Vimentin

Cells were lysed in RIPA buffer on ice. After centrifugation at 12000g for 15 min at 4°C, the supernatant was collected and the concentration was measured using BCA method. Fifty micrograms of total protein were separated by SDS/PAGE. Then, proteins were transferred on to PVDF membranes (Millipore). After blocking with TBST containing 5% milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. Next day, the membranes were washed with TBST four times. Then, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. The blots were developed by ECL detection reagents (GE Healthcare). The gray intensity of protein bands was quantitated with ImageJ and normalized to GAPDH. The MMP2, MMP9, collagen I, fibronectin, p-Akt (Thr³⁰⁸), Akt, p-PI3K (Tyr⁴⁵⁸), PI3K, p-mTOR (Ser²⁴⁸¹), mTOR, GAPDH antibodies were purchased from Cell Signaling. Insulin-like growth factor binding protein (IGFBP)3, IGFBP5, and IGFBP6 antibodies were purchased from Abcam. α-SMA, N-cadherin, E-cadherin, and p16 antibodies were purchased from BD Biosciences, and vimentin was purchased from Covance.

Paraffin tissues were embedded and sectioned at 5μM and dewaxed in xylene and rehydrated in alcohol with citrate antigen retrieval as previously described [5 (link)]. Standard Mayer's hematoxylin and eosin (H&E) was performed. Cleaved Caspase-3 (Cell Signaling Cat#9661, 1:200), Vimentin (Covance Cat#PCK-594P 1:500), BrdU (BD Cat#563445 1:100). pSmad1/5 (Cell Signaling Cat#9516 1:200 ), Snail (Santa Cruz Cat#28199 1:200), Slug (Santa Cruz Cat#166476 1:100), Ecadherin (BD Cat#610181 1:200), K8/18 (Fitzgerald Cat#20R-CP004 1:500), K5 (Covance Cat#PRB160P 1:500), pSmad2 (Cell Signaling Cat#3101 1:500), CollagenIV (Abcam Cat#19808 1:500) and p63 (Santa Cruz Cat#8344 1:200). Paraffin derived sections were counterstained with hematoxylin (Vector Labs QS) and mounted with Cytoseal. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 12% Fraction-V BSA (Pierce) and slides were mounted in SlowFade mounting medium containing DAPI (Invitrogen). All fluorescent secondary antibodies were highly cross-adsorbed, produced in goat and used at a dilution of 1:200 for 20 min (Molecular Probes). Quantification of IHC and IF was performed using NIH ImageJ (http://rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html) as previously described [43 (link)].