Pol 2 promoter

A 10 mM stock solution of vorinostat (Cayman Chemical Company, Ann Arbor, MI) was prepared in dimethylsulfoxide (DMSO) and diluted with MEM to achieve a final concentration of 2.5 µM in the culture medium. The equivalent amount of DMSO (0.5 µl in 2 ml culture medium) served as controls. For the knockdown of TGIF1 and TGIF2 gene expression, prevalidated siRNA (Ambion Life technologies, Grand Island, NY) were used at a 30 nM concentration (Table 1). For the knockdown of Smad2/3/4, the prevalidated RNA interference (RNAi) oiligos (Table 1) were cloned into a pcDNA 6.2 miR RNAi expression vector under the control of Pol II promoter (Life Technologies, Grand Island, NY). The siRNA and RNAi plasmid transfections were performed using the commercially available transfection reagent lipofectamine3000 (Life technologies corporation, Grand Island, NY) as per the manufacturer’s instructions. Briefly, a transfection solution was prepared by adding 10 µl lipofectamine3000 in 200 µl of optiMEM medium to the appropriate amounts of siRNA/RNAi plasmid in 200 µl of opitiMEM. The mixture was incubated for 5 min at room temperature. The above transfection mixture was added to the human corneal fibroblasts in 1 ml of a serum-free culture medium.