Cyclind

RIPA lysis buffer (with 1% phenylmethanesulfonyl fluoride) (Beyotime, Shanghai, China) was used to lyse the cells. After determination of protein concentration with a BCA protein concentration determination kit (Beyotime), the protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transferation onto polyvinylidene fluoride membranes (ThermoFisher). Following blocking with 5% bovine serum albumin, the membranes were incubated with antibodies against NR1D1 (1:1000; Abclonal, Wuhan, China), cyclinD (1:1000; ABclonal), cyclinE (1:1000; Proteintech, Wuhan, China), SOCS3 (1:1000; ABclonal), JAK-1 (1:1000; Affinity, Changzhou, China), p-JAK1 (Tyr 1034/Tyr 1035; 1:1000; Affinity), JAK2 (1:500; Affinity), p-JAK2 (Tyr 1007/Tyr 1008, 1:1000; Affinity), STAT3 (1:500; Affinity), p-STAT3 (Tyr 705, 1:500; Affinity), β-actin (1:2000; Proteintech) at 4 °C overnight. Thereafter, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:10000; Proteintech) at 37 °C for 40 min. Blots were visualized with an enhanced chemiluminescence substrate kit (7 Sea biotech, Shanghai, China).

Subconfluent cells were washed with PBS and lysed for 20 minutes on ice in a RIPA buffer containing 1% protease inhibitor cocktail (Beyotime). The protein was collected, and its concentration was determined by the BCA protein assay reagent kit (Thermo Fisher Scientific) then separated by SDS‐PAGE and transferred to nitrocellulose membrane. The membrane was then blocked with 5% non‐fat milk for 1 hour at room temperature and incubated with primary antibody overnight at 4°C. The following antibodies were used: GAPDH (Bioworld), MICAL1 (Proteintech) (Santa Cruz), NF‐kB (Santa Cruz), β‐catenin, p‐β‐catenin, p‐GSK‐3β, p‐S6K, histone‐H3, ERK, p‐ERK, Akt and p‐Akt (Cell Signaling), cyclin D (ABclonal), c‐myc, CDK (2, 4, 6) and cyclin (A, E) (Santa Cruz). The membranes were incubated with secondary HRP‐conjugated antibodies (Santa Cruz) and visualized with ECL reagent (Millipore). Digital images of immunoblots were obtained with a Chemidoc XRS and analysed using the image analysis program Quantity One (Bio‐Rad).