Fig 1: snoMEN knock-down delivered using a lentiviral vector.(a) Structure of lentiviral vector encoding snoMEN targeted to the endogenous miR21 primary transcript (Lenti-mCherry–pri-miR21 snoMEN). This construct has three snoMEN RNAs (blue pentagons) and an mCherry FP-marker cDNA as described in Fig 1A, except that the insert was sub-cloned into a vector that would produce the lentivirus particle (pLVX-puro, Clontech). (b) Micrographs show apoptosis induction by transducing with either Lenti-mCherry–pri-miR21-snoMEN, or Lenti-mCherry–Control-snoMEN virus particles. Images were taken 72 hours after transduction. Scale bar is 10 μm. (c) Total RNA from human lung primary (ATCC-CCL-75) and lung Cancer (ATCC-CRL-5868) cells was harvested 24 hours after transduction. Following cDNA synthesis, qPCR was performed using both a matured miR21 specific primer and a universal primer provided by the PerfeCta SYBR Green qPCR kit (Quanta Biosciences, see also methods). GAPDH was used as a control. Graph depicts mean and standard deviation from a minimum of 4 independent experiments. (d) Graph shows a time course of changes in the population of Annexin-V positive cells transduced with either lenti-pri-miR21-snoMEN (Green), lenti-pre-miR21-snoMEN (Blue) or Control (Red). The cell number was counted using FACS. The Annexin-V signal was detected using Guava Nexin Reagent (Guava technologies).