Fig. 4: Decrease of O-GlcNAcylation level induces differentiation and cell death. | Nature Communications

Fig. 4: Decrease of O-GlcNAcylation level induces differentiation and cell death.

From: FBXO31-mediated ubiquitination of OGT maintains O-GlcNAcylation homeostasis to restrain endometrial malignancy

Fig. 4

a Representative bright-field images of endometrial cancer organoids (EC-Os) treated with 50 µM OSMI-1 at day 1 and day 3. Scale bar: 50 µm. b Immunoblot assessing O-GlcNAcylation level in EC-Os treated with 25 µM or 50 µM OSMI-1 for 48 h. Actin was used as the loading control. c Comparison of the numbers of EC-Os at day 3 after treatment with OSMI-1 versus the control DMSO. d Cross-sectional area of EC-Os at day 3 after OSMI-1 treatment compared to control (DMSO). (n = 101 and 57 organoids). e TUNEL staining showing apoptotic cells in EC-Os after 50 µM OSMI-1 treatment. Nuclei are visualized with DAPI (blue). Scale bar: 50 µm. f Representative immunofluorescence images of control and OSMI-1 treated EC-Os. Mitotic cells (PH3, red), tubulin (green), DAPI-labeled nuclei (blue), and Phalloidin labeled F-actin (magenta). Scale bar: 10 µm. g Quantification of the number of PH3+ cells. (n = 42 and 46 organoids). h Representative immunofluorescence images of control and OSMI-1 treated EC-Os showing ciliated epithelial cells (Ac-tubulin, green), secretory cells (PAEP, red), DAPI (blue), and F-actin (magenta). Scale bar: 50 µm. Insets show magnification of the area in the white box, scale bar: 5 µm. i Quantification of the number of Ac-tubulin+ cells. (n = 40 and 28 organoids). (j–l) qPCR analyses of stemness (j), EMT (k), and angiogenesis (l) markers in EC-Os treated with OSMI-1 or DMSO, normalized to actin mRNA. m Measurement of cell viability in patients-derived endometrial organoids with the indicated inhibitors. RLU represents relative light units. 5 replicates per each patient-derived organoid. Results in (a, b) show a representative example from n = 3 independent experiments. Results in (c, d) represent n = 6 biologically independent experiments, and results in (g, i-l) represent n = 3 biologically independent experiments, with p-values calculated by unpaired two-tailed Student’s t-test and data presented as mean ± SD. The source data for (b–d, g, i–m) are provided in the Source Data file.

Back to article page