Sirna duplex oligos

pENTER-HDAC2 and YY1 or p300 and empty vector (pENTER) plasmids encoding a FLAG or HA tag were purchased from Vigene (Rockville, MD, USA). Ablation of HDAC2, YY1 or p300 was performed by transfection with siRNA duplex oligos, which were synthesised by Genepharma Company (Shanghai, China). The sequences of the siRNAs were as follows: (sense strand) Scrambled siRNA, HDAC2 siRNA: 5’-AAGCATCAGGATTCTGTTA-3ʹ, YY1 siRNA: 5ʹ-CGAGGATCAGATTCTCATC-3ʹ^{41 (link)}, and p300 siRNA: 5ʹ-AACCCCUCCUCUUCAGCACCA-3ʹ. Cell transfection was performed with Lipofectamine TM 2000 (Invitrogen) as described in the manufacturer’s protocol.

Ablation of FOXK1 was performed by transfection with small interfering RNA (siRNA) duplex oligos, which was synthesized by Genepharma Company (Shanghai, China). Control siRNA (Scrambled RNA, Shanghai, China) and FOXK1-specific siRNA 1 (sense: 586- CCAUCAAGAUCCAGUUCAC (dTdT) -605, antisense: 605- GUGAACUGGAUCUUGAUGGdTdT -586 and FOXK1-specific siRNA 2 (sense: 889- GAGACAGCCCCAAGGAUGA (dTdT) -908, antisense: 908- UCAUCCUUGGGGCUGUCUC (dTdT) -889 were transfected using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection and Western blot analysis were performed.

Small interfering RNA (siRNA) duplex oligos targeting TAZ and LATS1 mRNA (siTAZ and siLATS1) and non-targeting duplex oligos, which served as a negative control (siCON), were synthesized by Shanghai Genepharma Corporation, China. After seeding for 24 h, cells were transfected with 2 μg/ml of siRNA using RNAi-Mate (Shanghai Genepharma Corporation, China) as recommended by the manufacturers’ instructions and cultured in osteogenic medium. Given that the calcium depositions of BMSCs lasted for 14 days, cells were subjected to two rounds of transfection prior to performing the experiments. The siRNA sequences are listed below:
siTAZ: 5′-GGCCAGAGAUAUUUCCUUATT-3′, 5′-UAAGGAAAUAUCUCUGGCCTT-3′;
siLATS1: 5′-GUCGAUACGAAAGCUUUGUTT-3′, 5′-ACAAAGCUUUCGUAUCGACTT-3′;
siCON: 5′-UUCUCCGAACGUGUCACGTT-3′, 5′-ACGUGACACGUUCGGAGATT-3′.