Plko lentiviral vectors

siRNA against WASF3 and the pLKO lentiviral vectors containing an shRNA against SHOX2 or STAT3 were purchased from Open Biosystems (Huntsville, AL). Coding sequences of human HA-tagged WASF3 and FLAG-tagged SHOX2 were respectively cloned into pCDH-CMV-MCS-EF1 lentivector (System Biosciences, Mountain View, CA) as described previously [8 (link), 13 (link)]. The pGL-WASF3 promoter constructs (-350/+494, -650/+494, -1250/+494) were generated as described previously [16 (link)]. The following primary antibodies were used in Western blot assays: anti-WASF3, anti-STAT3, anti-pSTAT3 (Tyr 705), and anti-E-cadherin antibodies (Cell Signaling Technology, MA); anti-HA, anti-FLAG, and anti-β-Actin antibodies (Sigma-Aldrich, MO); and anti-SHOX2 antibody (Abcam, MA). The STAT3 inhibitor S3I-201 was obtained from Selleckchem (Houston, TX).

The pLKO lentiviral vectors to knockdown CUL1 (#1: sense, 5′- GATTTGATGGATGAGAGTGTA-3′; #2: sense, 5′-GCCAGCATGATCTCCAAGTTA-3′) were obtained from the Dana Farber/Harvard Cancer Center DNA Resource Core. The shRNA sequences of pLKO vectors to deplete FBW7 are: #1: sense, 5′-AACCTTCTCTGGAGAGAGAAA-3′; #2: sense, 5′- CCAGAGACTGAAACCTGTCTA-3′. The pLKO shCK1 and shGFP constructs were obtained from Dr. J Wade Harper (Harvard Medical School, MA). The pLKO-lentiviral vectors to deplete endogenous BRG1 (#1: TRCN0000015551, #2: TRCN0000015552) were from OpenBiosystems. The corresponding lentiviruses were packaged and generated by transfecting each pLKO lentiviral plasmid with pCMV-VSV-G and pCMV-dR8.9 into 293T cells using a polyethyenimine (PEI) transfection protocol.

Downregulation of Atg genes were performed with pLKO lentiviral vectors (Open Biosystems/Dharmacon, CO, USA) expressing rat-specific shRNA sequences from TRC (the RNAi consortium) library as described previously^{62 (link)}. A combination of TRCN0000092163 and TRCN0000092166 for Atg7 (GenBankTM NM_001012097), TRCN0000033552 for Becn1 (GenBankTM NM_053739.2) and a pLKO vector containing scrambled shRNA (Open Biosystems/Dharmacon) as control vector were used. Primary cortical neuron cultures were infected at DIV7 with 50 ng of the viral capsid protein p24/ml culture medium for each vector.