Propidium iodide

HeLa cells were cultured in full medium dissolved with various doses of flavonoids in A. conyzoides (50, 100, 200, 300, and 400 μg/mL) or sterile ddH₂O (control) for 24 h. For cell cycle analysis, HeLa cells were then collected and fixed with 70% ethanol overnight at 4°C, washed with PBS, and incubated with propidium iodide (PI, Sangon Biotech Co., Shanghai, China) for 10 min. For apoptosis analysis, HeLa cells were harvested and incubated with Annexin-V-fluorescein isothiocyanate (FITC)/PI (Sangon Biotech Co.) at room temperature in the dark for 15 min. Analyses were performed with a flow cytometer (Beckman Coulter, Miami, USA). Each experiment was repeated three times.

Cell membrane damage was analyzed using the method described by Collado et al. (Collado et al., 2017 (link)). The bacterial cell suspension was double dyed using propidium iodide (PI) (final concentration 10 mM) (Sangon, China) and 5(6)-carboxydiacetate fluorescein succinimidyl ester (CFDA) (final concentration 10 μM) (Beijing Solarbio Science & Technology Co. Ltd., China). Bacterial cell forward scatter, lateral astigmatism, and fluorescent channels FL1 (green) and FL2 (red) were determined using a flow cytometer BD FACSVerse™ (Becton, Dickinson and Company, USA). 1,000 cells were detected in each sample.
The cell membrane hydrophobicity and fluidity assays were performed as described by Pelletier et al. (Pelletier et al., 1997 (link)), and Voss and Montville (Voss and Montville, 2014 (link)), respectively.

Fresh spores were incubated in PDB medium (1.0 × 10⁶ spores mL⁻¹) with or without 0.25 mg L⁻¹ cinnamon oil for 6 h at 25 °C under 200 rpm shaking condition. The spores were collected by centrifugation, washed by phosphate buffer solution (PBS, 20 mmoL L⁻¹, pH 7.4), and stained by 5μmoL L⁻¹ fluorescein diacetate (No. A600202, Sangon, Shanghai, China), 5 μmoL L⁻¹ MitoTraker Orange (Invitrogen, Carlsbad, CA, USA), 50 mg L⁻¹ 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI, No. A606584, Sangon, Shanghai, China), and 20 mg L⁻¹ propidium iodide (PI, NO. E607306, Sangon, Shanghai, China). For PI staining, half of the cinnamon oil-treated spores were incubated in boiling water for 10 min, which was as a positive control. The staining was performed following the product instruction. Then, stained spores were observed microscopically and photographed by a Nikon Eclipse Ni-U microscope with individual filter sets.

Bcap-37 cells (2×10⁴ cells/well) were seeded into 6-well plate (Corning, Elmira, NY) overnight. The cells of apoptosis assay were treated with different concentrations (30, 60 and 90 µg/mL, respectively) of TAT-BIR(T34A) for 36 h or 60 h, then the cells were harvested by centrifugation at 1000 rpm for 8 min. After a twice-wash step with PBS, the cells were re-suspended in 500 µL of Binding Buffer. Staining was done by incubating cells with with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI) (50 µg/mL) (Sangon Biotech, China) for 15 min at room temperature in the dark. The samples were analyzed by flow cytometry assay. The samples for cell cycle assay were prepared by following subsequent procedures: treatment with 30 µg/mL of TAT-BIR(T34A), TAT-CC(T117A), and TAT-Survivin(T34/117A) for 48 h, fixation with ice-cold 70% (v/v) ethanol in PBS (pH7.4) at 4°C overnight after centrifugation at 300 g for 5 min, staining with 50 µg/mL PI ( including 50 μg/mL RNase A) for 30 min. The prepared samples were analyzed by flow cytometry (Becton Dickinson, USA).

CLSM assays were conducted as previously described [43 (link)]. Briefly, overnight cultures of S. aureus diluted 1:100 in TSB + G medium were inoculated into the glass-bottom cell culture dish (15 mm in diameter; Nest, Wuxi, China), and cultivated without shaking at 37 °C for 24 h. After washing with PBS and fixed with 4% polyoxymethylene, the biofilms were stained with 50 μg/mL FITC-conjugated Concanavalin A (FITC-ConA) (Sigma-Aldrich, St. Louis, MI, USA) and 5 μg/mL propidium iodide (PI) (Sangon Biotech, Shanghai, China) at room temperature in the dark, respectively. The biofilms were then visualized with a LSM800 CLSM (Zeiss, Jena, Germany) with 488 nm excitation and 537 nm emission wavelengths for FITC-ConA, 535 nm and 615 nm for PI, respectively. A series of optical sections were observed and rendered in three-dimensional (3D) mode using ZEN 2012 lite software.
To determine the direct effect of Sak on biofilm formation, S. aureus strain XN108 was cultivated in TSB + G medium supplemented with rSak at a concentration of 0, 5, and 10 µg/mL, respectively. The biofilms were statically formed in the glass-bottom cell culture dish at 37 °C for 24 h, and analyzed by CLSM as mentioned above.

Fresh spores of P. digitatum were treated by 0 or 0.03% CBO in PDB medium for 4 h and 8 h at 25 °C under oscillating condition. According to the product instructions, the harvest spores were suspended in 20 mmoL L⁻¹ phosphate buffer solution (pH 7.4), and stained by fluorescein diacetate (FDA) (Sangon, Shanghai, China) with a work concentration of 5μmoL L⁻¹, MitoTraker Orange (MTO) (Invitrogen, Carlsbad, CA, USA) with a work concentration of 5 μmoL L⁻¹, and propidium iodide (PI) (Sangon, Shanghai, China) with a work concentration of 20 mg L⁻¹. The spores, which were treated with 0.03% CBO for 4 h and then incubated in boiling water for 10 min, were set to the positive control in the experiment of PI staining. Afterward, the spores were photographed by a fluorescence microscope (Eclipse Ni-U, Nikon, Tokyo, Japan).