Oligofectamine reagent

Cyclin A1 and luciferase control siRNAs were produced by in vitro transcription using Silencer siRNA construction kit (Thermo Fisher Scientific, MA, USA). The siRNA sequences were designed using the computational algorithm available at the vendor’s website. The cyclin A1-specific siRNA sequences are 5′-AACCAGAATAACACCTGATTCCCTGTCTC-3′ and 5′-AAGAATCAGGTGTTATTCTGGCCTGTCTC-3′. The luciferase-control siRNA sequences are 5′-AATCGAAGTATTCCGCGTACGCCTGTCTC-3′ and 5′-AACGTACGCGGAATACTTCGACCTGTCTC-3′. siRNAs were synthesized and purified according to the manufacturer’s manual. For cyclin A1 knockdown experiment, cells were seeded in six-well plates (2 × 10⁵ cells/well) and were transfected on the following day with 12 nM siRNA using Oligofectamine reagent (Qiagen Inc, CA, USA). The concentration of siRNA used was based on the recommendation from the manual of Silencer siRNA construction kit. Cyclin A1 expression was induced by tetracycline (1μg/ml) addition, whereas equal volume of the solvent ethanol was added to the control. The siRNA containing media were replaced with normal growth media 24 hr after transfection and the cells were harvested 24 hrs after medium replacement.