Caspase 3 8g10

Protein was isolated and concentration quantified using a BSA Protein Assay Kit (ThermoScientific). Ten-20 μg of total protein was separated on a Mini-PROTEAN TGX, Any kDTM gel followed by transfer onto PVDF membranes (Bio-Rad). The membranes were blocked (5% non-fat dry milk in TBS + 0.5% Tween20) prior to overnight incubation at 4 °C with primary antibodies to β-catenin (BD), Bcl-2 (D17C4), Caspase-3 (8G10), or GAPDH (D16HH, Cell Signaling). Following washes, membranes were incubated for 1 h at room temperature with secondary HRP-conjugated goat antibodies to mouse or rabbit IgG (Cell Signaling). Chemiluminescence was performed using Western ECL Blotting Substrate (Bio-Rad) followed by X-ray film-based imaging. Films were scanned and quantified for integrated optical density (IOD) using ImageJ software. To remove antibodies, membranes were incubated for 15 min at room temperature in Restore Western Blot Stripping Buffer (ThermoScientific).

Non-small cell lung cancer (A549, NCI-H292, NCI-H1299 and NCI-H358), hepatocellular cancer (SNU449, HuH1, HuH7, Chang, Hep3B, HepG2), colon cancer (WDr) and breast cancer (MCF-7) cell lines, obtained from American Type Culture Collection (ATCC), were cultured in complete RPMI 1640 medium (Gibco) supplemented with 10% FBS (Thermo Scientific) and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Polyinosinic-polycytidilic acid (polyI:C), is a synthetic short-chain analog of dsRNA from InvivoGen. Actinomycin D, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Stattic were from Sigma. Tyrphostin AG 490 was from Calbiochem. Antibodies used were rabbit anti-IL6 polyclonal antibody (ab6672, Abcam), ChromPure rabbit and mouse IgG control, whole molecule (Jackson ImmunoReseach), functional grade purified anti-human TLR3 (e-Bioscience), mouse IgG1 (ICIG1) (FITC) isotype (ab91356, Abcam), anti-TLR3 antibody (40C1285.6) (FITC) (ab45053, Abcam), anti-STAT3 (phospho Y705) antibody and anti-STAT3 antibody (Abcam), anti-JAK2 and Phospho-JAK2 (Cell Signalling), Caspase-3 (8G10, Cell Signalling).

Equal amounts of protein were used for total extracts or for immunoprecipitation with specific antibodies, followed by SDS-PAGE and Western blot analysis with the indicated antibodies (carried out using the ECL™ Western Blotting Analysis System; Amersham Pharmacia Biotech Ltd., Buckinghamshire, UK), as previously described. Antibodies against p-Stathmin 1 (p-OP18 S16, sc-12948-R), Stathmin 1 (OP18, sc-55531), alpha-tubulin (sc-5286), JAK2 (sc-294), STAT3 (sc-7179), PARP1 (sc-56197) and actin (sc-1616) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against p-JAK2 Y221 (#3774S), p-STAT3 Y705 (#9131S) and caspase 3 (#8G10) were from Cell Signaling Technology (Danvers, MA, USA). The antibody against acetyl-alpha-tubulin L40 (ab24610) was from Abcam (Cambridge, MA, USA).

Lung tissues were fixed in 2% PFA overnight, washed with 70% ethanol, embedded in paraffin and sectioned (5 µM). Tissue sections were de-paraffinized followed by antigen retrieval, blocked with 3% BSA and 1% donkey serum and then incubated antibodies. For TUNEL assays, sections were stained with terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) as per the manufacturer’s guidelines (Apo Tag Peroxidase In Situ Apoptosis Detection Kit, S7100, Millipore/Sigma). DAPI was used to counter stain the nuclei. For Ki-67 staining, de-paraffinized lung sections were incubated with anti-Ki-67 antibody (ab16667, Cell Signaling Technology, MA) overnight. Sections were incubated with HRP-conjugated secondary anti-rabbit antibody and developed by DAB kit (Invitrogen) and images were scanned using the Aperio ScanScope slide scanner (Leica Biosystems, IL). TUNEL-positive and Ki-67 positive cells in the entire section were enumerated and both total and percent positive cells are presented. Caspase-3 (8G10) (9665P, Cell Signaling Technology) antibody was used to detect apoptotic cells.