Antibodies against γ h2ax

After treatment, iCMs were washed twice with PBS and then fixed for 5 min at RT using a PFA-4%/sucrose-2% solution. Fixed cells were incubated for 10 min with glycine 0.1 M and then washed with PBS for 5 min. Cells were then permeabilized with 0.3% Triton X (Triton X detergent, Sigma-Aldrich) in PBS for 30 min, followed by a 5-min wash with PBS. Cells were then blocked with 2% bovine serum albumin (BSA) (Merck) for 1 h at RT. Subsequently, they were incubated with PBS containing 0.1% Tween, 1% BSA, and the primary antibody overnight at 4°C. To assess cardiac differentiation, cells were stained using antibodies against Sarcomeric Actin (α-S-Actin) (Abcam 9465), cardiac Troponin I (cTnI, AbCam 47003), cardiac Troponin T (cTnT) (13-11 Thermo Fisher Scientific), Myosin light chain 2 atrial (MLC2a, Synaptic System 311011), and Myosin light chain 2 ventricular (MLC2v, Proteintech 10906) to assess presence of DNA breaks, cells were stained using antibodies against γ-H2AX (9718 Cell Signaling), to assess cytotoxicity cells were stained with cleaved Caspase-3 Ab (Cell Signaling Technologies 9664). To assess senescence induction cells were stained with P16 and P21 (Proteintech 10355) Antibodies.

The apoptosis detection kit, cell cycle detection kit and comet assay kit were bought from KeyGen Biotech company (Nanjing, China). MTT (Thiazolyl blue tetrazolium bromide) and DAPI (DAPI, dihydrochloride) were obtained from Sigma Chemical Co. (MO, USA). Antibodies against GADD45A, ERK, p-ERK and β-actin were obtained from Proteintech Group Inc. (Chicago, USA). Antibodies against γH2AX was from Cell Signaling Technology (MA, USA) while the enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science, Inc. (United States). A specific inhibitor of MEK1/2 (PD0325901) was purchased from Selleck (Shanghai, China).

NSCLC cells were treated with 10 mM metformin and/or 25 μM celecoxib for 48 h. Cells were washed twice with PBS and then fixed in 4% (v/v) paraformaldehyde and permeabilized with 0.2% (v/v) Triton X-100 in PBS for 5 min. The TUNEL assay was performed by using a One Step TUNEL Apoptosis Assay Kit (Beyotime Inst Biotech, China). In brief, TUNEL detection solution was added to each sample and incubated at 37°C for 60 min. Then, the genomic DNA of apoptotic cells was broken, and the exposed 3′-OH was catalyzed by terminal deoxynucleotidyl transferase (TdT) to add dUTP labeled by FITC. After washing with PBS, the cells were restained with propidium iodide (PI). The fluorescent photos of the cells were captured by a fluorescence microscope (Zeiss, Jena, Germany). For immunofluorescence staining, antibodies against γ-H2AX (1:500 dilutions; Cell Signaling Technology, MA, USA) were used and then conjugated with Alexa Fluro^® 488 goat anti-rabbit IgG (1:2000 dilutions; Invitrogen, Carlsbad, CA). The fluorescent photos of the cells were captured by a fluorescence microscope (Zeiss, Jena, Germany).