Amd3100 octahydrochloride hydrate

AMD3100 octahydrochloride hydrate (A5602; Sigma-Aldrich, UK) stock solution was prepared by dissolving 5 mg lyophilized product in 0.5 mL sterile water, then added to 4.5 mL phosphate buffered saline (PBS) to produce a 1 mg/mL injection solution, which was aliquoted and stored at −20°C until needed. Rat VEGF 165 (400-31; PeproTech, Rocky Hill, NJ) was prepared by dissolving the lyophilized product in sterile water to make a 0.1 mg/mL stock solution and then 1 mL of stock solution was added to 4 mL of sterile PBS +0.1% bovine serum albumin (BSA) (A9418; Sigma-Aldrich) to achieve 100 μg/mL injectable solution, which was aliquoted and stored at −20°C until needed. Recombinant human IGF1 (100-11; PeproTech) and murine GCSF (250-05; PeproTech) were prepared in the same manner. Finally, PBS +0.1% BSA, “sham growth factor,” to determine the effects of AMD3100 alone was also prepared.

The following antibodies were used in this study: anti-USP33, anti-slug, and anti-twist1 antibodies were purchased from ProteinTech Group (Chicago, IL, USA); anti-CXCR4 antibody was purchased from BD Bioscience (Franklin Lakes, NJ, USA); anti-HA and anti-Flag M1 antibodies were from Sigma-Aldrich (St. Louis, MO, United States); anti-ppERK, anti-ERK, and anti-β-arrestin2 antibodies were from Cell Signaling Technology (Boston, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-snai1, and anti-β-actin antibodies were purchased from Santa Cruz (Dallas, TX, USA).
N-ethylmaleimide (NEM, deubiquitinase inhibitor), SDF-1 (SDF-1α, CXCR4 agonist), AMD3100 octahydrochloride hydrate (1,1′-[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride, CXCR4 antagonist), dynasore hydrate (3-Hydroxy-naphthalene-2-carboxylic acid (3,4-dihydroxy-benzylidene)-hydrazide hydrate, dynamin inhibitor), HA affinity beads, and Flag affinity beads were all purchased from Sigma-Aldrich.

Transforming growth factor-β1 (TGF-β1, Sigma) was added in 5 ng/mL final concentration to the cells. AMD3100 octahydrochloride hydrate (2 μg/mL, Sigma) was used to inhibit CXCR4 receptor. During cell migration assessment cells were treated with Mitomycin C (10 μg/mL, Sigma, Part No.: M4287) to prevent cell proliferation. BSA (bovine serum albumin, Sigma, Part No.: A3294) was used for aspecific blocking in several methods.

Whole lateral walls were dissected and fixed as described before (Mirzadeh et al, 2008 (link); Monaco et al, 2019 (link)). In short, the mice were killed by CO₂ inhalation and subsequent cervical dislocation (adult) or by decapitation (E18), and the brain was dissected in a buffered sucrose solution. The lateral wall was thinly removed and either directly fixed in ice-cold 3% PFA, 4% sucrose in PBS, or incubated at 37°C overnight in 1 ml Euromed-N (#ECM0883L; Euroclone) containing 2% B27 (#17504044; Invitrogen) and, when indicated, recombinant human GDF15 (10 ng/ml; #Q99988; R&D Systems), human recombinant EGF (20 ng/ml; #AF-100-15; Peprotech), PD158780 (20 μM; #513035; Calbiochem/Merck), AMD3100 octahydrochloride hydrate (6 μM; #A5602; Sigma-Aldrich), TBA (10 μM; #S8049; Selleckchem), NKY80 (200 μM; #116850; Sigma-Aldrich), U0126 (10 μM; #BML-EI282-0001; Enzo Life Sciences), or SAG (200 nM; #11914; Cayman Chemicals). For immunofluorescence, the incubated lateral walls were fixed the next day, and all dissections were left in the 3% PFA, 4% sucrose in PBS solution at 4°C overnight, and then kept in PBS containing 0.01% azide until immunostaining.