Fig 1: Binding of [125I] – MN20054 to Fibroblasts.HUVEC and WI-38 fibroblasts (CCL-75™; ATCC) were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [125I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, or a 100-fold molar excess of MN10021. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.
Fig 2: 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).
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