Anti ki67

Cell or tissue lysates and immunoblot analysis were performed as described previously [11 (link), 12 (link)]. Densitometric analysis was conducted using ImageJ software. The primary antibodies used include: anti-STIP1 (1:200, Boster Biotechnology, Wuhan, China), anti-ALK2 (1:400, Boster Biotechnology, Wuhan, China), anti-SMAD1/5 (1:200, Boster Biotechnology, Wuhan, China), anti-CTSK (1:500, Boster Biotechnology, Wuhan, China), anti-ERK1/2 (1:500, Boster Biotechnology, Wuhan, China), anti-Hsp90 (1:500, Boster Biotechnology, Wuhan, China), anti-Ki67 (1:500, Boster Biotechnology, Wuhan, China) and anti-GAPDH (1:500, Boster Biotechnology, Wuhan, China).

The collected tumor tissues were fixed with 4% polyoxymethylene and made into paraffin-embedded sections, which were then cultured with the primary antibody anti-Ki67 (dilution ratio of 1 : 1500, BOSTER, Biological Technology Co., Ltd, Wuhan, Hubei, China) at 4°C overnight. The following day, the sections were cultivated with the corresponding secondary antibody labeled with horseradish peroxidase. The sections were subsequently assessed under a microscope after the visualization of Ki67 signal with the aid of 2,4-diaminobutyric acid. The sections were examined by two independent pathologists who were blinded to the experiment protocols. Ki67 staining was scored as the result of staining intensity multiplied percentage, with staining intensity graded four levels (no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3), and the percentage of positive cells was classified into the following four grades: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%).

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.

MicroRNA in situ hybridization (ISH) Buffer and Controls kit (Exiqon, Vedbaek, Denmark) were used for ISH. The double (5′–3′) digoxigenin (DIG)‐labeled miR‐200a probe and U6 probe were purchased from Boster (Wuhan, China) and ISH was conducted according to the manufacturer's protocol of the microRNA ISH Optimization Kit (Boster, Wuhan, China). The expression of BRD4, Ki‐67, and caspase‐3 were detected by immunohistochemistry in the specimens which were fxed in 10% neutral‐buffered formalin and subsequently embedded in paraffn. After a brief proteolytic digestion and peroxidase blocking of the tissue slides which were cut to a thickness of 4 µm previously, the slides were incubated overnight at 4°C with the anti‐BRD4 (1:1000, Abcam, USA), anti‐ki67, and anti‐caspase‐3 (All 1:100, Boster, China). In situ hybridization and IHC were scored in a semiquantitative manner. The scoring system was conducted as follows: 0 (<5%), 1 (5%‐30%), 2 (31%‐70%), and 3 (≥71%). Additionally, the staining intensity was stratified as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Based on the intensity score, the percentage of positive cells were multiplied to obtain the final immunoreactivity scores (IRS) for Ki‐67. Apoptosis ability in the tumor xenografts was also determined from the number of Caspase‐3‐positive cells.