Axio observer fluorescence microscope system

According to the manufacturer’s instructions, the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, Wisconsin, WI, USA) was used for the TUNEL assay to detect apoptotic cells. Lung cancer cells (2 × 10⁴ cells/well) were cultured on 8-chamber slides. The cells were washed with PBS and fixed by 4% paraformaldehyde in PBS for 20 min at room temperature. They were then permeabilized by 0.1% Triton X-100 in PBS for 5 min at room temperature. The slides were incubated with mounting media for fluorescence-containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) for 15 min, at room temperature and in the dark. After this, the cells were visualized using the ZEISS Axio Observer fluorescence microscope system (Carl Zeiss, Oberkochen, Germany). Digital images were analyzed using ZEN 2.1 software (Carl Zeiss).

According to the manufacturer’s instructions, the DeadEnd^TM Fluorometric TUNEL System (Promega, Madison, Wisconsin, WI, USA) was used for the TUNEL assay to detect apoptotic cells. Lung cancer cells (2 × 10⁴ cells/well) were cultured on 8-chamber slides after being were treated with ebractenoid F. The cells were washed with PBS and fixed by 4% paraformaldehyde in PBS for 20 min at room temperature. They were then permeabilized by 0.1% Triton X-100 in PBS for 5 min at room temperature. The slides were incubated with mounting media for fluorescence-containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) for 15 min, at room temperature and in the dark. After this, the cells were visualized using the ZEISS Axio Observer fluorescence microscope system (Carl Zeiss, Oberkochen, Germany). Digital images were analyzed using ZEN 2.1 software (Carl Zeiss).

The cells were fixed using 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were then permeabilized with 100% cold-methanol for 5 min and blocked using 4% bovine serum albumin in PBS with 0.1% Triton X-100 for 1 h. The primary antibodies were added, and the cells were incubated overnight at 4°C; then, Alexa Fluor 488 (#A32723, #A32731, Invitrogen, Carlsbad, CA) or Texas Red (#T-862, #T-2767, Invitrogen)-conjugated secondary antibodies were added and the cells were incubated for 1h at room temperature. The cells were then incubated with 1 μg/ml 4',6-diamidino-2-phenylindole (#D9542, Sigma-Aldrich, St. Louis, MO) for 5 min at room temperature and subsequently mounted using the Fluoromount-G Mounting Medium (#0100-01, Southern Biotech, Birmingham, AL). The cells were visualized using the ZEISS Axio Observer fluorescence microscope system (Carl Zeiss, Oberkochen, Germany). Digital images were analyzed using ZEN 2.1 software (Carl Zeiss). For quantitative analysis, 100 cells were counted in 10 random fields and performed at least 3 independent experiments. The size of the autophagosome was 0.5 to 2 μm, and the number of LC3 puncta (0.5 to 2 μm puncta size) per cell was counted.