Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 1. AMD070 and ACT-1004 reduce B16-F10 migration in wound assays. B16-F10 melanoma cells were plated in 6-well plates along with either a 30 µM concentration of their respective inhibitor or 20 µL of DMSO and left in a 37˚C incubator for 24. After 24 hours, the cells were scratch wounded with a 200 µL pipette tip, and images were taken of wounds at 0, 4, 8, and 18 hours after wounding. The photos taken at 18 hours post-wounding show the wound had healed entirely in our control cells, while our control + DMSO cells were about 80% healed. The CXCR4 inhibitor (AMD070) and CXCR7 inhibitor (ACT-1004) still showed a large wound.

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Migration, Concentration Assay, Transferring, Control

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 2. CXCR inhibitors decreased melanoma wound healing by ~60%. Bar graphs showing progressive healing of melanoma wounds at each time point over an 18-hour time frame after CXCR inhibition. The data starts at 0% of the wound area healed at 0 hr, and as time progresses, cells invade the wound and make the area smaller, healing the wound. A difference was observed between the control and the control with DMSO, but an even more significant difference was observed between our controls and our AMD070 and ACT-1004 inhibited cells. *p < 0.05, **p < 0.005, ****p < 0.0001. N = 4.

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Inhibition, Control

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 3. CXCR4 and 7 inhibition decreases melanoma cell chemokinesis. Box plot graphs show how melanoma migration decreased after CXCR4 and CXCR7 inhibition. Data from 4 experiments was normalized to the control values. The number of asterisks present identifies ANOVA significant p-values and are in comparison to the Control + DMSO values. There was no significant difference between the CXCR4 and CXCR7 inhibitors. ***p < 0.001, ****p < 0.0001. N = 4. These results with the two inhibitors match what we know about SDF1 binding to CXCR4/7. Experimental studies on the thermodynamics and kinetics of recep- tor interactions with SDF1 have shown that CXCR7 has a higher affinity for SDF1 than CXCR4 [32]. Our structural modeling of the CXCR4:SDF1 and CXCR7:SDF1 complexes and their relaxation in the lipid bilayer environment; the binding free energy analysis of these interaction partners is shown in Appendix 2, along with experimental Kd values. We find that CXCR7 binds with a stronger affinity to SDF1 (more negative DG) than CXCR4, which is consistent with the above exper- imental observations. The structures provide a biophysical basis for CXCR7,

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Inhibition, Migration, Control, Comparison, Binding Assay

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 4. Effect of CXCR4/7 KD in melanoma wound healing. Bar graphs showing progressive healing of melanoma wounds at each time point over an 18-hour time frame for each set of transfections. The data starts at 0% of the wound area healed at 0 hr, and as time progresses, cells invade the wound and make the area smaller, healing the wound. Normal B16-F10 cells were compared to A. Scramble siRNA, B. CXCR4 siRNA, C. CXCR7 siRNA and D. CXCR4 and CXCR7 siRNAs. The only significant difference observed was between our control cells and CXCR4 and CXCR7 siRNA knockdown cells in the presence of SDF1. *p < 0.05.

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Transfection, Control, Knockdown

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 5. CXCR4/7 KD decreases melanoma cell migration. Box plot graphs show how melanoma cells decrease migration rates after CXCR4 and CXCR7 KD. Data from 5 experiments was normal- ized to the control values. ANOVA significant p-values are identified by the number of asterisks present: *p < 0.00001. Cell velocity is one of the critical indicators of increased/decreased cell migra- tion and is necessary for successful metastasis. We wanted to determine if CXCR4/7 influences melanoma migration. B16-F10 cells were transfected with scramble, CXCR4 KD, CXCR KD, or a combination of CXCR4 and 7 KD siRNA to measure a change. B16-F10 cells were then plated either in the presence of or without SDF1. Cell velocity was measured by manually tracking at least 15 cells at a time from each experiment using an ImageJ plug-in. The formula used to meas- ure velocity in ImageJ was the following:

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Migration, Control, Transfection

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 6. CXCR4/7 KD decreases B16-F10 melanoma cell velocity. Graphs represent the velocity, in µm per second, after melanoma transfection with A. CXCR4 siRNA, B. CXCR7 siRNA, and C. CXCR4 and CXCR7 siRNAs. siRNAs were compared to untreated and scrambled random siRNA. Significant p-values are identified by the number of asterisks present: *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Transfection

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 7. CXCR4/7 KD + SDF1 decreases B16-F10 melanoma cell velocity. Graphs repre- sent the velocity, in µm per second, after melanoma transfection with A. CXCR4 siRNA, B. CXCR7 siRNA, and C. CXCR4 and CXCR7 siRNAs and the addition of SDF1. siRNAs were compared to untreated + SDF1 and scramble random siRNA +SDF1. Significant p-values are identified by the number of asterisks present: *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Transfection

Journal: OALib

Article Title: The Complementary Roles of CXCR4 and CXCR7 in Melanoma Migration

doi: 10.4236/oalib.1112617

Figure Lengend Snippet: Figure 8. CXCR7 KD decreases melanoma cell size. A) Images of rat B16-F10 melanoma cell line stained with phalloidin-488 after siRNA transfection for CXCR4 or CXCR7. Arrows in CXCR7 KD point to long stress fibers in melanoma cells. B) We plotted the cells’ cell size area after transfection. The ** shows the significant reduction of CXCR after siRNA (Anova less than p < 0.0001). The number below boxplots corresponds to many cells traced (N = 100).

