Histone h2b gfp lv gfp

LOXL3 shRNAs were obtained from the MISSION TRC shRNA human library (Sigma-Aldrich) in pLKO.1-puro vector as well as MISSION pLKO.1-puro non-mammalian shRNA (non-targeting, NTC) control plasmid (SHC002 SIGMA). shRNA scramble control (Crtl) and indicated LOXL3 shRNAs were subsequently cloned into Histone H2B-mRFP1 LV-RFP (Addgene 26001) or Histone H2B-GFP LV-GFP (Addgene 25999), respectively. LOXL3 and LOXL3Δ (lacking exons 4 and 5) FLAG-tagged cDNAs were subcloned into pWPI lentiviral vector (Addgene 12254) that allows simultaneous expression of the transgene and EGFP. Lentiviruses were packaged in HEK293T cells co-transfecting the lentiviral vector, psPAX2 (Addgene 12260) and pMD2.G (Addgene 12259) with Lipofectamine2000 DNA Transfection Reagent (Invitrogen). Melanoma cells were transduced with viral supernatant supplemented with polybrene (2 μg/mL) and either selected with 1 μg/ml puromycin for 2 days (pLKO based lentiviruses) or sorted for EGFP positive cells using a BD FACSVantage SE cell sorter (pWPI based lentiviruses). For LOXL3 transient silencing, 100 nM of LOXL3 ON-TARGETplus SMARTpool (siSMART-L3) or control scramble siRNA (Dharmacon) were transfected in melanoma cells with Lipofectamine2000 (Invitrogen).