L pgds

Immunofluorescence was used to detect the expression of L-PGDS in tumor tissue (1 : 50; Bioworld, Louis Park, MN). The secondary antibodies were Alexa Fluor 555-labeled donkey anti-rabbit IgG (1 : 300, Invitrogen, Carlsbad, CA). Images were acquired using microscopy (Nikon, Tokyo, Japan). For immunohistochemical analysis, the tumor tissues were fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and cut into 5 μm thick sections. Then, these sections were incubated with anti-PCNA (1 : 100; Bioworld, Louis Park, MN) overnight at 4°C and, subsequently, incubated with the secondary antibody at 37°C for 30 min. Finally, tissues were counterstained with 3,3′-diaminobenzidine (DAB) and photographed by microscopy.

Cell and tissue lysates were extracted in a lysis buffer (RIPA, Pierce) and proteinase inhibitor. Then, a total of 60 μg protein was separated in 12% SDS-polyacrylamide gels with 180 kDa prestained protein marker (MP102-02, Vazyme Biotech Co., Ltd.) and transferred to PVDF membranes. The membranes were blocked with 5% BSA and then incubated with primary antibodies against GAPDH (1 : 2000; Bioworld), CD9 (1 : 500; Cell Signal Technology), CD63 (1 : 500; Abcam), CD81 (1 : 500; Abcam), Calnexin (1 : 500; Cell Signal Technology), L-PGDS (1 : 500; Bioworld), Bax (1 : 400; Cell Signal Technology), Bcl2 (1 : 500; Cell Signal Technology), Oct4 (1 : 500; Cell Signal Technology), Nanog (1 : 500; Bioworld), Sox2 (1 : 800; Wanlei bio), p-STAT3 (1 : 500; Cell Signal Technology), and t-STAT3 (1 : 500; Cell Signal Technology) at 4°C overnight. The membranes were washed three times with Tris-buffered saline/Tween and incubated with goat anti-rabbit secondary antibody (1 : 2000, Invitrogen) at 37°C for 1 hour. The signals were visualized using Luminata crescendo western horseradish peroxidase substrate (Millipore) and image software of GE (ImageQuant LAS4000 mini).