Uhrf1

The cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St Louis, MO, USA) and protein concentrations were quantified by bicinchoninic acid (BCA) assay (Beyotime Institute of Biotechnology, Shanghai, China). The proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The PVDF membrane was then blocked in 5% skimmed milk and 0.1% Tween-20 at room temperature for 1.5 h and subsequently was incubated in primary antibody overnight at 4°C. The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature and again washed and developed using an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, Shanghai, China). The relative protein expression was analyzed by Quantity One v4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primary antibodies (UHRF1, Cyclind D1, PCNA, PPARγ, and β-actin) were purchased from Proteintech (Wuhan, China).

The effect of alisertib on Aurora A and the Jak/STATs signaling pathways was assessed by immunoblotting, as described previously 21 (link), 23 , using the following phospho-specific antibodies: p-Aurora A/B/C (Cell Signaling Technology, #2914), Aurora A (35C1) (Abcam, ab13824), p-Jak1 (T1022/1023) (Cell Signaling Technology, #3331), Jak1 (6G4) (Cell Signaling Technology, #3344), p-Jak2 (T1007/1008) (Cell Signaling Technology, #3776), Jak2 (D2E12) (Cell Signaling Technology, #3230), p-STAT1 (T701) (Cell Signaling Technology, #7649), STAT1 (D1K9Y) (Cell Signaling Technology, #14994), p-STAT2 (T690) (Cell Signaling Technology, #4441), STAT2 (D9J7L) (Cell Signaling Technology, #72604), PARP (46D11) (Cell Signaling Technology, #9532), DNMT3B (D7070) (Cell Signaling Technology, #67259), DNMT1 (Proteintech., 24206-1-AP), DNMT3A (Proteintech., 19366-1-AP), ubiquitin (Proteintech., 10201-1-AP), UHRF1(Proteintech., 21402-1-AP), flag (Abmart, M20008L) and β-actin (Proteintech.,66009-1-Ig).