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: Evaluation of Fas expression on Nthy-ori 3–1 thyrocyte surface with glycosylation inhibitors for 72 h. Analysis was performed using PE-conjugated anti-Fas IgG1 by flow cytometry. (A) Representative overlay and single histograms. (B) Cumulative bar graph for MFI values. Results are expressed as mean ± SD. The statistical significance of the data was assessed using one-way ANOVA with Bonferroni correction. Statistical significance between IFNγ-treated cells and NT cells is indicated with asterisks as follows: ****p ≤ 0.0001.

Fig 4: 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 5: 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.