Anti serpine1

Sarcoma samples were fixed in 10% formalin, embedded in paraffin, and processed as 5-μm continuous sections. Samples were dewaxed with ethanol and blocked to inhibit endogenous peroxidase. They were then heated in a microwave to retrieve antigens, cooled to room temperature, and then blocked by incubation in goat serum for 30 min at 37°C. Samples were incubated in rabbit anti-ISG15, anti-NUP50, anti-PTTG1, anti-TSR1, and anti-SERPINE1 (Abcam, Cambridge, UK; 1:1,200) overnight at 4°C, followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody at 37°C for 30 min and stained using 3,3′-diaminobenzidine. The cell nucleus was stained blue by hematoxylin. Sections were then dehydrated, cleared by xylene, and mounted. ISG15, NUP50, PTTG1, TSR1, and SERPINE1 expression was detected by IHC using a streptavidin peroxidase method, with adjacent tissues as control groups. The experimental procedure was performed according to the manufacturer's instructions. The Image-ProPlus 6.0 Software (MediaCybernetics, USA) was used to analyze the expression of proteins and statistics on the results of immunohistochemistry.

Total protein was extracted using the RIPA buffer with 1% PMSF (Beyotime Biotechnology). For the Western blot analysis, PVDF membranes were incubated with the following primary antibodies overnight at 4°C: anti-CTCF (Cell signaling Technology, Danvers, MA, USA), anti-Histone3 (Beyotime Biotechnology), anti-SRC (Abcam), anti-CTBP1 (Abcam), anti-SERPINE1 (Abcam) and anti-β-actin (Beyotime Biotechnology). After the samples were washed with 0.1% TBST, they were incubated with the appropriate secondary antibodies (Beyotime Biotechnology), and the Chemilmanger™ 5500 system (Alpha Innotech, San Jose, CA, USA) was used to visualize the results. The gray value was calculated using the Image J software (http://rsb.info.nih.gov/ij/).

HPCs were incubated in media containing different concentrations of PAI-039 in DMSO for 48 h, fixed with 100% methanol for 5 min at −20°C, and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% bovine serum albumin, and 0.1% Tween for 2 h at room temperature. The HPCs were then incubated with rabbit anti-SERPINE1 (diluted 1 : 100, Abcam, Cambridge, UK) at 4°C overnight. After washing three times in PBS, the primary antibodies were reacted with the corresponding Alexa Fluor 488-conjugated anti-IgG (diluted 1 : 500, Abcam, Cambridge, UK) at 37°C for 30 min. Sections were examined under an Olympus CX41 fluorescence microscope (Olympus, Japan).