Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: ChIP-SICAP identifies the transcription factor CUX1 as a positive regulator of human adipocyte differentiation. (A) Schematic of C/EBPβ ChIP-SICAP experiments in hAPCs. (B) C/EBPβ ChIP-SICAP was performed at day 0 (undifferentiated hAPCs) and one day (day 1) after inducing adipocyte differentiation. Proteins enriched at day 1 vs. day 0 (FC > 3, P value < 0.0005) were annotated. (C) Expression heatmap shows mRNA levels of adipocyte markers in hAPCs in which individual genes (from B) were knocked-out (deleted genes on x-axis) relative to control cells at day 3 of differentiation. (D) SNPs near CUX1 that are associated with altered susceptibility to type 2 diabetes (data from T2D knowledge portal). (E) CUX1 mRNA levels in subcutaneous adipose tissue from non-diabetic (n = 31) and T2D (n = 39) subjects. GADPH was used for normalization. Values represent mean ± SEM. (F) Western blot analysis of CUX1 and GAPDH (loading control) protein levels in control (ctrl) and CUX1 knock-out (KO) hAPCs. CUX1 forms are labelled (see details in ): Full length (FL) and Cut-associated splicing product (CASP). (G) mRNA levels of CUX1 and indicated adipocyte marker genes in ctrl and KO cells on day 14 of differentiation. Values represent mean ± SEM, n = 3 biol. repl. per condition. (H, I) Bodipy staining (H) and TG levels (I) in ctrl and KO cells. Values represent mean ± SEM, n = 3 biol. repl. per condition. E,I, unpaired two-tailed Student's t tests; G, one-way ANOVA followed by Dunnett multiple comparisons. ∗P < 0.05.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Expressing, Control, Western Blot, Knock-Out, Marker, Staining, Two Tailed Test

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: The full-length isoform of CUX1 mediates its pro-adipogenic function in hAPCs. (A) Gene structure of human and mouse CUX1 isoforms: CUX1 full-length ( CUX1 -FL) and alternatively spliced product ( CUX1 -CASP). (B, C) mRNA levels of FL and CASP isoforms in hAPCs (B) and mAPCs (C) at day 3 of differentiation. (D, E) mRNA levels of FL and CASP isoforms in undifferentiated hAPCs and hAPC-derived adipocytes (D); and mAPCs and mAPC-derived adipocytes (E). (F) Western blot analysis of CUX1 and GAPDH (loading control) in hAPCs with CRISPR-mediated knockout (KO) of CUX1-CASP only or both CASP and FL isoforms. (G) Bodipy staining of triglycerides in Control (ctrl), CASP KO, and CUX1 KO cells at day 14 of differentiation. (H) mRNA levels of adipocyte genes in cells from (G). (I) Western blot analysis of CUX1 and GADPH (loading control) in hAPCs transduced with ctrl or CUX1-expressing lentivirus and differentiated for 14 days. (J) Oil-red-O staining of triglycerides in ctrl- and CUX1-FL-expressing cells at day 14 of differentiation. (K, L) mRNA levels of CUX1 and adipocyte genes in ctrl- and CUX1-FL-expressing hAPCs at day 3 (K) or day 14 (L) of differentiation. Gene expression values (B-E, H,K,J) represent mean ± SEM, n = 3 biol. repl. per condition. B-E, unpaired two-tailed Student's t tests; H, two-way ANOVA followed by Sidak's test; K,L, one-way ANOVA followed by Dunnett multiple comparisons test. ∗P < 0.05, #P < 0.05.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Derivative Assay, Western Blot, Control, CRISPR, Knock-Out, Staining, Transduction, Expressing, Gene Expression, Two Tailed Test

