Hoechst dye

Immunocytochemical staining of cultured cells was carried out following previously described procedures.^{18 (link),50 (link)} Briefly, neuronal cells were grown on glass coverslips, fixed with 4% PFA, blocked with 3% goat serum in PBST for 1–2 h and then incubated overnight with primary antibody in blocking solution. After washing, the coverslips were incubated with secondary antibody for 1 h and then incubated with Hoechst dye for nuclear staining and imaged under fluorescence microscopy (Leica, Wetzlar, Germany) with a digital spot camera.

To monitor HeLa cell viability and proliferation we used the xCELLigence Real-Time Cell Analyzer Dual Plate (RTCA DP, ACEA Biosciences, San Diego, CA, USA) as described in our previous study (Adamska et al. 2019 ). Briefly, the cells were seeded in E-plates 16 (ACEA Bioscences) at densities of 2 × 10⁴ cells/well. After 24 h, bersaldegenin-1,3,5-orthoacetate was added to the cells at a concentration range of 0.1–20.0 µg/mL for 24 h. Additionally, DMSO – as a solvent of the compound, was added to HeLa cells (a control sample) at a concentration of 0.25% (v/v). Vinblastine sulphate was used as a positive control. The RTCA software v. 1.2.1. calculated IC₅₀ values. All the experiments were performed in duplicate, in three independent repeats (n = 6).
To show the effect of bersaldegenin-1,3,5-orthoacetate in HeLa cell nuclei, we stained the cells with the blue fluorescent Hoechst 33342 dye (Life Technologies, Carlsbad, CA, USA). HeLa cells were seeded in 12-well plates at a density of 1 × 10⁵ cells/well. After 24 h, the cells were treated with bersaldegenin-1,3,5-orthoacetate at concentrations of 0.5, 1.0, 2.0, and 5.0 µg/mL for another 24 h. The concentration of the solvent (DMSO) in a control sample was 0.25% (v/v). Then, the cells were stained with the Hoechst dye (1.0 µg/mL) and observed under a fluorescent microscope (Leica, Heerbrugg, Switzerland).

The blue fluorescent Hoechst 33342 dye (Life Technologies, Carlsbad, CA, USA) was used to analyze the effect of α-HN on nuclei in SKOV-3 cells. The cells were seeded at a density of 5 × 10⁵ cells/well in 6-well plates and then treated with α-HN dissolved in DMSO at a concentration of 0.5, 2, and 10 µg/mL. DMSO concentration did not exceed 0.1% (v/v). Then, 24 h later, SKOV-3 cells were stained with 0.5 µg/mL of the Hoechst dye (dissolved in PBS) for 25 min in a CO₂ incubator and observed under a fluorescent microscope (Leica, Heerbrugg, Switzerland).