Anti anxa2

Cells were seeded in 12-well plates containing glass coverslips, then cultured and treated with EGF as described above for 72 h. After washing with PBS three times, cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized in freshly prepared 0.15% triton X-100 in PBS for 10 min, blocked in 3% BSA/PBS at room temperature for 1 h, and incubated with anti-E-cadherin (BD Biosciences), anti-Vimentin (abcam), anti-Slug (Cell Signaling Technology), anti-Anxa2 (Santa Cruz Biotechnology), anti-STAT3 (Cell Signaling Technology), and anti-p-STAT3 (Y705, Cell Signaling Technology) antibodies in a humid chamber at 4°C overnight. After repeated washing with PBS, cells were stained with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 633 conjugated secondary antibodies (Invitrogen) at room temperature for 1 h, followed by staining nucleus by DAPI. Finally, the coverslips were mounted with Mowiol-based anti-fading medium and visualized under a laser scanning confocal microscope (Leica TCS SP5).

Cells (3 × 10⁵) were plated into six-well culture plates to achieve 70%–80% confluency, and were washed in PBS and suspended in 100 μL of RIPA buffer (Pierce, Dallas, TX, USA). Supernatant protein concentrations were determined using the BCA protein assay kit (Pierce). Supernatant samples containing 30 μg total protein were resolved by 10% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) depending on the molecular weights of the target proteins, and were transferred to immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) by electroblotting, and then probed with anti-E-cadherin (Cat# sc-21791, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-N-cadherin (Cat# sc-53488, Santa Cruz Biotechnology), anti-Vimentin (Cat# gtx100619, GeneTex, Irvine, CA, USA), anti-SNAIL1 (Cat# gtx125918, GeneTex), anti-Twist (Cat# gtx127310, GeneTex), anti-ZEB1 (Cat# gtx105278, GeneTex), anti-STC2 (Cat# sc-14352, Santa Cruz Biotechnology), anti-ANXA2 (Cat# sc-9061, Santa Cruz Biotechnology), anti-NRP1 (Cat# 3725, Cell Signaling Technology, Danvers, MA, USA), anti-SMAD2 (Cat#: 3103, Cell Signaling Technology), or anti-GAPDH (Cell Signaling Technology) antibodies. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Merck Millipore, Billerica, MA, USA).

MSC was lysed by RIPA lysis buffer. Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. After incubation in 5% skim milk for 1 h, the membrane was incubated with anti-osteocalcin, anti-osterix, anti-ANXA2, and anti- β-actin at 1:1000 (Santa Cruz Biotech, USA) at 4 °C for overnight. The membrane was then washed and incubated with HRP-conjugated secondary antibody at 1:5000 (Sigma, USA) at 25 °C for 1 h. ECL kit (Pierce Chemical, USA) was used to visualize the blots.