Fig 2: Effect of glycosylation inhibitors on the morphology of cell nuclei of thyrocytes. Human recombinant IFNγ (20 ng/mL) stimulated Fas expression and human recombinant FasL (20 ng/mL) induced thyrocyte apoptosis. Cells were stained with DAPI and analyzed in fluorescence microscopy. Red arrows show fragmented cell nuclei and apoptotic bodies, green arrows indicate cellular divisions of the nucleus.

Fig 3: Detection of apoptosis in Nthy-ori 3–1 cell line treated with glycosylation inhibitors for 72 h. Human recombinant IFNγ (20 ng/mL) stimulated Fas expression, and human recombinant FasL (20 ng/mL) induced thyrocyte apoptosis, which was determined by annexin V-FITC staining in flow cytometry. (A) Representative dot plots for all tested variants. (B) Stacked bar graph for % Annexin V-FITC+ PI- (early apoptosis) and Annexin V-FITC+ PI+ (late apoptosis) cells. The results are shown as mean values ± SD. The statistical analysis was performed using one-way ANOVA with Bonferroni correction. Statistical significance between IFNγ + FasL-treated cells vs NT cells is marked with asterisks as follows * p ≤ 0.05, and between Kf- and Sw-treated cells in apoptotic variants (IFNγ +FasL + Kf; IFNγ +FasL + Sw) relative to apoptotic cells with unmodified glycosylation (IFNγ +FasL) is marked with crosses as follows #p ≤ 0.05.

Fig 4: Proposed mechanism of swainsonine regulation of Fas-mediated apoptosis in Nthy-ori 3–1 thyrocytes. (A) In Sw-untreated thyrocytes, N-glycosylation reaches the final stage of complex-type structures, which are attached to Fas predominantly. Complex-type N-glycans on Fas are crucial to generating intracellular signals resulting in apoptosis. (B) Sw blocks α-Man II at the rough endoplasmic reticulum which leads to the attaching of oligomannose and hybrid-type N-glycans to Fas. The signal generated by Fas-FasL interaction is significantly lowered which prevents thyrocytes from apoptosis. Created in BioRender. Trzos, S. (2025) https://BioRender.com/s03e383.

Fig 5: MALDI-Tof mass spectrometry analysis of N-glycans from Nthy-ori 3–1 thyrocytes with induced apoptosis. N-oligosaccharides were analyzed in positive ion reflectron mode. (A) Heatmap showing quantitative analysis of all structures detected by MALDI-Tof MS analysis. (B) Quantitative analysis for common structural features of N-glycans: fucosylation, galactosylation, α2,3-sialylation, and α2,6-sialylation. (C) Quantitative analysis for major glycan types: oligomannose, hybrid, and complex. The results are presented as mean values. The statistical significance was assessed using the Brown-Forsythe and Welch one-way ANOVA test with Dunnett T3 correction. Significance levels between treated cells (IFNγ and IFNγ + FasL) versus NT control are marked with asterisks as follows *p ≤ 0.05 and **p ≤ 0.01. Comparisons for treated cells IFNγ +FasL versus IFNγ are marked with crosses as follows #p ≤ 0.05 and ##p ≤ 0.01.