Shmed12

To stably knock down endogenous MED12 expression, cells including A549, H1299, H1975 and GLC-82 were grown to 30% confluency in 6-well culture plates and then infected with lentivirus carrying an shRNA targeting MED12 (ShMED12) or a negative control vector (ShCtrl) (Sigma). After 48 h of infection at 37 °C, the medium was replaced with fresh medium and incubated further for 72 h before analysis using RT-qPCR and western blotting for MED12 expression. The shRNA sequences used were 5′-GGTACTTCATACTTTGGAA -3′ (ShMED12^1#) and 5′-AGAGAAATTACGTTGTAAT -3′ (ShMED12^2#).

Human breast cancer cell lines T47D and MCF-7 were obtained from the ATCC and cell authentication was conducted at the ATCC using short tandem repeat DNA profiling. MISSION shRNA plasmids, shMEN1 (TRCN0000040141), shKMT2A (TRCN0000005956), shMED12 (TRCN0000018578), shWAPL (TRCN0000167548), and shGATA3 (TRCN0000019301), were obtained from Sigma-Aldrich (sequences listed in Table S2). Vehicle plasmid pLKO.1-puro (Addgene plasmid #8453) was obtained from Addgene. Plasmids were purified by QIAprep Spin Miniprep kit (Qiagen), and 293T cells were cotransfected with 3^rd Generation Packaging Mix (Applied Biological Materials, Richmond, BC, Canada) combined with either the shRNA plasmid or the vehicle plasmid for lentivirus production. The lentivirus-containing medium was harvested after 48 h of transfection, and cells were infected with lentivirus for 24 h. The infected cells were selected using puromycin at 4 μg/mL for 1 week. MI-503 (Selleckchem, Huston, TX, USA) was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock and diluted to the working concentrations in the medium.

shRNA transfer vectors were purchased from Sigma (Sigma; catalog numbers: shControl- SHC002, shMed12- TRCN0000096467, shCdk8- TRCN0000023107, shCyclinC- TRCN0000077830, shMed1- TRCN0000099578, shLamc1- TRCN0000055420, shSdc2- TRCN0000311814, Itgb5- TRCN0000067123). Each lentiviruses were produced by transient cotransfection of HEK293T cells with shRNA transfer vectors with pMD2G and psPAX2 (AddGene) using X-tremeGENE9 (Roche Applied Science). For lentivirus infection, mNS-5 cells were seeded in 60 mm tissue culture plate at a density of 1 × 10⁶ cells per plate. Twenty-four hours later, cells were infected with lentiviruses in the presence of 8 μg/ml of polybrene (Millipore). Twenty-four hours after lentivirus infection, the medium was removed and replaced with fresh medium containing 600 ng/ml puromycin (Gibco) for selection.