Fig. 3: Increase of O-GlcNAcylation level promotes proliferation and stemness. | Nature Communications

Fig. 3: Increase of O-GlcNAcylation level promotes proliferation and stemness.

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

Fig. 3

a Bright-field images of endometrial epithelial organoids (EE-Os) depicting responses to Thiamet G (TMG) at day 1 and day 3. Representative images from control dimethyl sulfoxide (DMSO) and 10 µM TMG treated EE-O groups are presented. Scale bar: 50 µm. b Immunoblot with RL2 antibody assessing O-GlcNAcylation level in EE-Os treated with DMSO, 5 µM, or 10 µM TMG for 48 h. Actin was used as the loading control. c Comparison of the EE-O numbers at day 3 of culture after 10 µM TMG or DMSO treatment. d Measurement of cross-sectional area of EE-Os at day 3 after treatment with 10 µM TMG versus DMSO. (Organoids derived from 6 biological replicates, n = 94 and 165 organoids). e Representative immunofluorescence images of control and TMG-treated EE-Os stained with PH3 (red), Tubulin (green), DAPI (blue), and F-actin labeled by Phalloidin (magenta). Scale bar: 5 µm. f Quantification of phospho-histone H3 (PH3) positive cells in each EE-O. (n = 31 and 22 organoids). g Representative immunofluorescence images of control and TMG-treated EE-Os. Ciliated epithelium is labeled by acetylated alpha-tubulin (Ac-tubulin, green), secretory cells by 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. h Quantification of the number of ciliated cells (Ac-tubulin + ) in each EE-O (n = 23 and 25 organoids). i qPCR analysis of stemness markers’ expression in EE-Os treated with TMG or DMSO, normalized to actin mRNA level. j Minimum-Distortion Embedding (MDE) projection of scRNA-seq data of DMSO and TMG treated EE-Os. k Subclustered epithelial populations of EE-Os (left), and the proportion of each subcluster in control and TMG-treated groups (right). Results in (a, b) show a representative example from n = 3 independent experiments. Results in (c, d) were derived from n = 6 biologically independent experiments, and results in (f–i) were derived from 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, f, h, i, k) are provided in the Source Data file.

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