expression constructs yepcrarl  (Federation of European Neuroscience Societies)

Journal: Nature Communications

Article Title: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network

doi: 10.1038/s41467-026-72077-5

Figure Lengend Snippet: a Scheme for the generation of an inducible β-cell-specific TBL1X and TBL1XR1 knockout. b Random fed blood glucose levels over time in iTBL/RβKO ( n = 9) and control ( n = 9) mice on HFD. c Total pancreas insulin content normalized to protein levels in iTBL/RβKO and control mice 4 and 11 weeks after tamoxifen administration. n = 3 for 4w control and iTBL/RβKO mice, n = 6 for 11w control mice, n = 4 for 11w iTBL/RβKO mice. d , e Blood glucose ( d ) and plasma insulin ( e ) levels during an oral glucose tolerance test after 22 weeks of HFD. Corresponding area under the curve in iTBL/RβKO ( n = 8) and control ( n = 9) mice ( d , right). f , g Relative mRNA expression determined by qPCR in pancreatic islets of iTBL/RβKO and control mice of Tbl1x and Tbl1xr1 ( f ) and islet genes ( g ). Control mice Tbl1x , Tbl1xr1 , Ins1 , Ins2 , Nkx6.1 , Slc2a2 , Mafa , Pdx1 , Pax6 , Ucn3 : n = 9, control mice ChgA , Ngn3 , Ldha , Hk1 : n = 8, iTBL/RβKO mice Tbl1x , Ngn3 , Ldha , Hk1 : n = 7, iTBL/RβKO mice Tbl1xr1 , Ins1 , Ins2 , Slc2a2 , Mafa n = 6, iTBL/RβKO mice Nkx6.1 , Pdx1 , Pax6 , Ucn3 , ChgA : n = 5. h α/β-cell mass ratio of pancreatic islets from iTBL/RβKO ( n = 4) and control mice ( n = 4) on HFD, 24 weeks after knockout induction. i Representative immunofluorescent staining of insulin + (blue, β-cells) and glucagon + (red, α-cells) cells of paraffin-embedded pancreas from iTBL/RβKO and control mice on HFD, 24 weeks after knockout induction. Each point represents one mouse. Data are represented as mean ± SEM. The following statistical tests were applied: two-sided student’s t test ( d – Area under the curve, f – h ) and 2-way ANOVA with Šidák’s multiple comparison post hoc test ( b – d – time course, e ). Source data are provided as a Source Data file.

Article Snippet: For this, INS1E cells were transfected with control siRNA or siRNA against TBL1X and TBL1XR1 for 72 h. After a medium change, cells were transfected with PAX6 expression constructs (purchased from Origene, BC011272 ), reporter gene constructs, and respective controls using Lipofectamine 2000 (Invitrogen, Cat# 11668027) for 48 h. For HDAC3 inhibition, expression vector transfected cells were exposed to either 10 μM BRD3308 (Sigma Aldrich, Cat# SML1639) or 5 μM RGFP966 (Cayman Chemicals, Cat# BYT-ORB636772) for 24 h. Firefly luciferase activity was normalized to renilla luciferase.

Techniques: Knock-Out, Control, Clinical Proteomics, Expressing, Staining, Comparison

Journal: Nature Communications

Article Title: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network

doi: 10.1038/s41467-026-72077-5

Figure Lengend Snippet: 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.

Article Snippet: For this, INS1E cells were transfected with control siRNA or siRNA against TBL1X and TBL1XR1 for 72 h. After a medium change, cells were transfected with PAX6 expression constructs (purchased from Origene, BC011272 ), reporter gene constructs, and respective controls using Lipofectamine 2000 (Invitrogen, Cat# 11668027) for 48 h. For HDAC3 inhibition, expression vector transfected cells were exposed to either 10 μM BRD3308 (Sigma Aldrich, Cat# SML1639) or 5 μM RGFP966 (Cayman Chemicals, Cat# BYT-ORB636772) for 24 h. Firefly luciferase activity was normalized to renilla luciferase.

Techniques: Immunoprecipitation, Negative Control, Transfection, Activity Assay, Knockdown, Over Expression, Luciferase, Gene Expression, Control, Comparison

Journal: Nature Communications

Article Title: TBL1X/TBL1XR1 govern β-cell identity through a PAX6-containing gene regulatory network

doi: 10.1038/s41467-026-72077-5

Figure Lengend Snippet: a IgG (negative control), H3K27ac (positive control), TBL1X, TBL1XR1, and PAX6 ChIP-qPCR in EndoC-βH1 cells for the human INS locus. The enrichment is calculated over the negative locus and the IgG control. n = 3 individual experiments. b Endogenous TBL1X, TBL1XR1, HDAC3, and SPT16 were immunoprecipitated in EndoC-βH1 lysates with rabbit IgG as negative control. Whole cell lysates (Input) and immunoprecipitated proteins were immunoblotted using the indicated antibodies. Representative blot is shown for 4 independent experiments. c Human INS ( hINS ) promoter activity in INS1E cells upon TBL/R1 knockdown. Luciferase activity normalized to renilla luciferase. n = 4 wells/condition. d Insulin secretion relative to insulin content in EndoC-βH1 cells upon TBL/R1 knockdown. After glucose starvation, insulin secretion was stimulated using 2.8 mM glucose (2.8 G) and 2.8 mM glucose supplemented with KCl (KCl). n = 4 wells/condition. e Scatter plot depicting the relationship between HbA1c [%] and relative TBL1X mRNA levels determined by qPCR, adjusted for age, body mass index (BMI), and disease state. Each dot represents an individual data point. n = 31. f , g Manhattan plot showing an association between elevated HbA1c levels and single nucleotide polymorphisms in the genetic region of TBL1X ( f ) and TBL1XR1 ( g ). Data are represented as mean ± SEM. The following statistical tests were applied: two-sided student’s t test ( a , c , d ), and linear regression ( e ). Source data are provided as a Source Data file.

Article Snippet: For this, INS1E cells were transfected with control siRNA or siRNA against TBL1X and TBL1XR1 for 72 h. After a medium change, cells were transfected with PAX6 expression constructs (purchased from Origene, BC011272 ), reporter gene constructs, and respective controls using Lipofectamine 2000 (Invitrogen, Cat# 11668027) for 48 h. For HDAC3 inhibition, expression vector transfected cells were exposed to either 10 μM BRD3308 (Sigma Aldrich, Cat# SML1639) or 5 μM RGFP966 (Cayman Chemicals, Cat# BYT-ORB636772) for 24 h. Firefly luciferase activity was normalized to renilla luciferase.

Techniques: Negative Control, Positive Control, ChIP-qPCR, Control, Immunoprecipitation, Activity Assay, Knockdown, Luciferase