Bca analysis

Cells were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China), and protein was harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, USA). After the incubation with a high affinity anti-p21 antibody (1:1000), anti-PTEN antibody (1:1000), anti-β-actin antibody (1:2000) (Cell Signaling Technology, USA), the membranes were then incubated with a secondary antibody (1:5000, Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.

Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extracts were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After incubation with a high-affinity anti-KRT6B antibody (1:2000, Proteintech, USA), anti-vimentin antibody (1:1000, Proteintech, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washing, the signals were detected using a chemiluminescence system (Millipore, USA) and analysed using Image Lab Software (Bio-Rad, USA).

Cells were lysed in RIPA lysis buffer. The protein was prepared and quantified using a bicinchoninic acid (BCA) analysis (Beyotime, China). Identical amounts of proteins were separated in 10% SDS–PAGE gels and transferred to PVDF membranes (Millipore, Germany). The proteins were blocked using 5% skim milk powder and incubated with the primary antibodies anti-GAPDH (ab9485) and anti-HMGA2 (ab207301) at 4°C for 12 h. Subsequently, the membranes were incubated with secondary antibody for 2 h. Finally, the blots were detected with an enhanced chemiluminescence kit (Pierce, USA), and the relevant information was obtained using Image Lab Software.

Cells were lysed in RIPA lysis buffer (RIPA, Beyotime, China). The protein was prepared and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). The same amounts of protein were extracted by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Schwalbach, Germany). The blocked protein with 5% skim milk powder was incubated with primary antibody anti-PTEN (#3285, Cell Signaling Technology), anti-YYM (#66281–1-Ig, Proteintech), anti-GAPDH (#ab181602, Abcam).at 4 °C for 12 h.Then the prepared membranes were incubated with secondary antibody (1:5000) for 2 h. Finally, the blots were detected by enhanced chemiluminescence kit (Pierce, Waltham, MA, USA) and related data was analyzed by Image Lab Software.

After the MDA-MB-231 cells were treated with XLLXF (50, 100, and 200 µg/mL) for 24 and 48 h, total cell protein lysates were extracted using RIPA lysis buffer that contained protease and phosphatase inhibitor cocktails. Protein lysates (20 µg), which were determined by BCA analysis (Beyotime, China), were loaded onto 10% SDS-PAGE gels. The protein bands were transferred onto NC membranes and blocked with 5% non-fat milk for 1 h at room temperature. The NC membranes with proteins were incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies used in the analyses were as follows: VEGFA (1:1,000, Proteintech), MMP2 (1:1000, Proteintech), MMP9 (1:1000, Cell Signaling Technology), Vimentin (1:1000, Proteintech), VE-cadherin (1:1,000, Cell Signaling Technology), TIMP-1 (1:1000, Proteintech), TIMP-3 (1:1,000, Proteintech), and Twist1 (1:1000, Proteintech). The membranes were incubated with relative sources of secondary antibodies (1:5000) at room temperature for 1 h. Specific protein bands were recognized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA). Image J software was used for image analysis.

Total proteins from cell lines and tissues were lysed with cold RIPA buffer (Beyotime) containing protease inhibitors (Sigma‐Aldrich). The protein concentration was measured by bicinchoninic acid (BCA) analysis (Beyotime). Total protein was separated by SDS‐PAGE gel, and transferred onto polyvinylidene difluoride membrane (Millipore). The membrane was blocked in 5% (w/v) non‐fat milk, and incubated with the primary antibodies for anti‐METTL3 (Abcam 1:1000), anti‐CCNE1 (Abcam 1:2000), anti‐GAPDH (Abcam 1:1000) at 4°C overnight, followed by incubation with horseradish peroxidase‐conjugate secondary antibody (Proteintech, 1:5000) for 1 hour at room temperature. After washes, signals were detected using a chemiluminescence system (Bio‐Rad) and analysed using ImageJ software (NIH).

Cells or tissues were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China). Bicinchoninic acid (BCA) analysis (Beyotime, China) was used to qualify the concentration of total proteins. Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, United States). The membranes were then blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h, followed by incubating with primary antibodies at 4°C overnight. After washing thrice by TBST, the membranes were incubated with secondary antibodies at room temperature for 1 h and washed thrice by TBST. Finally, the signals were detected using FDbio-Femto ECL (Fudebio, Hangzhou, China) and a chemiluminescence system (Bio-Rad, United States). The images were analyzed using Image J software (NIH).