Sc 38637

siRNA pools of gene-specific 19–25 nt sequences targeting YAP and MRTF-A designed to knock down mRNA were purchased from Santa Cruz Biotechnology (sc-38637 and sc-43944, respectively) along with a nontargeting 20–25-nt siRNA designed as a negative scramble control (sc-44236). Primary human NP cells, cultured no longer than passage 1, were tryspinized, counted, and centrifuged at 0.5 × 10⁶ cells per siRNA construct. Nucleofection was used to deliver the siRNA using the Amaxa Nucleofector II (Lonza, Basel, Switzerland) using a modified protocol from the manufacturer for primary human chondrocytes. Cells were resuspended in 100 ml of room-temperature Human Chondrocyte Nucleofector Solution (Lonza). Five micrograms of DNA was added and immediately transferred to a cuvette for nucleofection (program T-030). Following nucleofection, 500 ml of prewarmed 20% FBS containing F-12 medium was added and gently transferred to a prewarmed 6-well plate. Following 24 h incubation, medium was replaced with 10% FBS F-12 medium. All siRNA experiments were conducted 48 h postnucleofection.

A549 cells were transfected with siRNA using Lipofectamine 2000 reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. siRNAs specific for P53, YAP and SIRT1 were purchased from Santa Cruz (sc-29435, sc-38637, sc-40986, America). Control siRNA of P53, YAP and SIRT1 were purchased from Santa Cruz (sc-37007).

Small interfering RNA (siRNA) against the YAP gene (sc-38637; Santa Cruz Biotechnology Inc, Dallas, TX, USA) was used for loss-of-function experiments. The control siRNA sc-37007 (Santa Cruz Biotechnology) was used as a negative control. Each siRNA (37.5 nM) was transfected into ovarian cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The knockdown of a target gene was verified by Western blotting.