Fig. 7: O-GlcNAcylation controls the myofibroblastic BNC2/TEAD4/YAP1 transcriptional regulatory complex.

A LX-2 cells were treated or not with 50 µM of the OGT inhibitor OSMI-1 (referred to as OGTi) for 24 h. Western blot assays and their quantifications of BNC2, TEAD4, YAP1, and phospho-YAP1 (S127) protein levels are shown. HSP90 was used as protein loading control. The presented images are representative of 4 biologically independent experiments. MW, molecular weight markers. Log₂ fold changes (log₂ FC) between OGTi treatment and control conditions (right panel) are shown. Two-sided one-sample t-test with Benjamini–Hochberg correction for multiple testing was used to determine if the mean log₂ FC between OGTi and control conditions was statistically different from 0. B LX-2 cells were treated as in A. RT-qPCR was used to monitor gene expression (n = 4 biologically independent experiments). Log₂ FC between OGTi treatment and control conditions are shown. Two-sided one-sample t-test with Benjamini–Hochberg correction for multiple testing was used to determine if the mean log₂ FC between OGTi and control conditions was statistically different from 0. C. LX-2 cells were pre-treated with 20 µg/mL cycloheximide (CHX) for 1 h and then treated with CHX combined or not with OGTi (50 µM OSMI-1) for an additional 24 h. Western blot and simple western immunoassays for BNC2, TEAD4, YAP1, CCND1, and protein O-GlcNAcylation levels together with quantifications of BNC2, TEAD4, YAP1, and O-GlcNAcylation levels are shown. HSP90 was used as protein loading control. The presented images are representative of at least 4 biologically independent experiments. MW, molecular weight markers. Log₂ FC between CHX + OGTi and CHX conditions are shown. Two-sided one-sample t-test with Benjamini-Hochberg correction for multiple testing was used to determine if the mean log₂ FC between CHX + OGTi and CHX conditions was statistically different from 0. D LX-2 cells were treated as in A. Nuclear extracts were subjected to immunoprecipitation with an antibody against TEAD4 (ab58310, Abcam). Input and immunoprecipitated materials were analyzed by western blot and simple western immunoassay using antibodies directed against BNC2, TEAD4, or YAP1. LMNA was used as protein loading control. The presented data are representative of two biologically independent experiments. E LX-2 cells were treated as in A and sub-cellular fractionation was performed to obtain cytosolic and chromatin fractions. Western blot and simple western immunoassays of BNC2, TEAD4, YAP1, and P-YAP1 (S127) levels and quantifications of chromatin-bound BNC2, TEAD4 and YAP1 are shown. HSP90 and LMNA were used as protein loading controls. The presented images are representative of at least 4 biologically independent experiments. MW molecular weight markers. Two-sided one-sample t-test with Benjamini–Hochberg correction for multiple testing was used to determine if the mean log₂ FC between OGTi and control conditions was statistically different from 0. F LX-2 cells were transfected with a control luciferase reporter vector (pGL3-basic (pGL3b) promoter) or a TEAD-responsive luciferase reporter vector (pGL3b-8xGTIIC-luciferase plasmid) for 68 h and then treated or not with OGTi (50 µM OSMI-1) for an additional 24 h (n = 3 biologically independent experiments). Luciferase activities relative to that obtained with the pGL3b-8xGTIIC untreated (control) condition arbitrarily set to 100 are shown. Two-way ANOVA with Sidak multiple comparison post-hoc test was used. For all panels, the bar graphs show means + SD together with individual biological replicates.