Propidium iodide

A total of six groups of cells from the two cell lines were washed twice with cold PBS and resuspended in a 1 × 10⁶/ml cell suspension. Then, 100 μl of the cell suspension was incubated with 5 μl of FITC-Annexin V (Shanghai Yaji Biotechnology Co., Ltd., Shanghai, China) and 5 μl of propidium iodide (Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China) at room temperature in the dark for 15 min. SA-FLOUS solution was added, and the mixture was incubated at 4 °C for 20  min. Flow cytometry was used to detect the apoptosis rate. The experiment was repeated thrice, and the average value was calculated.

Cell apoptosis was performed on HepG2 and MHCC97H cells by flow cytometry. The cells were washed twice using washing buffer, and the suspensions were cultured with Annexin V-FITC and propidium iodide (Yeasen Biotechnology, Shanghai, China) in the dark at room temperature for 20 min. Next, binding buffer was added into each well. The samples were finally analyzed using FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) within 1 h.

To analyze septation and subcellular localization, fresh conidia and mycelia of the Fon strains were washed with sterile ddH₂O₂ and co-stained with CFW or Hoechst 33342, respectively [9 (link)]. For apoptotic cell death analysis, the germ tubes of the Fon strains were treated with/without apoptosis-inducing compound farnesol (FOH; Shanghai Aladdin Bio-Chem, Shanghai, China) at 25 or 50 μM for 4 h at 26 °C. Necrotic cells and nuclei were co-stained with propidium iodide (PI; Shanghai Yeasen Biotech, Shanghai, China) and Hoechst 33342 (Shanghai Yeasen Biotech, China), respectively [35 (link),36 (link)]. To examine autophagy, the Fon strains were cultivated in liquid CM for 12 h, and the collected mycelia were rinsed with sterile ddH₂O₂. After washing, the mycelia were transferred into nitrogen-starved MM medium (MM-N) with/without 4 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. The mycelia were stained with the fluorescent dye monodansylcadaverine (MDC; Sigma-Aldrich, St. Louis, MO, USA) for 30 min in the dark, followed by ddH₂O₂ washing [37 (link),38 (link)]. All aforementioned samples were observed under a Zeiss LSM 780 Meta confocal microscope (Gottingen, Niedersachsen, Germany) with the appropriate excitation and emission filters for corresponding dyes and signals.