed, stained with AViD dye (Invitrogen), and fluorescently conjugated mAbs to CD14-Alexa700, CCR5-APC-Cy7 (BD), and CXCR4-FITC (R&D).

Techniques: Infection, Inhibition, Incubation, Luciferase, Activity Assay, Standard Deviation, Sequencing

Journal: PLoS ONE

Article Title: Silibinin Inhibits HIV-1 Infection by Reducing Cellular Activation and Proliferation

doi: 10.1371/journal.pone.0041832

Figure Lengend Snippet: PBMCs were stimulated with PHA for 3 days prior to exposure to IL-2 and the indicated concentrations of SIL. Twenty-four hours later, cells were stained for CD4, CXCR4, and CCR5 and analyzed by flow cytometry. A, Y-axis represents the concentration of the indicated cell type. The cell concentration is expressed per µl of cell suspension and was determined using counting beads. Panel B shows the percentage of total CD4+ T cells that express one, both, or neither co-receptor.

Article Snippet: Cells were washed, stained with AViD dye (Invitrogen), and fluorescently conjugated mAbs to CD14-Alexa700, CCR5-APC-Cy7 (BD), and CXCR4-FITC (R&D).

Techniques: Staining, Flow Cytometry, Concentration Assay

Journal: International Journal of Biological Sciences

Article Title: Identification of Ghrelin Receptor Blocker, D-[Lys3] GHRP-6 as a CXCR4 Receptor Antagonist

doi:

Figure Lengend Snippet: DLS inhibits CXCL12-induced chemotaxis. Activated T cells were loaded with Calcein AM dye, treated with different concentration of DLS or the CXCR4 specific antagonist, AMD, for 30 minutes and were then examined for their ability to migrate in response to CXCL12 (10nM) in 24-well Transwell migration chambers. After 5 hours of incubation, the chambers were removed and the plates were read at 485/590 excitation/emission in a Cytofluor fluorimeter. The graph shows Migration Index or the ratio of migration of inhibitor plus CXCL12-induced cells over CXCL12-only induced cell migration. The results demonstrate that DLS inhibits CXCL12 induced activated T cell chemotaxis. Data shown are mean ±SEM and plotted as summary of three experiments, each done with three replicates. (* p<0.05)

Article Snippet: Cells were washed with cold PBS and then FITC conjugated anti-human CXCR4 (12G5) antibody (BD Biosciences) added and incubated on ice for 30min.

Techniques: Chemotaxis Assay, Concentration Assay, Migration, Incubation

Journal: International Journal of Biological Sciences

Article Title: Identification of Ghrelin Receptor Blocker, D-[Lys3] GHRP-6 as a CXCR4 Receptor Antagonist

doi:

Figure Lengend Snippet: DLS does not influence CXCL12-induced CXCR4 internalization. Molt-4 cells were treated with different concentrations of DLS for 30 minutes, then treated with or without CXCL12 (10nM final concentration) for 90min. The cells were then harvested in cold FACS buffer and stained with anti-CXCR4-PE (12G5) antibody for 45 minutes at 4°C. The stained cells were then washed and fixed with 2%PFA and samples acquired in FACScan. The graph shows average of two replicate's mean fluorescence intensity (MFI) for each cell treatment. The results demonstrate that DLS and AMD (data not shown) do not internalize CXCR4 by itself and do not interfere with CXCL12-induced CXCR4 internalization.

Article Snippet: Cells were washed with cold PBS and then FITC conjugated anti-human CXCR4 (12G5) antibody (BD Biosciences) added and incubated on ice for 30min.

Techniques: Concentration Assay, Staining, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: Identification of Ghrelin Receptor Blocker, D-[Lys3] GHRP-6 as a CXCR4 Receptor Antagonist

doi:

Figure Lengend Snippet: DLS decreases HIV1-IIIB propagation in vitro in activated PBMCs. Activated PBMCs were washed and treated with DLS or AMD3100 for 30 min at 37°C. 10ng (p24) of HIV1-IIIB (CXCR4 tropic) was added per 10 6 cells/ml, and then the cells were incubated at 37°C for 3hrs. Cells were washed to remove virus and resuspended to a concentration of 500,000 cells/ml. Antagonists were added to appropriate tubes and cells were plated in triplicate in 24-well plates and incubated at 37°C. Supernatants were collected from each well at day 3 (data not shown), 6 and 9 and p24 levels were measured using ELISA. The graphs show % of p24 level over virus only controls at day 6 & 9. The results reveal that DLS inhibits X4 tropic HIV1-IIIB propagation in PBMCs. Data shown are mean ±SEM and plotted as summary of three experiments, each performed using different donors with three replicates in all cultures.(* p<0.05)

Article Snippet: Cells were washed with cold PBS and then FITC conjugated anti-human CXCR4 (12G5) antibody (BD Biosciences) added and incubated on ice for 30min.

Techniques: In Vitro, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay