Chemicals and Reagents
HUVECs and the endothelial cell medium were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Recombinant MG53 was obtained from Novoprotein (Shanghai, China). Growth-factor-reduced Matrigel Matrix was purchased from BD Biosciences (San Jose, CA, USA). Anti-MG53 antibody used for western blotting was from Abcam (Cambridge, MA, USA) and anti-MG53 antibody used for immunostaining was from Atlas Antibodies (Stockholm, Sweden). Antibodies to phosphorylated FAKY397, FAK, phosphorylated SrcY416, Src, phosphorylated AktT308, Akt, phosphorylated ERK1/2 and ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA, USA). MβCD was from Absin Bioscience (Shanghai, China). Endothelial marker IB4 was from Invitrogen (Austin, TX, USA). Pierce classic IP kit was from Thermo Scientific (Rockford, IL, USA).
HUVECs were cultured in endothelial cell medium with 5% (v/v) fetal bovine serum (FBS), 1% (v/v) endothelia cell growth supplement and 1% (v/v) antibiotic solution. Cells used were passaged 4-10 times.
Cell scratch wound healing assay
HUVECs, cultured in 24-well plates, were allowed to grown to 90% confluence and scratched with a 200 μL pipette tip, followed by stimulation with rhMG53 (0, 5, 10 and 20 μg/mL) for 24 h. Photomicrographs were taken immediately after the scratch and after rhMG53 treatment. Image J software was used to measure the change in scratch area over time.
Cell migration assay
HUVEC migration was also performed using transwell chambers with an 8.0 μm-sized porous membrane. HUVECs (2×104/well) were added to the upper chambers and treated with rhMG53 (0, 5, 10 and 20 μg/mL). As a chemoattractant, the endothelial cell medium containing 2% FBS was added to the lower chambers. After 24 h, cells remaining in the upper chambers were removed and the cells that have migrated to the lower chambers were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, photographed with a microscope and counted.
Tube formation assay
HUVECs (1×105/well) were seeded onto 24-well plates, which were pre-coated with growth-factor-reduced Matrigel matrix (250 μL/per well), and exposed to 1% FBS growth medium containing rhMG53 (0, 5, 10 and 20 μg/mL) for 18 h. Then, tube formation was photographed and the total tube length in 5 fields was quantified using Image J software.
HUVECs were lysed in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with 1% protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were separated using SDS-PAGE and transferred to polyvinylidene fluoride membrane. Amongst the different experiments, the membranes were blocked and incubated with primary antibodies for MG53 (1:1000), phosphorylated FAKY397 (1:1000), phosphorylated SrcY416 (1:1000), phosphorylated AktT308 (1:1000), phosphorylated ERK1/2 (1:1000), total FAK (1:1000), total Src (1:1000), total Akt (1:1000) and total ERK1/2 (1:1000) overnight at 4 °C. Then, the membranes were exposed to HRP-conjugated species-specific respective goat IgG (Cell signaling technology) for 60 minutes at room temperature. The protein bands were visualized with chemiluminescence, and Image J software was used to measure the density of the bands.
In order to investigate the time course of rhMG53 uptake, HUVECs were stimulated with rhMG53 (10 μg/ml) for 0, 15, 30 and 120 min. In order to determine the effects of MβCD on rhMG53 uptake, HUVECs were pre-treated with MβCD (5 mM) for 1 h, followed by stimulation with rhMG53 (10 μg/ml) for 3 h. Then the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 for 15 min at 4 °C, blocked with 5% bovine serum albumin at 37 °C and incubated with a primary anti-MG53 antibody (1:50) at 4 °C overnight. After washing with PBS, the cells were incubated with rhodamine-conjugated secondary antibody (1:100) against the primary antibody applied and the nuclei was stained with 4’,6-diamidino-2-phenylindole (DAPI).
HUVECs were stimulated with rhMG53 (10 μg/ml) for 24 h and the protein samples were prepared. Classic IP kit was used to perform protein immunoprecipitation. In brief, equal amounts of protein were incubated with protein A/G plus agarose beads and anti-FAK antibody or IgG overnight at 4 °C, with gentle rotation. According to the IP kit instruction, the immune complexes were captured and solubilized by 50 μL SDS sample buffer. Then, the immune complexes were subjected to SDS-PAGE and immunoblotted with primary antibodies against MG53 and Src.
Postnatal retinal angiogenesis model was used in this study. All animal care and experimental procedures were approved by the Animal Care and Use Committee of Southern Medical University. Postnatal C57BL/6J mice were injected intraperitoneally with vehicle control or rhMG53 at 6 mg/kg body weight from postnatal day 5 (P5) to P12.
Retina whole-mount immunostaining
Mouse retina whole-mount immunostaining was performed as previously described . In brief, the eyes were harvested and immediately fixed with 4% paraformaldehyde for 2 h at room temperature. Then the eyes were washed 3 times with ice-cold PBS and the retinas were dissected and cut into four radial incisions under a stereomicroscope. After being fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 5% bovine serum albumin, the retinas were washed and incubated with endothelial marker IB4 (1:100) at 4 °C overnight. Following thoroughly washing, the retinas were transferred to the glass slide, flat-mounted and sealed under a glass coverslip in mounting media.
One-way ANOVA followed by post hoc comparison was performed to analyze the results and all of the data are shown as mean ± standard deviation (SD). A p value of <0.05 was considered as statistically significant.