Annexin 5 pe 7 add apoptosis detection kit

Flow cytometry was conducted based on the manufacturer’s instructions. Cells were measured with a BD Beckman cytometer (BD Biosciences) and analyzed using FlowJo software after incubating with the Annexin V-PE/7-ADD Apoptosis Detection Kit (Vazyme, A213-01). Similarly, cells were incubated with the PI/RNase Staining Kit (BD Biosciences) to analyze the cell cycle and were quantified with a BD Beckman cytometer.

Trametinib and U0126 were purchased from MedChemExpress (Monmouth Junction, NJ, United States). Dimethyl sulfoxide was used to dissolve Trametinib powder to an initial concentration of 10 mM and preserved in −80°C. The CCK-8 kit was obtained from Dojindo (Rockville, MD, United States). The Annexin V-FITC/PI apoptosis detection kit and Annexin V-PE/7-ADD apoptosis detection kit were purchased from Nanjing Vazyme Biotech (Nanjing, China). Penicillin and streptomycin were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, United States). DMEM were purchased from CORNING (Corning Life Sciences, Bedford, MA, Unied States). The fetal bovine serum (FBS) was purchased from TBD (Tianjin, China). Lipofectamine 3,000 and TRIzol™ Reagent were obtained from Life Technologies (Carlsbad, CA, United States). XF96 seahorse assay plate was purchased from Agilent (Santa Clara, CA, United States). The antibodies used were as follows: anti-PKM2 antibody (#15822-AP), anti-c-myc antibody (#67447-1-lg), anti-GLUT1 antibody (#21829-1-AP), anti-LDHA antibody (#19987-1-AP), anti-β-actin antibody (#66009-1-lg), MEK1/2 polyclonal antibody (#11049-1-AP), ERK1/2 polyclonal antibody (#16443-1-AP), and anti-PCNA antibody (10205-2-AP) from Proteintech (Wuhan, China); and anti-Ki67 antibody (ab279653) from Abcam (Cambridge, United Kingdom).

The manufacturer’s instructions were followed when performing flow cytometry. Apoptosis was detected with the Annexin V-PE/7-ADD Apoptosis Detection Kit (Vazyme, A213-01). The B525 nm wavelength was selected for the Fluorescein (FITC) signal channel, and the B610 nm signal channel was selected for the ECDPE-TR (ECD) signal channel.