MAGE-A3 fragment was amplified with forward primer 5′-CTGCTCACCCAACATTTCGT-3′,

reverse primer 5′-CACTCTTCCCCCTCTCTCAA-3′. MAGE-A3/PolyA fragment was amplified with the forward primer and Selleck AZD8931 reverse primer of PolyA 5′-GTGGTTTGTCCAAACTCATCAA-3′. PCR conditions were: 95°C for 15 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min; and 4°C hold. Ten microliters of PCR product was analyzed on 2% agarose gels. SYBR® Premix Ex Taq™ (Perfect Real Time) was used for real-time PCR (qPCR) of CALR. The Light Cycler PCR system (Roche Diagnostics, Mannheim, Germany) was used for qPCR amplification and cycle threshold (Ct) detection. The thermal cycling conditions comprised an initial denaturation step at 95°C for 30 s, 40 cycles at 95°C for 5 s, and 61°C for 30 s. Primers were 5′-GCACTTGGATCCACCCAGAA-3′ and 5′-GAAGTTGTCAAAGATGGTGCCAGA-3′. The melting curves were analyzed after amplification. Each PCR reaction was done in triplicate. Dinaciclib ic50 Relative changes in expression were calculated using the 2-ΔΔCt method (Reference), where ΔCt is the difference in threshold cycles for the target gene and reference (ACTB), and ΔΔCt is the difference between the ΔCts of the treated sample and control or calibrator. Thus, the expression levels

were reported as fold changes relative to the calibrator. The value was used to plot the expression of apoptotic genes with the formula 2-ΔΔCt. Western blot analysis Four sub-group U87 cells were lysed in radioimmunoprecipitation (RIPA) buffer and total protein concentration was determined with a bicinchoninic

acid (BCA) assay (Beyotime, China). Twenty micrograms of total protein were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The membranes were washed, blocked, and incubated sequentially with specific primary antibodies, namely: rabbit monoclonal anti-CALR (1:1000), rabbit polyclonal anti-MAGE-A3 (1:100), both from Abcam (MA, USA); anti-PI3K (1:200), PLEKHB2 anti-Akt (1:200)/phosphorylated (p)-Akt (1:200), anti-Erk1/2 (1:200)/p-Erk1/2 (1:200) from Santa Cruz (CA, USA); mouse monoclonal anti-matrix metalloproteinases (MMP)2 (1:1000), rabbit monoclonal anti-MMP9 (1:10000) and rabbit polyclonal anti-β-actin (1:1000) from Abcam. Incubation in primary antibodies was followed by horseradish peroxidase -conjugated anti-rabbit secondary Epacadostat datasheet antibody (Zhongshan, 1:2000). The reactions were detected by enhanced chemiluminescence assay. Each experiment was performed in triplicate. Cell proliferation assay Cell proliferation was detected by methyl-thiazolyl-tetrazolium (MTT) assay. U87 cells were seeded in 96-well plates at a density of 1 × 104 cells/well. After 24 h, the cells were transfected with null, Ad-vector, Ad-CALR or Ad-CALR/MAGE-A3 and cultured for 1-7 d. Cell proliferation was determined by adding MTT (5 mg/mL) and incubating the cells at 37°C for 4 h further. The precipitate was solubilized by the addition of 150 μL/well dimethyl sulfoxide (Sigma) and shaken for 10 min.