Hoechst 33342 solution

HCT116 cells were plated in 24-well plates at a density of 1×10⁴ cells/well and cultured for 24 h at 37°C. Cells were then treated with different concentrations of reagents and cultured at 37°C in a humidified incubator containing 5% CO₂ for 48 h. Subsequently, 300 µl of 5 µg/ml Hoechst 33342 solution (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) was added to each well and the cells were stained in the dark for 10 min at 37°C. The morphology of the cells was observed and images were captured using a fluorescence microscope (magnification, ×400).
Following treatment for 72 h with different concentrations of reagents (0.03, 0.06, 0.12, 0.25 µM M3) at 37°C, apoptosis was analyzed using the Annexin-V-FLUOS Staining kit (Roche Diagnostics, Basel, Switzerland). Briefly, the cells were harvested, washed with PBS, centrifuged with 300 × g for 5 min at 4°C and stained with 100 µl Annexin-V-FLUOS labeling solution. Cells were incubated for 10–15 min at 15–25°C and then analyzed using a flow cytometer.

Hoechst33342 staining was used to observe the nuclear morphology of the apoptotic cells. The esophageal cancer cells (KYSE450 and KYSE30) were seeded (3×10⁵ cells/well) in a 6-well plate and cultured at 37°C in a humidified incubator with 5% CO₂ overnight prior to phlorizin treatment (0.00, 0.20 and 0.80 mM). After 48 h, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature, followed by staining with 3 µg/ml Hoechst33342 solution (Beijing Solarbio Science and Technology Co., Ltd.) for 10 min at room temperature in the dark. The stained cells were then washed with PBS and observed using an IX71 fluorescent microscope at ×400 magnification (Olympus Corporation).
Annexin V staining was used to detect apoptotic cells using live cells. The KYSE450 and KYSE30 cells were seeded (3.0×10⁵ cells/well) in 6-well plates and cultured at 37°C in a humidified incubator with 5% CO₂ overnight. Subsequently, the cells were treated with 0.1% DMSO or phlorizin (0.20 or 0.80 mM) for 48 h. After washing with PBS, the cells were labeled with an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences) and were visualized using an IX71 fluorescent microscope (magnification, ×100; Olympus Corporation).

Proliferation of HTM cells was detected by using BeyoClick EdU Cell Proliferation Kit (C0071S; Beyotime, Shanghai, China) according to the manufacturer's protocols. HTM cells were cultured and treated in 96-well plates at 5 × 10³ cells per well (three replicates for each group). Briefly, 10-µM EdU was added followed by incubation for 12 hours at 37°C. After cells were fixed with 4% paraformaldehyde for 15 minutes, they were permeabilized with 0.3% Triton X-100 in PBS. Nuclei were dyed with Hoechst 33342 solution (B804; Solarbio, Beijing, China) and captured by a high-content imaging analysis system at 100× magnification. Calculation of the percentage of EdU-positive cells was performed using ImageJ (National Institutes of Health, Bethesda, MD).