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    r> In this study we investigated the role and the potential of targeting ABC transporters in PDAC therapy. We showe that a member of the ABCC family, ABCC3, is highly expressed in PDAC tumours and that its expression is dependent on mutation of TP53. We also show that ABCC3 is required for LPI-mediated PDAC progression via STAT3 and HIF1α signalling pathways, which have previously been shown to be involved in PDAC onset and progression (Corcoran et al., 2011; Hoffmann et al., 2008).
    2. Materials and methods
    2.1. Cell lines and plasmids
    2.2. RNA interference
    The siABCC3-3 sequence was used to generate pSuper retro–based vectors that express short hairpin RNA (shRNA). Control vector pSuper 4Mut contains a four-point mutated sequence unable to target the human ABCC3. Retroviral stocks were generated as pre-viously described (Sala et al., 2012) and infected CFPAC-1 SB 203580 were selected with 1 μg/ml of puromycin. Knockdown efficiency was determined by Western blotting. For proliferation analysis, stably transfected CFPAC-1 cells (shABCC3 and 4Mut) were seeded at a density of 10,000 cells/well in a 12-well plate in the presence of 1 μg/ml puromycin and incubated for 6 days. Cells were counted daily in duplicate with trypan blue exclusion.
    2.3. Cell viability and colony formation assays
    PDAC cell lines seeded at a density of 5 × 104 cells/well in 12-well or 2 × 104 cells/well in 24-well cell culture plates were treated in duplicate, DMSO was used as a negative control. After 72 h cells were counted with trypan blue exclusion.
    To validate the effects of therapies on the ability of cancer cells to form colonies in anchorage-independent condition, soft agar colony formation assay was performed (Domenichini et al., 2019). Colonies were grown for 4 weeks and then fixed in 10% Acetone/ Methanol, stained with a 0.05% crystal violet solution and counted.
    Total RNA was extracted using GeneJET TNA Purification (Thermo Scientific, #K0732) according to the manufacturer's
    A. Adamska, et al. Advances in Biological Regulation xxx (xxxx) xxxx
    instructions, followed by cDNA synthesis (Thermo Scientific, # EP0742). RT-qPCR was performed according to the manufacturer's instructions (Fermentas, #K0222) using an ABI 7500 RT-QPCR system. As a control, QARS cDNA was also amplified. Changes in gene expression, relative to control, was calculated using relative CT analysis.
    2.5. Protein analysis
    Following ABCC3 silencing, PDAC cells were incubated with Caspase 3/7 reagent (1:1000 dilution) (Essen Bioscience) accord-ingly to manufacturer's instruction and monitored for up to 72 h using IncuCyte Life Cell Analysis Imaging System (Sartorius).
    2.7. LPI stimulation and release analysis
    PDAC cells were serum-starved overnight before LPI stimulation. Cells were incubated with 1 μM LPI (MerckMillipore, cat# 440153) for 8 min (acute stimulation) or 0.5 μM LPI for 72 h (long-term stimulation). Cell viability and protein analysis were per-formed as described above.
    HPAFII cells were transfected with siRNAs targeting ABCC3 and cPLA2. Twenty-four hours post-transfection, cells were labelled with [3H]myo-Inositol for 48 h. Cells were then incubated with or without EGF (20 ng/ml) for 1 h. Lipids were extracted from cell supernatants by phase separation and radioactivity was assessed by scintillation counting.
    2.8. Radiolabelled LPI preparation
    HEK293T cells were fed tritiated myo-inositol to convert into 3H-LPI. The 3H-LPI released by the cells was separated by thin layer chromatography and recovered.
    Membrane vesicles were prepared as described previously (Byrne et al., 2002) from HEK293T (untransfected cells or cells transiently-transfected with pcDNA3-ABCC3). Vesicles containing 60 μg of membrane protein were incubated at 37 °C for 15 min in 150 μl total volume containing 50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM ATP, 10 mM MgCl₂, 100 μg/ml creatine kinase (Roche, UK), 10 μM creatine phosphate (Roche, UK) and 2 μM LPI (cold-LPI (Sigma-Aldrich) spiked with 0.5 nCi ³H-LPI prepared as described above) in the presence or absence of 100 μM vanadate (Sigma-Aldrich). The reaction was stopped by adding ice-cold buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.5). The vesicles were recovered by rapid filtration through cellulose nitrate discs (0.2 μm pore size, 25 mm diameter Whatman; Fisher, UK) using a 1225 Sampling Manifold (Millipore) and washed four times with of ice-cold transport buffer. The 3H-LPI accumulated in the vesicles was measured in a 1049 DSA scintillation counter (Wallac).