Fig. 2

Sensitivity of breast cancer MDA-MB-231 and T47D cells with varying expression of GAL3ST1 to apoptosis induced by doxorubicin and hypoxia. Cells grown in presence of doxorubicin at concentration of 0.5 μM (MDA-MB-231 cells) or 1 μM (T47D) and in hypoxic conditions (1% O₂) for 48 h. Cellular response measured by presence of active form of caspase-3 (a, b) or staining with Annexin V and propidium iodide (c, d). Statistically significant differences (**p < 0.01, *p <0.05). Flow cytometry dot plots show percentage of early apoptotic cells (Annexin V⁺/PI⁻, lower right) and late apoptotic cells (Annexin V⁺/PI⁺, upper right). Levels of galactosyloceramide (GalCer) and UGT8 in MDA-MB-231 and T47D cells with varying expression of GAL3ST1. Immunostaining of neutral glycolipids from MDA-MB-231, MDA.C and MDA.SUL cells (e) and from T47D, T47D.CRISPR.C and T47D.Δ.GAL3ST1.1 cells (f) separated by HP-TLC. For immunostaining, aliquots of neutral glycosphingolipids corresponding to 1 × 10⁷ cells applied to HP-TLC plate and stained with anti-GalCer rabbit polyclonal antibodies. g Real-time PCR used to analyse expression of UGT8 mRNA in MDA-MB-231 and T47D cell lines with varying expression levels of sulfatide. UGT8 levels normalised against β-actin and T47D.CRISPR.C cells assigned as calibrator sample. Results expressed as mean. h Western blotting analysis of UGT8 expression in breast cancer cell lines with different expression levels of sulfatide. Anti-UGT8 rabbit polyclonal antibodies used to detect UGT8 in cell lysates. FITC fluorescein isothiocyanate, GalCer galactosyloceramide, PI propidium iodide