Zeb1 sirna

Mouse ESCs (D3) were cultured in mouse ESC media (LIF/serum), which comprise DMEM containing 15% FBS, 100 mM Minimum Essential Medium with Non-Essential Amino Acids (Life Technologies), and 1,000 µ/mL mouse leukemia inhibitory factor (mLIF) (Millipore, USA) with 50 mM 2-mercaptoethanol (Life Technologies). For the EB formation, mESCs were harvested by trypsinization and transferred to bacterial culture dishes in ES medium without LIF. Mouse embryonal carcinoma cell lines (P19) were cultured in DMEM with 1% l-glutamine (Life Technologies) supplemented with 10% FBS. Cells were cultured in a standard humidified incubator at 37°C in a 5% CO₂ atmosphere.
Zeb1 siRNA was purchased from GenePharma Bio, Inc. (Shanghai, China). The sequence is as this: 5′-GACCAGAACAGUGUUCCAUGUUUAA-3′. As a negative control, the siRNA sequence is 5′-UAUAGCUUAGUUCGUAACC-3′. The cells were incubated with Zeb1 siRNA/negative control siRNA using TurboFect™ siRNA Transfection Reagent (Life Technologies) according to the manufacturer’s protocol. When the optimal confluency for adherent cells is 70–90%, transfection is performed. The transfection reagent/siRNA mixture is added into the cells and incubated at 37°C. Cells were harvested 24–48 h post-transfection for assay.