Imagequant software

ChIP assays were performed with LCLs from family 1. Cells in culture were cross-linked with formaldehyde (1%) during 15 min at room temperature. The cross linking reaction was stopped by addition of glycine (0.125 M) during 5 min. Cells were washed twice with cold PBS, collected by centrifugation and lysed in lysis buffer (1%SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0, protease inhibitors (P8340, Sigma)). Protein/DNA extracts were sonicated and centrifuged at 14,000 r.p.m. for 15 min. Protein concentration was determined (DC protein assay, Bio-Rad), and chromatin from 200 μg total protein extract was used per immunoprecipitation with 4 μl of either anti-POT1, anti-TRF2 or anti-TRF1 antibody (Supplementary Table 6) and protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, sc-2003)34 (link). The immunoprecipitated DNA was transferred to a Hybond N⁺ membrane using a dot-blot apparatus. The membrane was then hybridized with a telomeric probe containing 1.6 kb of 5′-TTAGGG-3′ repeats. Quantification of the signal was performed with ImageQuant software (Molecular Dynamics). The amount of telomeric DNA after ChIP was normalized to the total input telomeric DNA.

The cellular lysates were prepared and proteins were then resolved on SDS–PAGE and transferred to Immobilon polyvinyldifluoride (PVDF) membranes. The blots were blocked with 4% BSA for 1 h at room temperature and then probed with rabbit or mouse anti-human antibodies against periostin, vimentin, N-cadherin, E-cadherin, Twist, Snail, p-ERK, ERK, p-p38, p38, p-JNK, JNK, and β-actin (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence using ImageQuant LAS 4000 (GE Healthcare Life Sciences, Little Chalfont, UK). Quantitative data were obtained using a computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

For examining the effect of hypoxia on IGF1R expression, pancreas cancer cells were lysed 48 hours after incubation under normoxic or hypoxic conditions. Total protein (50 μg) was separated on polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) with subsequent antibody (anti-IGF1R antibody; ab16890, Abcam, or anti-β-actin antibody; Cell Signaling Tec, Danvers, CO.), incubation. The bands were detected using an enhanced chemiluminescence system (Wako). Densitometry quantification was performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) on a LAS 4000-mini Image Reader (GE Healthcare UK Ltd, Little Chalfont, UK).

The protein of anterior lens capsules and lens cortex was extracted in lysis buffer (1 M Tris-HCl at pH 7.5, 1% Triton X-100, 1% Nonidet p-40, 10% SDS, 0.5% sodium deoxycholate, 0.5 M EDTA, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Proteins were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride filter membranes (Millipore, Bedford, MA). Nonspecific protein binding to the membrane was blocked with blocking buffer (5% nonfat milk, 200 mM NaCl, 50 mM Tris, and 0.05% Tween 20). The blocked membrane was then incubated with mouse anti-human-WRN (1 : 800; Abcam, Cambridge, UK) and anti-GAPDH (1 : 1000; Santa Cruz, CA, USA) at 4°C for 18 h. The membrane was washed three times with TBST (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for 5 min each time, followed by incubating with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (1 : 2000; Santa Cruz, CA, USA) for 2 h. Detection was performed using an ECL chemiluminescence kit (Pierce, Rockford, IL) and the signal was exposed to an X-ray film that was scanned using Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA).