Annexin 5 fitc pi

HeLa and BMDMs were seeded in a 12‐well plate and grown to 60% confluence before transfection. Transfection of C. albicans purified Sce1 protein was performed by PULSin protein delivery reagent (Polyplus‐transfection) with a total concentration of 2 µg/ml Sce1. The effectiveness of the method was confirmed by transfection of R‐phycoerythrin, a red fluorescent protein used as a positive control (Figure S8C). Exogenous expression of Sce1 in HeLa cells was assessed by transfection of pCDH plasmids expressing Flag‐tagged full‐length or N/C terminus of Sce1 into HeLa cells. To evaluate Sce1‐induced mammalian cell death, SYTOX Green dye or PI (Thermo Fisher Scientific) was added after the indicated time points. After incubation in the dark for 10 min, micrograph images were collected using an Olympus fluorescence microscope (Olympus IX73) equipped with 20× and 40× objective. Results were presented as percentages of SYTOX Green‐ or PI‐positive cells versus total live cells. To evaluate cell apoptosis, an Annexin V‐FITC/PI (Transgene) apoptosis detection kit was used. The Annexin V‐FITC/PI stained apoptotic cells were quantified and grouped by flow cytometry (BD Fortessa).

HT-29 (80,000 cells/well) and LoVo (100,000 cells/well) cells were plated into 24−well plates and cultured overnight. Following this, the cells were treated with small compounds at concentrations of 20 µM and 30 µM for either 24 h or 48 h. Following treatment, the cells were harvested, rinsed with cold PBS, and subsequently stained with Annexin V FITC/PI (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Cell death was detected by flow cytometry (ThermoFisher Scientific, Waltham, MA, USA).