Journal: Bioscience Reports

Article Title: Biological and mutational analyses of CXCR4–antagonist interactions and design of new antagonistic analogs

doi: 10.1042/BSR20230981

Figure Lengend Snippet: Sequences and structures of eleven antagonists

Article Snippet: AMD11070 was obtained from MedChemExpress (Monmouth Junction, NJ, U.S.A.).

Techniques: Sequencing

Journal: Bioscience Reports

Article Title: Biological and mutational analyses of CXCR4–antagonist interactions and design of new antagonistic analogs

doi: 10.1042/BSR20230981

Figure Lengend Snippet: Competitive receptor binding activities of various antagonists for wild-type CXCR4 and its mutants ^*

Article Snippet: AMD11070 was obtained from MedChemExpress (Monmouth Junction, NJ, U.S.A.).

Techniques: Binding Assay

Journal: Bioscience Reports

Article Title: Biological and mutational analyses of CXCR4–antagonist interactions and design of new antagonistic analogs

doi: 10.1042/BSR20230981

Figure Lengend Snippet: The residues implicated in antagonist binding as determined from mutational experiments are shown for ( A ) CVX15, ( B ) LY2510924, ( E ) IT1t, ( F ) AMD3100, and ( I ) AMD11070. The residues are colored in the same way and meaning as described above in . Molecular docking calculations were performed for LY2510924, AMD3100 and AMD11070 to predict the CXCR4-antagonist complex structures. Using the co-crystal structure of CXCR4-CVX15 as a reference, the superposition of each of the above-mentioned antagonists (shown in blue) with CVX15 (red) is shown for ( C ) LY2510924, ( G ) AMD3100, and ( J ) AMD11070. The interactions of LY2510924, AMD3100, and AMD11070 with CXCR4 residues based on the docking results are illustrated for ( D ) LY2510924, ( H ) AMD3100, and ( K ) AMD11070. The molecular docking calculation was conducted using the crystal structure 3OE0 and the SYBYL program and displayed by PyMOL. The CXCR4 structure (in pale cyan) is shown in ribbons with important residues highlighted in colors: D262 (yellow), E288 (Violet), W94 (Magenta), D171 (Green), and D97 (Orange). The results were averages of at least three independent experiments.

Article Snippet: AMD11070 was obtained from MedChemExpress (Monmouth Junction, NJ, U.S.A.).

Techniques: Binding Assay

Journal: Bioscience Reports

Article Title: Biological and mutational analyses of CXCR4–antagonist interactions and design of new antagonistic analogs

doi: 10.1042/BSR20230981

Figure Lengend Snippet: Binding areas of the antagonists are shown by circles or ovals in different colors of deep blue for HC4319, pink for DV1, red for DV1 dimer, green for DV3, yellow for V1, magenta for vMIP-II, cyan for CVX15, black for LY2510924, orange for IT1t, gray for AMD3100, and deep sky blue for AMD11070. The CXCR4 helices in Figures 4–7 were generated and downloaded from GPCRDB.

Article Snippet: AMD11070 was obtained from MedChemExpress (Monmouth Junction, NJ, U.S.A.).

Techniques: Binding Assay, Generated

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