Plko 1 lentiviral shrna

Small interfering RNA (siRNA) with pre-designed sequences targeting human NFATc1, PPP3R1, ALDH1A1 and scramble siRNA were from Sigma-Aldrich (St Louis, MO). pGL3-NFAT luciferase (17870), two shRNA sequences targeting SOX2, pLKO.1 Sox2 3HM a (26353) and pLKO.1 Sox2 3 hr b (26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were purchased from Addgene (Cambrige, MA; http://www.addgene.org). The pLKO.1-lentiviral shRNA with different inserts specifically targeting NFATc2 were purchased from Sigma-Aldrich (TRCN0000016144, TRCN0000230218). Human full length NFATc2 were amplified by PCR, and the RFP-NFTAc2 plasmids were generated by cloning the sequences into PCDH-CMV-MCS-EF1-COPRFP vector (SBI, Mountain View, CA). For luciferase reporter construction, SOX2 regulatory regions were amplified by PCR from human genomic DNA and cloned into pGL3 (Promega) to generate the SOX2-luc constructs. Primers used for genomic DNA amplification were listed in Supplementary file 1A. Site directed mutagenesis of the consensus NFAT binding site (GGAAA to GACTA) were performed using QuikChange (Stratagene).