Gtx83114

HFL1 cells were grown on coverslips and treated with or without 100 ng/ml His‐PTX3 fusion protein for 24 h. Cells were fixed in 4% paraformaldehyde for 20 min and incubated with antibodies against His (sc‐803, Santa Cruz) and CD44 (GTX83114, GeneTex). For lung sections, samples were incubated with PTX3 (1:100, ab90806; Abcam), fibronectin (1:350 dilution, #15613‐1‐AP, ProteinTech) and α‐SMA (1:100 dilution, GTX100904, GeneTex) at room temperature for 1 h. Samples were incubated with Alexa 568‐ or 488‐conjugated secondary antibodies (Invitrogen) at a 1:200 dilution. The samples were washed with 0.1% Tween 20 in PBS and stained with DAPI (P36935, Invitrogen) on coverslips. The fluorophores were excited by a laser at 405, 488, or 543 nm and observed using an FV‐3000 confocal system (Olympus). Randomly chosen fields were photographed at ×200 magnification under a fluorescence microscope.

The membrane protein of MDA‐MB‐231 cell was extracted by Mem‐PER™ Plus kit (Thermo) following the manufacturer's steps. Collected the membrane protein and incubate with His‐PTX3, and then incubated with the specific antibody that recognises PTX3 (ab90806; Abcam) or CD44 (GTX83114; GeneTex) at 4°C overnight. After, agarose beads of protein‐A/G were supplemented into the lysates, and mixtures were centrifuged to collect the beads, and the beads were washed by RIPA buffer for three times. To elute the proteins which were bound to the beads by using electrophoresis buffer to combine with beads, and then perform by Western blot.

Tissues were embedded in paraffin and sliced for IHC experiment to verify the protein expression. The antibodies against Ki67 (550609, 1:50, BD Biosciences); PARP (ab32064, 1:100, Abcam); fibronectin (GTX61206, 1:250, GeneTex); MMP-9 (10375-2-AP, 1:250, Proteintech); TGF-β (ab190503, 1:100, Abcam); vimentin (550513, 1:50, BD Biosciences) and CD44 (GTX83114, 1:500, GeneTex) were used.