Journal: Annals of Clinical and Translational Neurology

Article Title: A CXCR4 receptor agonist strongly stimulates axonal regeneration after damage

doi: 10.1002/acn3.50926

Figure Lengend Snippet: CXCR4 and CXCL12α are expressed at the crushed site of the sciatic nerve. (A) Longitudinal cryo‐sections of control (top) and crushed (bottom) sciatic nerves show CXCR4 expression ( red ). Dotted lines define the crushed area (the proximal site of the injury is on the left). (B) Higher magnification of the crush site. Scale bars: 500 µm (A), 50 µm (B). (C) Cross‐sections of sciatic nerves from mice expressing cytosolic GFP ( green ) specifically in SC, of controls (top panels), and after 3 (middle panels) or 7 days (bottom panels) from nerve crush. Neurofilament (NF) staining ( blue ) identifies the axonal compartment; CXCR4 is in red . CXCR4 co‐localizes with NF, restricting its expression to the axonal compartment. Scale bars: 50 µm. Right panels show higher magnifications (scale bar: 10 µm). (D) Cross‐sections of sciatic nerves show CXCL12α expression ( red ) at the crushed site 3 and 7 days after crush. Right panels show higher magnifications. Scale bars: 50 µm (right panels: 10 µm). Images are representative of at least three independent sets of experiments.

Article Snippet: After surgery, mice were i.p. injected with the indicated treatments: NUCC‐390 26 mg/kg (daily), or AMD3100 4 mg/kg (twice daily), or AMD3100 plus NUCC‐390, or 100 µg neutralizing anti‐CXCL12α antibody (weekly, R&D MAB310).

Techniques: Expressing, Staining

Journal: Annals of Clinical and Translational Neurology

Article Title: A CXCR4 receptor agonist strongly stimulates axonal regeneration after damage

doi: 10.1002/acn3.50926

Figure Lengend Snippet: CXCR4 engagement promotes the recovery of neuromuscular activity after sciatic nerve injury. (A) Scheme of the experimental workflow. After sciatic nerve crush mice were treated either with an antibody neutralizing CXCL12α, or with AMD3100, or with vehicle. CMAP was recorded at day 14. (B) Histogram reports the area of CMAP traces 14 days after sciatic nerve crush. (C) Scheme of NUCC‐390 administration after sciatic nerve crush, and time‐course of CMAP recordings. (D) Histogram reporting the area of CMAP traces at the indicated time points after sciatic nerve crush (± NUCC‐390). (E) Representative CMAP traces at different time points after sciatic nerve crush (± NUCC‐390). Black arrows indicate the stimulation artifact. For each trace, the scale of CMAP amplitude is reported on the right. (F) Histogram showing the area of CMAP traces 14 days after sciatic nerve crush in mice treated either with vehicle, or with NUCC‐390 in combination with AMD3100. [Correction added on 06 December 2019 after first online publication: Figure 2 has been updated.]

Article Snippet: After surgery, mice were i.p. injected with the indicated treatments: NUCC‐390 26 mg/kg (daily), or AMD3100 4 mg/kg (twice daily), or AMD3100 plus NUCC‐390, or 100 µg neutralizing anti‐CXCL12α antibody (weekly, R&D MAB310).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: A new obligate CXCL4–CXCL12 heterodimer for studying chemokine heterodimer activities and mechanisms

doi: 10.1038/s41598-022-21651-0

Figure Lengend Snippet: ( a ) Structural model of the CXCL4–CXCL12 heterodimer. The CXCL4 monomer is shown in blue and the CXCL12 monomer is shown in red. First beta-strands forming the intermonomer interface are labeled. ( b ) The structure (top) and the amino acid sequences (bottom) of first beta-strands from CXCL4 (blue) and CXCL12 (red), representing the intermonomer interface, where several amino acids with side chains on the same side of beta-strands are labeled. The axis of symmetry is indicated by black lines on the amino acid sequences of shown beta-strands, and residues selected for mutation are colored in cyan. ( c ) Structural model of the CXCL4–CXCL12 heterodimer with amino acid residues selected for mutation labeled.

