ShRNAs synthesis and plasmids construction Single shRNA strands were 5′-GATCCCC-N21-TTCAAGAGA-N’21-TTTTTGGA-AA-3′ (sense) and 5′-AGCTTTTCCAAAAA-N21-TCTCTTGAAN’21-GGG-3′ (antisense). N21 was the sense sequence of MACC1 target oligonucleotides, N’21 was LY2603618 chemical structure antisense sequence of MACC1 target oligonucleotides. MK-0457 Three different template oligonucleotides targeting MACC1 [GeneBank, NM_182762.3] were as follow: MACC1-s1, 5′-AAAGACAGAAGGAGAAAGGAA-3′; MACC1-s2, 5′-AATCAAC- TGTCTGCTTCTAAC-3′; MACC1-s3, 5′-AATTATATGCCAGGACAGCTT-3′.

As a negative control, one scrambled sequence 5′-AACAGTTATCTATGCGACAGT-3′ (corresponding to MACC1-s3) was designed. These sequences were submitted to BLAST against human genome sequence to ensure that only MACC1 gene was targeted. All single shRNA strands were synthesized at Sangon Biotechnology Co., Ltd (Shanghai, China), and were

annealed and ligated into the BglII and HindIII sites of linearized psuper-EGFP plasmid. The four shRNAs INCB28060 inserted vectors were named as psuper-EGFP-s1, psuper-EGFP-s2, psuper-EGFP-s3, and psuper-EGFP-NC respectively. Cell transfection Human ovarian carcinoma OVCAR-3 cells (with high level of MACC1 expression measured in our preliminary study) were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China), and cultured in DMEM medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C with 5% CO2. Cells Thymidylate synthase were harvested in logarithmic phase of growth for all experiments described below. Cell transfection was performed following the protocol of Lipofectamine 2000 (Invitrogen, USA). The untransfected cells, empty vector (psuper-EGFP-neo) transfected cells, and nonspecific shRNA (psuper-EGFP-NC) transfected

cells were used as controls. Stably transfected OVCAR-3 cells were selected with 800 μg/ml G418 (Sigma, USA) after tansfection 48 h. After 12 days, resistant colonies were trypsinized and cultured in selective medium. Names of the stably transfected cells were OVCAR-3-neo, OVCAR-3-NC, OVCAR-3-s1, OVCAR-3-s2, and OVCAR-3-s3 respectively. RT-PCR Cell total RNA was isolated using Trizol Reagent (Invitrogen, USA), and first strand cDNA was synthesized from 1 μg total RNA according to the protocol of RevertAid first strand cDNA synthesis kit (Fermentas, EU). Primers used in RT-PCR were as follow: MACC1, 5′-CCTTCGTGGTAATAATGCTTCC-3′ (sense) and 5′-AGGGCTTCCATTGTATTGAGGT-3′ (antisense); β-actin, 5′-ACGCACC- CCAACTACAACTC-3′ (sense) and 5′-TCTCCTTAATGTCACGCACGA-3′ (antisense). PCR cycling parameters (19 cycles) were: denaturation (94°C, 30s), annealing (56°C, 30s) and extension (72°C, 30s). Equal amounts of PCR products were electrophoresed on 1.2% agarose gels and visualized by ethidium bromide staining. The specific bands of PCR products were analyzed by Image-Pro Plus 6.0 system, β-actin was used as a control for normalization.