Ab125075

RIPA lysis buffer (Beyotime Biotechnology, China) was utilized to extract total protein. The protein concentration was examined using BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein lysate were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Biotechnology, China) and transferred into a polyvinylidene fluoride membrane. After being blocked using skimmed milk, the membranes were incubated with primary antibodies including NGAL (ab125075, Abcam, 1 : 1000), KIM-1 antibody (ab228973, Abcam, 1 : 1000), Bax (ab32503, Abcam, 1 : 2000), Bcl-2 (ab32124, Abcam, 1 : 1000), GPNMB (ab227695, Abcam, 1 : 100), and GAPDH (ab9485, Abcam, 1 : 2500) at 4°C overnight. Then, the membranes were incubated with secondary antibodies at room temperature. Finally, the blots were visualized using the enhanced chemiluminescence solution.

Chrysophanic Acid (CHR) and CDDP were purchased from Sellect (Houston, TX, United States). Dulbecco’s modified Eagle medium F12 (DMEM/F12), Trypsin-EDTA (0.25%), and Antibiotic-Antimycotic solution were purchased from Gibco (United States). Fetal bovine serum (FBS) was supplied by CellMax (Beijing, China). Other consumable materials for cell culture were acquired from Greiner Bio-One (Essen, Germany). F4/80 (GB11027; Servicebio, Wuhan, China), c-caspase3 (GB11532; Servicebio, Wuhan, China), and Lipocalin-2/neutrophil gelatinase-associated lipocalin (NGAL; ab125075; abcam, Shanghai, China) antibody were used for immunohistochemistry (IHC). Antibody of Lipocalin-2/NGAL (NBP1-45682) was provided by Novus Biologicals (Littleton, United States). Antibodies of Bax (50599-2-Ig,) and Bcl2 (26593-1-AP) were provided by Proteintech (Wuhan, China). Antibodies of cleaved Caspase 3 (cst9664), NF-κB p65 (cst6956), Phospho-NF-κB p65 (cst 3033), IκBα (cst4814), phospho-IκBα (cst9246), IKKβ (cst2678), phospho-IKKα/β (cst2697), and GAPDH (cst2118) were provided by cell signaling technology Inc. (MSA, United States).

Primary human prostate specimens were collected from 118 PCa patients from Renji Hospital. Tumor differentiation was defined according to the Gleason grading system. Tumor staging was defined according to the 7th edition of the American Joint Committee on Cancer TNM staging manual [20 ].
Tissue slides were stained with primary antibody against AMACR (ab219309), CXCL1 (ab86436), LCN2 (ab125075) from Abcam (Cambridge, UK), Keratin18 (#4548, Cell Signaling Technology, Massachusetts, USA) and CD177 (LS-B1953, LifeSpan BioSciences, Washington, USA). IHC results were evaluated independently by two pathologists who were blinded to the patients’ details. The staining intensity was scored on the following scale: negative (0), weak (1), moderate (2), or strong (3). The staining extent score was on a scale of 0–3, corresponding to the percentage of immunoreactive cells (< 5, 5–25%, 26–50%, 50–100%, respectively). An aggregate score ranging from 0 to 6 was calculated by adding the intensity score and staining extent score together, resulting in a negative (0–1), low (2), moderate (3–4), and high (5–6) expression for each specimen. PerkinElmer Opal™ Tyramide Signal Amplification system was employed for multiplex protein co-staining and images were captured by VectraPolaris™ Quantitative Slide Scanner (PerkinElmer, Massachusetts, USA).