Enhanced chemiluminescence

Total proteins were obtained using RIPA buffer (Beyotime, China), and mitochondrial proteins were extracted with Mitochondrial Protein Extraction Kit (BOSTER, China). Then, protein concentrations were determined via a BCA Protein Assay Kit (Beyotime, China). Proteins were separated through SDS-PAGE and transferred to PVDF membranes. After blocking in 5% BSA, the membranes were subsequently incubated with primary antibodies, including Cyclin D1 (1:500; #2978, CST, USA), Cyclin E (1:500; #20808, CST, USA), Bax (1:5000; 50599-2-Ig, Proteintech, China), Bcl-2 (1:500; 12789-1-AP, Proteintech, China), Cytochrome C (1:5000; ab133504, Abcam, UK), Dickkopf-1 (DKK1) (1:1000; 21112-1-AP, Proteintech, China), tissue inhibitor of metalloproteinase-2 (TIMP2) (1:500; A1558, Abclonal, China), and β-actin (1:2000; 60008-1-Ig, Proteintech, China) overnight at 4 °C. Afterwards, the membranes were incubated with the secondary antibody (1:10,000; SA00001-1 or SA00001-2, Proteintech, China) for 40 min at 37 °C. Signals were detected with enhanced chemiluminescence (7 Sea biotech, China).

Western blot was employed to detect the protein expressions of related genes in patients with different MDS risks, as well as in the cells transfected by siRNA or lentivirus in combination with JQEZ5 and decitabine treatment. The expression of proteins in blood samples or treated cells were analyzed by Western-blot. PBS were lysed by sonication in RIPA buffer (the cells were lysed sonication in RIPA buffer). The cells were fully mixed and transferred to the new EP tube, and then were centrifuged at 12,000*g for 10 min at 4 °C. After centrifugation, the supernatant was mixed with loading buffer and stored at − 80 °C. After loading the same amount of protein (50–100 μg) with 10% SDS-PAGE, electrophoresis was separated and then was transferred to the PVDF membrane (Millipore Corporation, Milford, MA, USA). The protein PVDF was transferred to the TRIS buffer which contained 5% skim milk powder overnight. The membrane was blotted with relevant primary antibodies (1:1500) for 2 h. After being washed with PBS and 0.1% Tween-20, the blot was incubated with secondary antibody (1:2000). The expression level of related proteins was determined by enhanced chemiluminescence (7sea Biotech, Shanghai, China). Each experiments was conducted more than 3 times.

Cells from different groups were harvested, washed in PBS and lysed in RIPA buffer with 1 μM PMSF (Solarbio cience & Technology, Beijing, China), then stilled at 4°C for 30 min followed by centrifugation for 10 min. Supernatants were loaded on 10% SDS-PAGE gel and the separated proteins transferred onto polyvinylidene fluoride membrane (Millipore Corporation, Milford, Massachusetts, USA), which was then blocked in 10% nonfat milk in Tris buffer for 2 h with agitation and washed. Then, the membrane was blotted with primary antibodies for 2 h. After washing, the membrane was incubated with secondary antibodies (HRP-conjugated goat anti-rabbit or anti-mouse; Beyotime) for 45 min at room temperature. All protein bands were visualized with the use of the enhanced chemiluminescence (7Sea Biotech). According to manufacturer's instructions, cytoplasmic proteins were extracted using the Beyotime cytoplasmic protein extraction kit, and nucleoproteins were extracted using the Beyotime nucleoproteins protein extraction kit. Equal amounts of protein lysate were used for western blotting analysis. All tests were repeated three times. Grey value analysis was determined by Quantity One 4.6.2.