Fig. 1: O-GlcNAcylation is increased during myofibroblastic HSC activation.

A Representative images from co-immunostaining for the fibroblast marker PDGFRB (red) and O-GlcNAcylated proteins (green) in paraffin-embedded human liver sections from patients with alcohol-related cirrhosis (n = 10 biologically independent samples). White squares indicate regions for which a zoom image is provided on the right. Scale bars=5,000 µm for images showing entire sections and 500 µm for zoom image. Data obtained with livers from additional donors are shown in Supplementary Fig. 1A. B Graphical representation of experimental set-ups used in panels C–E to analyze O-GlcNAcylation in purified quiescent (Q-HSCs at 1 day (d) of culture) and spontaneously in vitro activated myofibroblastic HSCs (MF-HSCs at 7 d of culture). C Western blot and simple western immunoassays showing O-GlcNAcylation, OGT and ACTA2 protein levels in Q-HSCs and MF-HSCs. Ponceau S staining was used as protein loading control. The presented images are representative of 3 biologically independent experiments. MW, molecular weight markers. Log₂ fold changes (log₂ FC) between MF-HSCs and Q-HSCs are shown. D Immunofluorescence staining of protein O-GlcNAcylation with anti-O-GlcNAc antibody of Q-HSCs and MF-HSCs (shown images are representative of 4 biologically independent experiments). Scale bars=50 µm. Log₂ FC between MF-HSCs and Q-HSCs is shown. E Immunofluorescence staining of protein O-GlcNAcylation in Q-HSCs and MF-HSCs revealed by metabolic labeling with GlcAz for 24 h followed by click chemistry with an alkyne-containing dye (shown images are representative of 5 biologically independent experiments). Scale bars = 50 µm. Log₂ FC between MF-HSCs and Q-HSCs is shown. F Western blot assays showing O-GlcNAcylation, OGT and ACTA2 protein levels in LX-2 cells cultured in regular 2D conditions or grown as spheroids in 3D conditions for 72 h. A condition where spheroids were digested and cells put back into regular 2D culture plates (3D→2D) for 72 h is also shown. HSP90 was used as protein loading control. The presented images are representative of 5 biologically independent experiments. MW, molecular weight markers. Log₂ FC between 3D and 2D cells (left panel), and between 3D→2D and 3D (right panel) are shown on the right. For all panels, the bar graphs show means + standard deviation (SD) together with individual biological replicates. Two-sided one-sample t-test with Benjamini–Hochberg correction for multiple testing was used to determine if the mean log₂ FC was statistically different from 0.