After the adhesion of the cells, they were infected with Ad-bFGF-siRNA, meanwhile untreated cells and cells infected with Ad-GFP served as control and mock control. During consecutive selleck chemical seven days, 20 μl MTT solution (5 mg/ml) in PBS was added to each well for 4 h. After the culture medium was

drained out, 150 μl of DMSO was added into each well. Absorbance of each well was measured on a microplate reader. Three duplicate wells were set up for each group. RT-PCR Total RNA was extracted from cultured cells using TRizol reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s directions. First-strand cDNA was synthesized from total RNA(1 μg) using AMV reverse transcriptase (TaKaRa) with oligo(dT) primer at 42°C for 1 h in a 25 μl volume. RT product Lazertinib clinical trial (2 μl) with cDNAs was mixed with bFGF or β-actin specific primers in a PCR buffer containing 2.5 mM dNTP, 2.5 mM MgCl2 and 1 U Taq polymerase (TaKaRa). PCR amplification was performed over 31 cycles (45 sec at 94°C, 60 sec at 60°C, and 45 sec at 72°C) to amplify bFGF, and over 25 cycles (30 sec at 94°C, 30 sec at 57°C, and 90 sec at 72°C) to amplify β-actin. Primers used for amplifying bFGF included: forward-5′-CACCATGGCAGCCGGCAGCATCA-3′ and reverse-5′-TCAGCTCTTAGCAGACATTGG-3′. Primers used to amplify β-actin included: forward-5′-CCTCGCCTTTGCCGATC-3′ and reverse-5′-GGATCTTCATGAGGTAGTCAGTC-3′.

Amplified DNA fragments were separated in 2% agarose gels and visualized using ethidium bromide staining. Western blotting Western blot analysis was performed on whole cell extracts obtained by direct dissolution of cells in culture flasks using a whole cell protein extract reagent according to the manufacturer’s Rigosertib nmr directions (PIERCE). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit with bovine serum albumin

as a standard. Proteins (40 μg/lane) were separated on 12% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 3% fat-free milk in PBST (0.2% Tween-20 in PBS, pH 7.6) then incubated with primary antibody for 18-24 h at 4°C. Membranes were subsequently incubated with secondary antibodies conjugated to horseradish peroxidase (1:5000) for 1 h at RT. Bound antibody was visualized however using an Enhanced Chemiluminescence (ECL) western blot detection kit (Amersham Pharmacia Biotech). Primary antibodies used included: anti-bFGF (rabbit polyclonal, 1:1000, Santa Cruz), anti-Cx43 (rabbit polyclonal, 1:1000, Cell Signaling), anti-pCx43 for S368 (rabbit polyclonal, 1:1000, Cell Signaling), and anti-β-actin (mouse monoclonal, 1:1000, Santa Cruz). Immunofluorescence U251 cells grown on cover slips were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100/PBS (Sigma-Aldrich) for 20 min.