Article Snippet: Membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk at 4 °C and incubated with the primary anti-CXCL4 and anti-CXCL12 antibodies (MAB7952 and MAB310, respectively, R&D systems) overnight.

Techniques: Labeling, Mutagenesis

Journal: Scientific Reports

Article Title: A new obligate CXCL4–CXCL12 heterodimer for studying chemokine heterodimer activities and mechanisms

doi: 10.1038/s41598-022-21651-0

Figure Lengend Snippet: ( a ) Size-exclusion chromatograms. The elution profiles of lysozyme (14.4 kDa) and myoglobin (17 kDa) in blue and OHD 4–12 (15.9 kDa) in orange are shown. ( b ) WB analysis in non-reduced (left bands, N.R.) and reduced (right bands, R.) conditions demonstrates the presence of OHD 4–12 obtained following the mixing of CXCL4-S26C and CXCL12-L29C mutants in the presence of Cu 2+ detected with anti-CXCL4 (αCXCL4) or anti-CXCL12 (αCXCL12) antibodies (see also Supplementary Fig. S1). ( c ) NMR spectroscopic analysis of OHD 4–12 folding state. The 15N-HSQC NMR spectrum of the uniformly 15N-labeled 68 μM OHD 4–12 in 90% H2O/10% D 2 O at pH 6.9 in the presence of 20 mM NaCl, collected at 40 °C. ( d – f ) Expansions from the OHD 4–12 spectrum overlaid with CXCL4 wt (blue) and CXCL12 wt (red) 15N-HSQC NMR spectra. Known signal assignments for CXCL4 and CXCL12 are indicated. The 15N-HSQC NMR spectrum of the uniformly 15N-labeled 150 μM CXCL4 in 90% H2O/10% D 2 O at pH 5.0 in the presence of 20 mM NaCl was collected at 40 °C. The 15N-HSQC NMR spectrum of the uniformly 15N-labeled 51 μM CXCL12 in 20 MES buffer prepared with 90% H2O/10% D 2 O at pH 6.8 was collected at 25 °C. The difference in experimental conditions was due to the difference in solubility properties of CXCL4 and CXCL12.

Article Snippet: Membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk at 4 °C and incubated with the primary anti-CXCL4 and anti-CXCL12 antibodies (MAB7952 and MAB310, respectively, R&D systems) overnight.

Techniques: Labeling, Solubility

Journal: Scientific Reports

Article Title: A new obligate CXCL4–CXCL12 heterodimer for studying chemokine heterodimer activities and mechanisms

doi: 10.1038/s41598-022-21651-0

Figure Lengend Snippet: MDA-MB 231 breast cancer cells migration. ( a ) Migration of MDA-MB 231 cells treated with 100 nM of CXCL4 wt , its mutant CXCL4-S26C used as a CXCL4 counterpart to produce OHD 4–12 , CXCL12 wt , its mutant CXCL12-L29C used as a CXCL12 counterpart to produce OHD 4–12 , CXCL12 variants CXCL12 M (obligate monomer) and CXCL12 D (obligate dimer), and 50 or 100 nM of OHD 4–12 . Negative and positive controls were 0 and 10% FBS. Migration index was determined as a percentage of wound healing in the absence of chemokine treatment at 0% FSB. ( b ) Competitive inhibition of CXCL12-driven migration of MDA-MB 231 cells by OHD 4–12 at concentrations ranging from 1 to 200 nM. The OHD 4–12 inhibits CXCL12-induced migration of MDA-MB 231 cells in a dose–response manner. The migration of cells treated with 100 nM CXCL12 wt alone is shown for comparison. Negative and positive controls were as in panel ( a ). All presented data are means ± SEM (standard errors of the means) from n ≥ 3 independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001; analyzed by one-way ANOVA followed by a post-hoc Tukey multiple comparison test.

Article Snippet: Membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat milk at 4 °C and incubated with the primary anti-CXCL4 and anti-CXCL12 antibodies (MAB7952 and MAB310, respectively, R&D systems) overnight.

Techniques: Migration, Mutagenesis, Inhibition, Comparison