Rgd peptide

RGD peptide, Fibronectin, lipopolysaccharide (Escherichia coli 0111:B4), Cytochalasin B, Rhodamine-conjugated phalloidin and proteasome inhibitor MG132 were obtained from Sigma-Aldrich (Shanghai, China). NSC23766 was the product of Selleck Chemicals (Shanghai, China). Rabbit monoclonal antibodies for HMGN2 was from Cell Signaling Technology Inc. (Danvers, USA). Mouse monoclonal antibody for F-actin and Rabbit monoclonal antibodies for integrin α5, integrin β1 were purchased from Abcam (Cambridge, USA). Mouse and rabbit monoclonal antibodies for GAPDH, horseradish peroxidase (HRP)-conjugated secondary antibody and FITC fluorescent-labeled secondary antibody (goat anti-rabbit IgG, green) were provided by Beyotime Institute of Biotechnology (Haimen, China). Rabbit polyclonal antibody for NFI was provided by Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rac1 activation assay kit with anti-active Rac1 (Rac1-GTP) monoclonal antibody and anti-Rac1 rabbit polyclonal antibody were provided by NewEast Biosciences (Malvern, USA).

Astrocytes were collected from subcultures and 2×10³ cells/well were seeded to 96 well plates (MTS assay) or 1×10⁴ cells/well in 6 well plates in DMEM/F-12 with 10% FCS. At confluence, before treatment cells were starved for 24 h in serum-free DMEM/F-12. Then medium was changed to serum-free DMEM/F-12 containing one or more of the fowling substances: 1 ng/ml active TGF-β2 (R&D Systems, 302-B2-010/CF), 250, 1000 or 2000 ng/ml human recombinant OPN (R&D Systems, 1433-OP-050/CF), 100 nM RGD peptide (Sigma, A8052), or an anti-CD44 blocking antibody (1∶100, Abcam, ab41478). In each experiment, control cultures were incubated with the solvent in serum-free medium alone.

For AFM based RGD-integrin binding measurements, cells were cultured on stiff PA gels in the absence (i.e., DMSO only) and presence of GM6001 for 12–15 hrs duration. Spherical probes (67 kHz, Novascan), 4.5 μm in diameter were coated with 10 μg/ml solution of poly-L-lysine (Sigma, Cat # P4707) for 20 mins and then treated with 0.5% glutaraldehyde (Sigma, Cat # G7651) for 20 mins^{19 (link)}. Next, probes were dipped in 0.1 mg/ml solution of RGD peptide (Sigma, Cat # G4391) for 30 mins, washed 3–4 times in distilled H₂O, and then dried in a vaccum dessicator for 30 mins. After tip calibration using thermal noise method, RGD-integrin binding studies were performed. While indenting, probe was held at the cell surface for 10 secs to allow formation of integrin-RGD bonds, and then retracted at a tip speed of 3–4 μm/sec. Analysis of maximum adhesion force was performed using Igor Pro 6.22 A software.

For proteinase K treatment, placental EVs were treated with 10 μg/ml proteinase K in PBS; for controls, proteinase K was added to PBS alone. Samples and controls were incubated for 10 min at 37 °C before heat inactivation at 65 °C for 10 min followed by addition of 100 μM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor.
For inhibitory peptide experiments, RGD peptide was purchased from Sigma-Aldrich and HYD-1 (KIKMVISWKG) was synthesized (Genscript, Piscataway, NJ). All reagents were 95% or greater purity and resuspended in deionized water, aliquoted, and frozen. Labeled pEVs were incubated with 0.6 μM of inhibitory peptide and incubated at 37 °C for 30 min before intravenous administration into recipient mice.

A total of 10 mg of RGD^® peptide (Sigma Aldrich, A-8052, Darmstadt, Germany) was soaked in 11.5 ml distilled water, remaining under a magnetic stirrer after being dissolved. Then, 50 mg of Carbopol® was added and stirred. TEA drops were added until the needed viscosity was approximately reached (40,000–60,000 mPa-s).¹⁸

Ultrapure sodium alginate (PRONOVA UP LVG, Novamatrix, FMC Biopolymers) was covalently grafted with the cell-adhesion peptide (glycine)4-arginine-glycine-aspartic acid-serine-proline (hereafter abbreviated as RGD) using aqueous carbodiimide chemistry as previously described61 (link). Briefly, alginate solutions (1 wt.%) in MES buffer (0.1 M MES, 0.3 M NaCl, pH 6.5) were prepared and stirred overnight (ON) at room temperature (RT). N-Hydroxy-sulfosuccinimide (Sulfo-NHS, Pierce) and 1-ethyl-(dimethylaminopropyl)-carbodiimide (EDC, Sigma, 27.4 mg per g of alginate) were sequentially added at a molar ratio of 1:2, followed by 100 mg of RGD peptide (Genscript) per g of alginate. After stirring for 20 h at RT, the reaction was quenched with hydroxylamine (Sigma) and the solution was dialyzed against deionized water for 3 days (MWCO 3500). After purification with charcoal, RGD-alginate was lyophilized and stored at −20 °C until further use. The reaction yield was calculated using the BCA Protein Assay (Pierce), as previously described in62 (link).

The ROCK inhibitors Y-27632 (Rho Kinase inhibitor VI) and H-1152 were obtained from EMD Millipore (Billerica, MA, USA); Q-VD-OPh from MP Biomedicals (Eschwege, Germany); and GT from AppliChem (Darmstadt, Germany). The CNFy toxin (150 ng/ml) from Yersinia pseudotuberculosis and the C2I/C3 fusion toxin (Clostridium botulinum and Clostridium Limosum, respectively) combined with the C2II toxin from Clostridium botulinum (collectively termed C3 toxin) was produced and purified as described^{33 (link),34 (link)}. C3 toxin is a binary toxin consisting of C2II (200 ng/ml) and C2I/C3 (100 ng/ml) mixed in cell culture media. The FAK inhibitor 14 (FAK14) was purchased from Tocris (Bristol, UK), and Cilengitide, a cyclic pentapeptide RGD compound (cyclo-[RGDfN(Me)V])^{46 (link)}, the RGD peptide, DTT and iodocetamide were from Sigma (Taufkirchen, Germany).