The amount of target, normalized to the endogenous reference and relative to the control is given by 2-ΔΔCt (Relative Quantification, RQ). (ΔCt = Ct target gene – Ct endogenous reference; ΔΔCt = ΔCt transfected – ΔCt control). Western-blot analysis

Fifteen micrograms of total protein were loaded on 8% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman GmbH, https://www.selleckchem.com/products/GDC-0449.html Dassel, Germany). Blots were blocked with PBS containing 0.1% Tween-20 (PBST) and 5% powdered skim milk (PBSTM) 1 hour at room temperature and incubated overnight 4°C with rabbit polyclonal PARP3 antibody diluted 1:1000 in PBSTM (Alexis Biochemicals, San Diego, California; kind gift from Dr. Michèle Rouleau, Guy Poirier Laboratory, Québec, Canada). After washing with PBST, blots were incubated for 1 hour at room temperature with the secondary anti-rabbit antibody (Sigma-Aldrich, St Louis, Missouri) diluted at 1:1000 in PBSTM. After washing

with PBST, blots were developed using Pierce ECL 2 Western Blotting Substrate (Thermo Scientific, Waltham, Massachussets). β-actin was used as loading control. Cells that expressed at higher levels the short isoform (SK-N-SH), as verified by siRNA knock down, were used as reference (kind gift from Dr. Michèle Rouleau, Guy Poirier Laboratory, Québec, Canada) [8]. Intensity of individual bands Smad2 signaling was quantified using Image J densitometry software, and expressed relative to β-actin signal, as a measure of protein relative abundance in the different conditions. Telomerase activity assay Telomerase activity was determined in A549 transfected cells (24, 48 and 96 hours BI 2536 solubility dmso post-transfection) and in Saos-2 cells with the Cobimetinib mw highest ratio of genetic silencing, by TeloTAGGG Telomerase PCR ELISA (Roche Applied Science, Penzberg, Germany) as previously published [9]. This method is an extension of the original Telomeric Repeat Amplification Protocol (TRAP) [10]. Briefly, in a first step, a volume of cell extract containing 10 μg of total proteins was incubated with a biotin-labelled synthetic telomerase-specific primer, and under established conditions, telomerase present in cellular extracts

adds telomeric repeats (TTAGGG) to the 3′ end of the primer. In a second step, these elongation products were amplified by PCR using specific primers. An aliquot of the PCR products was denatured, hybridized to a digoxigenin labelled, telomeric repeat-specific probe, and bound to a streptavidin-coated microtiter plate. The immobilized PCR products were then detected with an antibody against digoxigenin that was conjugated to peroxidase. Finally, the probe was visualized by virtue of peroxidase-metabolizing TMB to form a coloured reaction product and semiquantified photometrically (450 nm). Thus, considering that the cut-off for telomeric repeat amplification protocol-ELISA negativity corresponds to optical density (OD)450 nm less than 0.2, all samples with OD450nm >0.2 were considered as telomerase positive.