C myc sirna

For gene knockdown experiments, c-Myc siRNA and non-targeting scrambled siRNA (NS) obtained from Santa Cruz Biotech were transfected into RPMI 8226 cells using PepMute™ siRNA transfection reagent (SignaGen Laboratories). For gene overexpression experiments, cells were transfected with pcDNA3.1-c-Myc, Flag-SUMO1 or HA-Ubiquitin plasmids (Addgene) using GenJet™ in vitro DNA transfection reagent (SignaGen Laboratories).

For KRT13 overexpression, a cDNA containing the full-length open reading frame of human KRT13 (NM_153490) was subcloned into pLVX-AcGFP1-N1 (pLV) (Clontech, CA) by introducing EcoR I and BamH I sites. The construct of pLVX-AcGFP1-N1-KRT13 (pLV-KRT13) was confirmed by DNA sequencing. The plasmid DNA was transfected to the 293 T cells to produce lentiviral particles, following the manufacturer’s instructions (System Biosciences, Mountain View, CA). MCF7 cells were transduced with the lentivirus and selected with puromycin (2 µg/ml for 2 weeks). For shRNA-mediated knockdown, non-targeting control or KRT13 shRNA lentiviral particles (Santa Cruz Biotechnology, Dallas, Texas) were used to infect the HCC1954 cells. The plasmid pcDNA3-c-Myc (Addgene, Cambridge, MA) was used to generate c-Myc-overexpressing cells. To silence c-Myc, cells were transfected with c-Myc siRNA (Santa Cruz Biotechnology) using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA).

PDGFRα siRNA duplex (5′-GGAGGAUG AUGAUUCUGCCAUUAUA-3′ ), PDGFRβ siRNA duplex (5′-UCACGGAAAUAACUGAGAUCACCAU-3′ ), and control (mock) siRNA duplex (5′-ACAUCACGUACGCG GAAUACUUCGA-3′) were obtained from Invitrogen. JAG2 siRNA, a pool of 4 siRNA duplexes (5′-GCAAGGAAGCUGUGUGUAA-3′, 5′-GCGUGUGC CUUAAGGAGUA-3′, 5′-GAACGGCGCUCGCUGC UAU-3′, 5′-GGUCGUACUUGCACUCACA-3′ ) were purchased from Dharmacon. c-MYC siRNA is a pool of 3 different siRNA duplexes (5′-CCCAAGGUAGUUAUCCUUAtt-3′, 5′-GGAAACGAC GAGAACAGUUtt-3′, and 5′-CCUGAGCAAUCA CCUAUGAtt-3′ ) were purchased from Santa Cruz. Transfection of siRNA was performed using Lipofectamine™ (Invitrogen) according to the company's instructions.