Fig. 5: TBL1X and TBL1XR1 are regulators of PAX6 mediated gene expression.
From: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network
a, b 5 selected metabolic processes identified via gene ontology analysis using significantly enriched TBL1X (a) and TBL1XR1 (b) interaction partners with a unique peptide count > 2 identified in the interactome screen. c, Endogenous TBL1X, TBL1XR1, HDAC3, and SPT16 was immunoprecipitated in INS1E cell lysates with rabbit IgG as negative control. Whole cell lysates (Input) and immunoprecipitated proteins were immunoblotted using the indicated antibodies. d TBL1X, TBL1XR1, and vinculin protein levels in INS1E cells after siRNA transfection. e Murine Ins2 (mIns2) promoter activity in INS1E cells upon TBL/R1 knockdown or/and PAX6 overexpression. Luciferase activity normalized to renilla luciferase. n = 4 wells/condition. f Endogenous HDAC3 and SPT16 was immunoprecipitated in INS1E cell lysates upon TBL/R1 knockdown with rabbit IgG as a negative control. Whole cell lysates (Input) and immunoprecipitated proteins were immunoblotted using the indicated antibodies. g Murine Ins2 (mIns2) promoter activity in INS1E cells upon TBL/R1 knockdown and/or PAX6 overexpression with/ without HDAC3 inhibitor (BRD3308, 1 µM, 24 h). Luciferase activity normalized to renilla luciferase. n = 4 wells/condition. h Relative Ins2 gene expression in INS1E cells upon TBL/R1 knockdown determined by qPCR. n = 4 wells/condition. i Insulin secretion relative to insulin content in INS1E cells upon Tbl1xr1 overexpression. Insulin secretion was stimulated using 2 mM glucose (2 G), 20 mM glucose (20 G), and 2 mM glucose supplemented with KCl (KCl). Control 2 G n = 3 wells/condition, all other data n = 4 wells/condition. j Insulin secretion in murine pancreatic islets upon Tbl1xr1 overexpression. Insulin secretion was stimulated using 2.8 mM glucose (2.8 G), 16.8 mM glucose (16.8 G), and 2.8 mM glucose supplemented with KCl (KCl). n = 5. k Ins2 gene expression determined by qPCR in murine pancreatic islets upon adenovirus-mediated Tbl1xr1 overexpression. n = 3 wells/condition. Data are represented as mean ± SEM. The following statistical tests were applied: 1-way ANOVA with Dunnett’s multiple comparison post hoc test (e), 2-way ANOVA with a Tukey’s multiple comparison post hoc test (g), and a two-sided student’s t test (h–k). ns = no significance,*p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.
