Ab206655

We collected 160 human epithelial ovarian cancer samples with accompanying patient follow-up information between January 2010 and January 2015 in the Department of Pathology of Peking Union Medical College Hospital. Follow-up was performed until January 1, 2020. Pathological diagnoses were reconfirmed by a pathologist. The project was approved by the Ethics Committee (Peking Union Medical College Hospital), and informed consent was obtained from patients or their family members. The details of immunohistochemical staining (IHC) and scoring were described previously (Li et al., 2010 (link); Zhang et al., 2015 (link)). The following antibodies were used: anti-EZH2, 1:200, Abcam ab191080; anti-CYP27B1, 1:500, Abcam ab206655. The intensity of immunostaining was scored as follows: 1 +, weak; 2 +, moderate; 3 +, strong; or 4 +, very strong. The area of positive staining in each microscopic field was scored as follows: 1 +, 0–25%; 2 +, 25–50%; 3 +, 50–75%; or 4 +, 75–100%. A final score between 5 and 80 was obtained by multiplying the product of the two scores by 5. A final score between 0 and 42 was considered “low expression” and a final score between 43 and 80 considered “high expression.” All pathological diagnoses were confirmed in a blinded manner by three expert pathologists.

A Minute™ Cytoplasmic and Nuclear Fractionation kit (SC-003, Invent, USA) was used for tissue protein extraction. Aliquots were assayed for protein content using the BCA method. Samples were separated by 12% SDS-PAGE under reducing and electroblotted onto a PVDF membrane. They were then blocked in TBS+ 0.1% TBST containing 5% non-fat powdered milk and shaken for 60 m. Membranes were incubated with VDR (14526–1-AP, Proteintech, USA), CYP27B1 (ab206655, Abcam, UK), CYP19 (DF3564, Affinity, China), HOXA10 (26497–1-AP, Proteintech, USA), GAPDH (ab9485, Abcam, UK) and H3 (ab1791, Abcam, UK) primary antibodies at 4 °C overnight. The resulting antibody solution was a 1:1000 dilution. Membranes were then bathed for 5 min in TBST three separate times, then incubated with horseradish peroxidase (HRP), a conjugated secondary antibody (Servicebio, China), at a dilution of 1:3000, for 30 min. Membranes were then washed. Enhanced chemiluminescence was used for exposure imaging. Photos of the bands were captured and the density of each band was determined using Image Alpha software. The ratio of each cytoplasmic protein /GAPDH and nuclear protein/Histone H3 band density was calculated and considered as an indicator for expression level in the targets.

Cell or tissue lysates were extracted for loading into 10% SDS-PAGE gels and immunoblotting was performed as previously described 34 (link). Primary antibodies, including rabbit anti-Cyp27b1/1α(OH)ase (Abcam, ab206655), rabbit anti-VDR (Abcam, ab3508), rabbit anti-Sirt1 (Millipore, 07-131), rabbit anti-p16 (Proteintech, 10883-1-AP) and rabbit anti-β-actin (Cell Signaling Technology, 8457S) were used for immunoblotting. Immunoreactive bands were visualized with ECL chemiluminescence (Bio-Rad) and analyzed by Image J.