Article Snippet: CXCR Inhibition The CXCR4 inhibitor AMD070 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and the CXCR7 inhibitor ACT-1004-1239 (MedChemExpress, Monmouth Junction, NJ, USA) were diluted to 2 mg/mL and 5 mg/mL with dimethyl sulfoxide (DMSO), respectively, according to manufacturer protocols.

Techniques: Staining, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The CXCR4–CXCL12 -Axis Is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects In Vitro

doi: 10.3390/ijms20194740

Figure Lengend Snippet: Growth inhibition and apoptosis of B cell lymphoma cell lines upon treatment with CXCR4 antagonists. ( a ) Structure of the CXCR4 antagonists AMD3100, AMD070, and WK1. ( b ) Cell growth of SuDHL4 (as GCB-DLBCL model), RI-1 and U2932 (as NGCB-DLBCL model), and BL2 (as Burkitt model) cell lines in the presence of increasing concentrations (range: 1–90 µM) of the CXCR4 antagonists AMD3100, AMD070, its niacin derivative WK1 and niacin, respectively, as determined by the EZ4U proliferation assay and expressed by percentage of normal absorption. ( c ) Annexin V positivity of BL2 (as Burkitt model) and SuDHL4 (as GCB-DLBCL model) cells treated with AMD3100, AMD070 and its niacin derivative WK1 (concentration: 40 µM; for 48 h) as determined by flow cytometry and compared to the DMSO treated control cells. The treatments and Annexin V staining were performed in triplicate and the medians ± standard deviations are depicted. ( d ) Percentage of cleaved caspase 3 positive BL2 (as Burkitt model) and SuDHL4 (as GCB-DLBCL model) cells treated with 40 µM of AMD070 or 20 µM and 40 µM of its niacin derivative WK1 for 24 h as determined by flow cytometry and compared to the DMSO treated control cells. The treatments and cleaved caspase staining were performed in triplicate and the medians ± standard deviations are depicted.

Article Snippet: AMD070 (MedChemExpress, Sollentuna, Sweden) (40 mg, 0.12 mmoL, 1 eq) was dissolved in MeOH (800 μL).

Techniques: Inhibition, Proliferation Assay, Concentration Assay, Flow Cytometry, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: The CXCR4–CXCL12 -Axis Is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects In Vitro

doi: 10.3390/ijms20194740

Figure Lengend Snippet: Growth inhibition and apoptosis of B cell lymphoma cell lines upon treatment with CXCR4 antagonists. ( a ) Structure of the CXCR4 antagonists AMD3100, AMD070, and WK1. ( b ) Cell growth of SuDHL4 (as GCB-DLBCL model), RI-1 and U2932 (as NGCB-DLBCL model), and BL2 (as Burkitt model) cell lines in the presence of increasing concentrations (range: 1–90 µM) of the CXCR4 antagonists AMD3100, AMD070, its niacin derivative WK1 and niacin, respectively, as determined by the EZ4U proliferation assay and expressed by percentage of normal absorption. ( c ) Annexin V positivity of BL2 (as Burkitt model) and SuDHL4 (as GCB-DLBCL model) cells treated with AMD3100, AMD070 and its niacin derivative WK1 (concentration: 40 µM; for 48 h) as determined by flow cytometry and compared to the DMSO treated control cells. The treatments and Annexin V staining were performed in triplicate and the medians ± standard deviations are depicted. ( d ) Percentage of cleaved caspase 3 positive BL2 (as Burkitt model) and SuDHL4 (as GCB-DLBCL model) cells treated with 40 µM of AMD070 or 20 µM and 40 µM of its niacin derivative WK1 for 24 h as determined by flow cytometry and compared to the DMSO treated control cells. The treatments and cleaved caspase staining were performed in triplicate and the medians ± standard deviations are depicted.

Article Snippet: All cell lines were treated with the commercially available CXCR4 antagonists (MedChemExpress, Sollentuna, Sweden) AMD3100 and AMD070 [ ], and the novel niacin derivative of AMD070 called WK1, which was generated by us, in a range from 1 µM to 90 µM.

Techniques: Inhibition, Proliferation Assay, Concentration Assay, Flow Cytometry, Control, Staining