7 cells. (A) expression of microRNas in eV shed fetal bovine serum, 2 glutamine, and 1 antibifrom DU145-DIaPh3 KD cells was measured by qRT-PcR analysis, in the presence or otics (Invitrogen) under a humidified atmosphere absence of RNase a or Triton X-100. (B) RaW264.7 cells were transfected with miR-125a or of 5 CO2 at 37 . The cells were subcultured at control miR (miR-ctrl). Western blot was performed to assess aKT1 or -actin expression. a density of 6 103 cells/cm2. For cell transfections (C) Overexpression of miR-125a, but not a control miRNa, in RaW264.7 cells reduced cell proliferation, as assessed by crystal violet analysis (student t test, *P 0.05). of synthetic miRNA oligonucleotides, RAW264.7 cells were grown to 80 confluence in 60-mm culture plates, at which time they were transiently transfected with the 150 pmol miRNAs (hsa-miR-125a and miR Measurement of EV using nanoparticle tracking analysis negative control, Thermo Scientific) using Lipofectamine 2000 Cells were washed twice with PBS, and then incubated in (Invitrogen) according to the manufacturer’s instructions. serum-free medium for an additional 24 h. Conditioned medium Immunofluorescence microscopic analysis was harvested, and cell debris were removed by centrifugation Cell membranes were labeled with FITC-conjugated CTxB at 300g for 15 min. Supernatants were applied to nanoparticle (green), and imaged using an Axioplan 2 microscope (Zeiss) as tracking analysis using the NanoSight system. previously described.23 In vitro gelatin degradation assay Isolation of EV from culture medium Shed EV were isolated from DU145 stimulated with SB203580 Shed EV were collected from conditioned medium and puri- (10 M) and HB-EGF (100 ng/ml), in the presence or absence of fied as previously described.23 Alternatively, where indicated, EV pretreatment of PD98059 (10 M), as described above. The gelashed from DU145 were collected from conditioned medium from tin degradation assay was performed as previously described.10 cells stimulated with SB203580 (10 M) and HB-EGF (100 ng/ In brief, EV were seeded onto FITC-labeled gelatin-coated covml) for 16 h, in the presence or absence of a 30 min pretreat- erslips, and incubated for 1 h at 37 . Trypsin and protease ment with PD98059 (10 M). Briefly, conditioned medium was inhibitor were used as positive and negative controls, respectively. subjected to two successive, rapid centrifugations at 300g and Western blotting analysis 800g to eliminate cells and debris, respectively. The resultCells were lysed in RIPA buffer (50 mM TRIS-HCl, pH 7.4, ing supernatants were subjected to ultracentrifugation for 2 h 150 mM NaCl, 5 mM EDTA, 1 NP-40, 0.4-Thiouridine 5 sodium deoxyat 100 000g to pellet the EV.Omburtamab Recovered material was resus- cholate) supplemented with 1 SDS, boiled at 95 with conpended in 100 l of ice-cold phosphate-buffered saline (PBS) per stant shaking for 5 min, frozen at -20 , homogenized through tube.PMID:24179643 Recipient cells (LNCaP, DU145, T24, or RAW264.7) were a 26G1/2 needle (BD Biosciences), frozen again at -80 , and serum-starved for 16 h and then treated with 20 g of EV for the upon thawing debris were removed by centrifugation. Equivaindicated times. lent protein amounts (25 g) were resolved by SDS-PAGE,www.landesbiosciencecancer Biology Therapy014 Landes Bioscience. Do not distribute.transferred to a nitrocellulose membrane, and specific antibodies used to determine protein expression. Imaging of DU145 morphology Control or DIAPH3-sil.
