Anti nkd1

ISH and IHC were performed as previously described. In brief, the double (5′–3′) digoxigenin (DIG)-labeled miR-744 probe and U6 probe were purchased from Boster (Wuhan, China) and ISH was conducted according to the manufacturer's instructions of the microRNA ISH Optimization Kit (Boster, Wuhan, China). IHC was carried out with appropriate primary antibodies according to their manufacturer's instructions. These antibodies included anti-NKD1 (1:100, Abcam, USA), anti-ki67, anti-caspase-3, anti-CD31, and anti-CD34 (All 1:100, Boster, China). ISH and IHC scores were performed using a semiquantitative grading system as our previous study [43 (link)]. Sections with no labeling or with fewer than 5% labeled cells were scored as 0. Sections with 5%–30% of cells labeled were scored as 1, with 31%–70% of cells labeled as 2, and with labeling of ≥ 71% as 3. The staining intensity was scored similarly, with 0 used for negative staining, 1 for weakly positive, 2 for moderately positive, and 3 for strongly positive. Each sample was examined separately and scored by two pathologists. Cases with discrepancies in the scores were discussed to reach a consensus.

Cells were lysed in RIPA buffer (25 μM Tris-HCl (pH 7.6), 150 μM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1% proteinase inhibitors (Cat. No: P2850, Sigma) for total protein preparation. Nuclear protein extracts were obtained with Nuclear Extraction Kit (Active Motif) according to the manufacturer's instructions. Briefly, 40 μg protein samples were analyzed by 10% SDS-PAGE and the gels were transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with 5% non-fat dried milk in TBST (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20) for 1 h at room temperature, and incubated overnight at 4°C with specific primary antibodies. Membranes were washed three times with TBST buffer, then incubated for 1 h with 1:2000 secondary antibodies, and developed with enhanced chemiluminescence (ECL, Millipore, USA). The primary antibodies included anti-NKD1 (1:1000, Abcam, USA), anti-SFRP1 (1:1000, Abcam, USA), anti-GSK3β antibody (1:1000, Abcam, USA), anti-TLE3 antibody (1:1000, Abcam, USA) and anti-β-catenin (1:1000, Abcam, USA) antibody. Anti-GDPAH (1:5000, Beyotime, China) and anti-p84 (1:1000, Abcam, USA) antibodies were used as the loading control.