Antifade mounting medium with dapi

Localization and expression of Smad9 and P-smad9 proteins in the GCs was evaluated by immunofluorescence (IF). Cells growing on the glass slide were washed in PBS, fixed in Immunol Staining Fix Solution (Beyotime Biotechnology) for 10 min, permeabilize with 0.5% Triton for 10 min, after that, blocked for 1 h at room temperature with 5% BSA. Then the GCs were incubated with antibodies against Smad9 (1:1000), P-smad9 (1:800) at 4°C for overnight. Cells were rinsed three times in PBS after primary antibody incubation and then were sequentially incubated with Alexa Fluor 555 donkey anti-rabbit secondary antibody (Beyotime Biotechnology, 1:1000) (Nie et al. 2012 (link)) for 1 h at room temperature. Finally, cells washed in PBS to omitting the antibodies mixture. After that, we used anti-fade Mounting Medium with DAPI (Solarbio, Beijing, China) to mount slides. Fluorescence images were acquired with an Olympus BX61 fluorescence microscope. Fluorescence images were recorded using a 40× objective lens.

RPMI 1640 medium, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco, USA. Minimum Eagle’s medium (MEM) was obtained from HyClone, USA. Antibiotic/antimycotic solution, kanamycin, puromycin, and dimethyl sulfoxide (DMSO) were obtained from SolarBio, China. DSF was purchased from Selleck, China, whereas CuCl₂ was obtained from J&K, China. Phenylmethanesulfonyl fluoride (PMSF), radioimmunoprecipitation assay (RIPA) buffer, and bicinchoninic acid (BCA) reagent were obtained from Beyotime, China. Cell counting kit-8 (CCK-8) reagent was purchased from Dojindo, Japan, whereas AnnexinV-FITC (AV-FITC), propidium iodide (PI) and PI kit were purchased from Roche, Germany. Electrochemiluminescence (ECL) was obtained from Biosharp, China. Lipofectamine 2000 (Lip2000) was purchased from Invitrogen, USA, whereas ROS assay kit was obtained from NJJCBIO, China. Seahorse XF Glycolysis Stress Test and XF Cell Mito Stress Test kits were purchased from Agilent, USA. DeadEnd™ Fluorometric TUNEL System was obtained from Promega, USA, whereas antifade mounting medium with DAPI was purchased from SolarBio, China.

The chromatin condensation was analyzed using DAPI staining^{43 (link)}, specifically, 1 × 10⁵ melanoma A375 cells were seeded into a well in a six-well plate placed with a sterilized glass coverslip in the well, and cultivated in the presence of 5% CO₂ gas at 37 °C for 24 h. The cells were subdivided into control group, CBP siRNA group (with 70% efficiency of CBP mRNA depletion), Ku70 siRNA group (with 70% efficiency of Ku70 mRNA depletion), CBP–Ku70 siRNA co-transfection group (with 60% efficiencies of CBP mRNA and Ku70 mRNA depletion, respectively), and NC siRNA group. The cells of each group were processed using 1 ml fix solution containing 4% paraformaldehyde (Beyotime Biotechnology, Nantong, China) for 10 min, when the cultivations were at 0, 8, 12, 16, 20, and 24 h, respectively. The fixed cells in each well were washed twice with PBS, and then further processed by adding 1 ml of Immunostaining Permeabilization Buffer containing Triton X-100 (Beyotime Biotechnology, Nantong, China) into the wells for 10 min at room temperature. The cells were then stained in 10 μl of Antifade mounting medium with DAPI (Solarbio, Beijing, China). The chromatin condensation was analyzed under a fluorescence microscope (Olympus, BX61; ×1000).