Igfbp 3

Fixed liver tissues were embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) for light microscopy. TUNEL staining was performed with commercial kits (Promega, Madison, WI, USA). Five to six random sections were investigated per slide to determine the necrotic area and percentage of apoptotic cells. To measure the necrotic area, sections were observed under an Axiovert 40 CFL microscope (Carl Zeiss, Oberkochen, Germany) and measured using iSolution DT 36 software (Carl Zeiss). For immunohistochemistry, sections were immunostained with antibody against IGFBP-3 (Santa Cruz Biotechnology) or 4-hydroxynonenal (4-HNE, Abcam).

Liver homogenates containing 10 μg of whole cell lysate were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the blot was probed with primary antibodies for IGFBP-3, survivin, SOCS-3, NOX4, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, STAT-3, p-STAT-3, catalase (Cell Signaling Technology, Beverly, MA, USA), and NOX2 (Abcam, Cambridge Science Park, Cambridge, UK).

Western blot analysis was performed as described previously [37 (link)]. Briefly, 30 μg of protein were run on 10% SDS-PAGE, transferred to nitrocellulose membrane (BioRad) and immunoblotted with the following antibodies: GRP78 (1:1000 BD Bioscience, San Jose, CA, USA), IGFBP-3 (1:1000 Santa Cruz, Dallas, TX, USA) and α Tubulin (1:5000 Merck Millipore, Burlington, MA, USA). After incubation with specific secondary antibodies conjugated to peroxidase (Sigma, St. Louis, MO, USA), proteins were visualised by Clarity ECL substrate (BioRad) using BioRad Chemidoc XRS + system and analysed using Image lab software (BioRad).

Cells were lysed in buffer containing 137 mmol/L NaCl, 15 mmol/L EGTA, 0.1 mmol/L sodium orthovanadate, 15 mmol/L MgCl₂, 0.1% Triton X-100, 25 mmol/L MOPS, 100 mmol/L phenylmethylsulfonyl fluoride, and 20 mmol/L leupeptin, adjusted to pH 7.2. Lysis of tumor specimens was performed using Omni Tissue Homogenizer (TH; Omni International, Kennesaw, GA). Antibodies specific for p-EGFR (Tyr1173), EGFR, Akt, Erk, IGFBP3, IGF1R and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and antibodies for p-ErbB2 (Tyr1221/1222), ErbB2, p-ErbB3 (Tyr1289), ErbB3, p-IGF1R (Tyr1135/1136), p-Akt (Ser473), p-Erk (Thr202/Tyr204), caspase-3 and PARP-1 were purchased from Cell Signaling Technology (Beverly, MA). Proteins were detected with an enhanced chemiluminescence Western blotting kit (Amersham Biosciences) according to the manufacturer's instructions.

Monolayer cultures of respective cell lines at an 80–90% confluence were lysed using 100 μl of RIPA buffer (Thomas Scientific). Tris-glycine (Bio-Rad) gels were loaded with 50–100 μg of lysates. After running gel electrophoresis, the gel was transferred to a nitrocellulose membrane for 2 hours. The membrane was blocked for 1 hour in 5% BSA or 5% skim milk at 4°C. The membrane was then washed 3 times with 1xTTBS and incubated overnight with the primary antibody at 4°C. Primary antibodies of coagulation factor III, IGFBP-3, DPP IV, PDGF-A, endothelin I, CXCL16, TIMP-1, amphiregulin, endostatin, angiogenin and GM-CSF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies for STAT3, pSTAT3, E-cadherin, Vimentin, Oct-4, VEGF and β-actin were purchased from Cell Signaling Technology (Danvers, MA). After incubation with the secondary antibodies conjugated with horseradish peroxidase (HRP), the protein bands were developed with the chemiluminescent reagents. The blot images were taken by using the Bio-rad molecular imager gel doc XR system. Western blot protein band density was measured by Image J software and presented after normalized by the house keeping protein β-actin. Normal FHC (CRL-1831) colon cell line was used as a reference for the protein expression comparison by measuring band intensity as a 1.0.

MCF-7, SKBR-3, MCF-7/T7-MTA1, MEF WT, MEF MTA1^−/−, HCT-116 cells were used in the study. RPMI 1640 (Invitrogen, CA) supplemented with 10% FBS and 1X antibiotic/antimycotic (15240062, Gibco, CA) was used to maintain the cells in a humidified 5% CO₂ at 37 °C. Antibodies used were MTA1 (#5447, Cell Signaling Technology), DNMT3a (ab188470, Abcam), IGFBP3 (sc-9028, Santa Cruz), HDAC1 (ab46985, Abcam), HDAC-2 (sc-7899, Santa Cruz), Vinculin (sc-73614, Santa Cruz), RNA Polymerase II (A300-654A, Bethyl labs), DNMT3a (sc-20703, Santa Cruz), YY1 (#2185, Cell Signaling Technology) and β-Actin (sc-47778, Santa Cruz). Secondary antibodies conjugated to Horseradish peroxidase were obtained from Sigma-Aldrich. Chemiluminescence detection reagents were obtained from Millipore (WBKLS0100). All other reagents were purchased from Sigma-Aldrich, if not mentioned.