Tured in RPMI 1640 medium with L-glutamine (Cellgro Mediatech, Inc. Herndon, VA) supplemented with 10 fetal bovine serum (FBS, Sigma, St. Louis, MO). The cells were maintained at 37uC in a humidified atmosphere of 95 air and 5 CO2.and resuspended in RPMI 1640 containing 0.4 agarose (Cambrex Bio Science, Rockland, ME) and 5 FBS. Using a 12-well plate, cells were 478-01-3 manufacturer overlaid onto a bottom layer of solidified 0.8 agarose in RPMI 1640 containing 10 FBS at a concentration of 26103 cells/well and incubated for 25033180 3 weeks. Colonies were stained with 0.005 crystal violet dissolved in 70 methanol before being photographed and quantified.VEGF ELISAThe supernatant from cultured cells was collected at the indicated time points and VEGF levels were measured using human VEGF ELISA kit (R D systems Inc. Minneapolis, MN) according to the manufacturer’s instructions. All experiments were performed on at least two separate occasions.Plasmids, siRNAs and transfectionsPlasmid pCDNA3.1 was obtained from Invitrogen (Carlsbad, CA), and pcDNA3-Myr HA-AKT2 (Addgene plasmid 9016) was purchased from Addgene (Cambridge, MA). Plasmid pLKO.1shAKT2 (human NM_001626) and its control vector SHC002 (shCON) were purchased from Sigma. siRNA pools targeting AKT1 (siAKT1), AKT2 (siAKT2), AKT3 (siAKT3), and nontargeting control siRNA (siNTC) were purchased from Dharmacon, Inc (Lafayette, CO). Human neuroblastoma cells were transfected with plasmids or siRNA using Lipofectamine 2000 (Invitrogen) as previously described [3,5]. BE(2)-C/shCON and BE(2)-C/shGRP-R cells were stably transfected with shRNA as described [13]. Stably-transfected BE(2)-C/shAKT2 cells were selected with puromycin (Sigma) at 2.5 mg/ml for one week. GRP and IGF-1 were purchased from Bachem (Torrance, CA). GRP (100 nM) and IGF-1 (100 nM) stimulations were performed following overnight serum starvation, and samples were collected at the indicated time points.Migration and invasion assaysFor transwell migration, transwell filters (8 mm; Corning, Lowell, MA) were coated on the lower chamber with 5 mg/ml collagen type I (BD Biosciences) overnight and then blocked with 2.5 BSA/PBS for 1 h. 16105 cells in serum-free media were added to the upper chamber and 10 FBS containing RPMI media into bottom well, and incubated for 6 h. Cells were fixed with 4 paraformaldehyde, stained with DAPI, and counted. For invasion assay, the transwell filters were coated with 1/30 diluted Matrigel (BD Biosciences). Cells (1.56105) in serum-free media were added to the upper well and 10 FBS containing RPMI media was added to the bottom well. After 48 h incubation, cells were fixed with 4 paraformaldehyde, stained with DAPI, and counted. Assay was performed in duplicate, and cells were counted from five randomly selected microscopic fields.Western blot and RT-PCR analysesPrimary antibodies against GRP-R (Abcam, Cambridge, MA), AKT1, AKT2, AKT3 and N-myc (Cell Signaling, Danvers, MA) were used. Horseradish peroxidase-conjugated secondary antibodies against mouse and rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). b-actin MedChemExpress ITI007 antibody was from Sigma-Aldrich. Western blot analysis was performed to evaluate the efficacy of transfection with plasmid, shRNA, and siRNA in cells, while using b-actin as a loading control. Total RNA was isolated using the RNAqueousTM kit (Ambion, Austin, Texas) according to the manufacturer’s instructions. Isolated RNA was used to synthesize cDNA using the High-Capacity cDNA.Tured in RPMI 1640 medium with L-glutamine (Cellgro Mediatech, Inc. Herndon, VA) supplemented with 10 fetal bovine serum (FBS, Sigma, St. Louis, MO). The cells were maintained at 37uC in a humidified atmosphere of 95 air and 5 CO2.and resuspended in RPMI 1640 containing 0.4 agarose (Cambrex Bio Science, Rockland, ME) and 5 FBS. Using a 12-well plate, cells were overlaid onto a bottom layer of solidified 0.8 agarose in RPMI 1640 containing 10 FBS at a concentration of 26103 cells/well and incubated for 25033180 3 weeks. Colonies were stained with 0.005 crystal violet dissolved in 70 methanol before being photographed and quantified.VEGF ELISAThe supernatant from cultured cells was collected at the indicated time points and VEGF levels were measured using human VEGF ELISA kit (R D systems Inc. Minneapolis, MN) according to the manufacturer’s instructions. All experiments were performed on at least two separate occasions.Plasmids, siRNAs and transfectionsPlasmid pCDNA3.1 was obtained from Invitrogen (Carlsbad, CA), and pcDNA3-Myr HA-AKT2 (Addgene plasmid 9016) was purchased from Addgene (Cambridge, MA). Plasmid pLKO.1shAKT2 (human NM_001626) and its control vector SHC002 (shCON) were purchased from Sigma. siRNA pools targeting AKT1 (siAKT1), AKT2 (siAKT2), AKT3 (siAKT3), and nontargeting control siRNA (siNTC) were purchased from Dharmacon, Inc (Lafayette, CO). Human neuroblastoma cells were transfected with plasmids or siRNA using Lipofectamine 2000 (Invitrogen) as previously described [3,5]. BE(2)-C/shCON and BE(2)-C/shGRP-R cells were stably transfected with shRNA as described [13]. Stably-transfected BE(2)-C/shAKT2 cells were selected with puromycin (Sigma) at 2.5 mg/ml for one week. GRP and IGF-1 were purchased from Bachem (Torrance, CA). GRP (100 nM) and IGF-1 (100 nM) stimulations were performed following overnight serum starvation, and samples were collected at the indicated time points.Migration and invasion assaysFor transwell migration, transwell filters (8 mm; Corning, Lowell, MA) were coated on the lower chamber with 5 mg/ml collagen type I (BD Biosciences) overnight and then blocked with 2.5 BSA/PBS for 1 h. 16105 cells in serum-free media were added to the upper chamber and 10 FBS containing RPMI media into bottom well, and incubated for 6 h. Cells were fixed with 4 paraformaldehyde, stained with DAPI, and counted. For invasion assay, the transwell filters were coated with 1/30 diluted Matrigel (BD Biosciences). Cells (1.56105) in serum-free media were added to the upper well and 10 FBS containing RPMI media was added to the bottom well. After 48 h incubation, cells were fixed with 4 paraformaldehyde, stained with DAPI, and counted. Assay was performed in duplicate, and cells were counted from five randomly selected microscopic fields.Western blot and RT-PCR analysesPrimary antibodies against GRP-R (Abcam, Cambridge, MA), AKT1, AKT2, AKT3 and N-myc (Cell Signaling, Danvers, MA) were used. Horseradish peroxidase-conjugated secondary antibodies against mouse and rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). b-actin antibody was from Sigma-Aldrich. Western blot analysis was performed to evaluate the efficacy of transfection with plasmid, shRNA, and siRNA in cells, while using b-actin as a loading control. Total RNA was isolated using the RNAqueousTM kit (Ambion, Austin, Texas) according to the manufacturer’s instructions. Isolated RNA was used to synthesize cDNA using the High-Capacity cDNA.
