Padeasy

Adenovirus vectors ADV2 (U6/CMV-RFP) encoding Rattus HDAC2 and c-Myc-specific short hairpin RNA (shRNA) were obtained from Shanghai GenePharma Co., Ltd. The following sequences were included in the present study: Adenovirus sh-HDAC2 (Ad-shHDAC2), 5′-GGTATAGATGACGAGTCATAT-3′; Ad-shc-Myc, 5′-GAATTTCTATCACCAGCAACA-3′; and Ad-sh negative control (NC), 5′-ACTACCGTTGTTATAGGTG-3′. The cytolysate products were centrifuged in a table centrifuge at 1,006.2 × g for 15 min at room temperature to harvest the viral supernatant. The AdEasy™ system (Shanghai GenePharma Co., Ltd.) was used for adenoviral vector construction. The pAdEasy (Stratagene; Agilent Technologies, Inc.) was used to package the plasmid, and the ratio of the ADV shuttle vector and packaging plasmid was 3~4:1. Subsequently, production and cell transduction of the adenovirus in 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) was performed, and cells were flow-cytometrically sorted to maintain a GFP positivity rate >95%. Next, H9C2 cells cultured in a 25-cm² culture flask were incubated with Ad-shHDAC2, Ad-shc-Myc or Ad-shNC (the titer of the virus was 1×10⁹ PFU/ml) at a multiplicity of infection of 100. DMEM without FBS was used to dilute adenovirus. After incubation for 6 h, the medium was changed to serum-containing medium for 24–48 h, followed by treatments.