Basic fibroblast growth factor (bfgf)

BC MCF7 cells were fostered in high-glucose DMEM (Abcam, Shanghai, China) added with 100 U/mL penicillin, 100 mg/L streptomycin, and 10% FBS (Abcam, Shanghai, China) under 5% CO₂, floating in serum-free DMEM/F12 culture medium (Abcam) with 20 ng/mL EGF, 20 ng/mL b-FGF, and 2% B27 (Abcam). Then, we spent 10 days in culturing them within ultra-low attachment plates. miR-30d-5p mimics were synthesized within the GenePharma company (Shanghai, China). Additionally, they were transfected into cells using Lipofectamine 2000 reagent in Invitrogen (USA) in light of the methods of Liu, et al. [16 (link)].

Isolation and culturing of adherent GBM-derived MSCs (GB-MSCs) were described in detail previously [15 (link)]. In the present experiments, the cells were cultured in DMEM/F-12 medium (PAN-Biotech, Germany), containing 20 ng/ml bFGF (Abcam, UK), 10% (v/v) fetal bovine serum (FBS) (PAA Laboratories), and antibiotic/antimycotic 100 IU/l (PAA Laboratories, Austria). After 10-14 days, they formed monolayer-consisting fibroblast-like cells.

Human cancer cell lines A549 and HeLa cells were purchased from RIKEN CELL BANK and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Wako, Japan) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS; HyClone, USA) and were maintained at 37 ℃ in an atmosphere of 5% CO₂-95% air. Rho 0 A549 (ρ⁰ A549) and Rho 0 HeLa (ρ⁰ HeLa) cells were established as previously described by culturing in the presence of ethidium bromide[26 (link)] (EtBr; Sigma, USA) (50 ng/mL), 1 × sodium pyruvate solution (Wako, Japan) (110 μg/mL), and uridine (Sigma, USA) (50 μg/mL) in RPMI 1640 supplemented with 10% FBS for more than 20 passages. Control parental A549 and HeLa cells were maintained in standard culture medium for the same period. We prepared three clones of BMSCs, MSC 1, 2, and 3 using BMSCs from Lonza (Basel, Switzerland). Human BMSCs were isolated as described previously [24 (link)] and cultured in DMEM/Ham’s F-12 medium (Wako, Japan) containing 15% FBS, 1% penicillin–streptomycin, and 10 ng/mL basic fibroblast growth factor (bFGF; Abcam; USA) (in 5% CO₂, at 37 °C) until 80% confluent.

Platelets from 3 mice were pooled and activated as indicated above. Platelets were fixed for 15 minutes by adding 1 vol of 4% formaldehyde, placed onto poly-L-lysine-coated coverslips and allowed to settle for 50 minutes. Attached platelets were permeabilized with 0.5% Triton X100 and blocked overnight in PBS with 1% BSA. Incubation with primary antibodies: VEGF (Abcam, ab1316), bFGF (Abcam ab8880), endostatin (Thermo Scientific, PA1-601), TSP-1 (Thermo Scientific MA5-13398), C3G (#1008 [33 (link)]), and VAMP-7 (anti-SYBL1, Abcam, ab36195), was performed at RT for 2h, followed by incubation (1 h at RT) with secondary antibodies: Cy3-conjugated Goat anti-rabbit (Abcam) for bFGF, endostatin and C3G; Cy5-conjugated Goat anti-mouse (Jackson Immunoresearch) for VEGF, TSP-1 and VAMP-7.
Immunofluorescence was quantified with ImageJ software. Colocalization was determined by Manders coefficient (M) analysis, using ImageJ with the JACoP plugin, as described [23 (link), 52 (link)].

The total protein concentrations in cells and sEVs were determined using the BCA Protein Assay kit. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with the appropriate primary and secondary antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific) and quantified using ImageJ software. Primary antibodies against CD63, CD81, TSG101, Calnexin, OCN, OPN, Runx2, VEGF, bFGF, ANG-1, and CD31 were obtained from Abcam (Cambridge, United Kingdom).

AMSCs treated with or without hypoxia were lysed in Laemmli Sample Buffer (Bio-Rad). Proteins were collected by centrifugation and concentrations were determined by BCA Protein Assay Kit (Thermo Scientific). Proteins were loaded on sodium dodecyl sulfate polyacrylamide gel for electrophoresis. After proteins were transferred to nitrocellulose membranes; primary antibodies against VEGF (abcam), bFGF (abcam), HIF-1α (Cell signaling technology) or BNDF (abcam) were incubated over night at 4°C. Then, corresponding secondary antibodies were incubated for 1 h at room temperature. GAPDH was used as internal standard.