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: CUX1 activates PPARG and adipogenic genes in hAPCs. (A) Top enriched pathways (Biological Processes (BP) database) identified from genes that are: downregulated by CUX1 knockout (KO) and upregulated by CUX1-overexpression in hAPCs at day 3 of differentiation. (B) Expression heatmap of adipocyte genes and precursor marker genes ( DPP4 , ICAM1 ) in CUX1 KO and OE hAPCs, relative to control cells (Ctrl) at day 3 of differentiation. (C) ChIP-seq tracks for CUX1 at COPS8 , PPARG, and EBF2 in Ctrll and CUX1 KO hAPCs one day after treatment with differentiation cocktail (day 1). (D) Motif analysis of CUX1 binding regions identified in Ctrl hAPCs. (E) ChIP-qPCR analysis of CUX1 binding at the +66 kb region (labeled in (C)) of PPARG in control and KO hAPCs stimulated with differentiation cocktail for 1 day. Values represent mean ± SEM, n = 3 biol. repl., unpaired two-tailed Student's t test. (F) Transcription assay showing activity of the +66 kb region of PPARG in immortalized hAPCs transfected with control or CUX1-expressing vector. Values represent mean ± SEM, n = 3 biol. repl., one-way ANOVA followed by Dunnett multiple comparisons test. (G) mRNA levels of adipocyte genes in control and KO cells treated with vehicle (control) or 1 μM rosiglitazone at day 14 of differentiation Values represent mean ± SEM, n = 3 biol. repl., two-way ANOVA followed by Sidak's test. (H) Plot showing correlation between CUX1 and PPARG mRNA levels in subcutaneous adipose tissue from healthy, nondiabetic female individuals (n = 39). ∗P < 0.05, ns, not significant.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Knock-Out, Over Expression, Expressing, Marker, Control, ChIP-sequencing, Binding Assay, ChIP-qPCR, Labeling, Two Tailed Test, Transcription Assay, Activity Assay, Transfection, Plasmid Preparation

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: Cux1 represses mouse adipocyte differentiation. (A – D) APCs were isolated from inguinal white adipose tissue (iWAT) or epididymal WAT (eWAT) of control and APC-selective Cux1 KO mice (male, age 6–8 weeks) 5 days after tamoxifen injection and then induced to differentiate for 9 days. (A, C) Western blot analysis of CUX1 and GADPH (loading control) in cells from iWAT (A) and eWAT (C). (B, D) mRNA levels of adipocyte genes in cells from iWAT (B) and eWAT (D). (E) Western blot analysis of CUX1 and GADPH (loading control) in control and CUX1-overexpressing (OE) hAPCs that were differentiated for 9 days. (F) mRNA levels of adipocyte genes in cells from (E). (G) ChIP-seq tracks for CUX1 in APCs isolated from inguinal WAT of control and KO mice. (H) Motif analysis of CUX1 binding regions in mAPCs stimulated with differentiation cocktail for 1 day. B, D, F, values represent mean ± SEM, n = 3 biol. repl. per condition, one-way ANOVA followed by Dunnett multiple comparisons test. ∗P < 0.05.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Isolation, Control, Injection, Western Blot, ChIP-sequencing, Binding Assay

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: Loss of Cux1 in mouse APCs enhances high fat diet-induced adipocyte differentiation. Control and KO mice harboring a tdTomato reporter gene were injected with tamoxifen (TMX) and fed a high fat diet (HFD) for 8 weeks. (A) Body weights. (B) Adipose tissue depot weights. (C) Immunofluorescence staining of PLIN1 (green) and tdTomato (new adipocytes, red) in iWAT (Top) and eWAT (Bottom). Scale 50 μm. (Right panels) Quantification of new adipocytes (tdTomato+; PLIN1+) as proportion of total adipocytes (PLIN1+). Values represent mean ± SEM, n > 5 sections from n = 5 animals per genotype. ( D , E ) H&E staining of iWAT (D) and eWAT (E). Scale 50 μm. (Right panels) Quantification of average adipocyte size in adipose tissue depots. Values represent mean ± SEM, n > 5 sections from n = 5 animals per genotype. Unpaired two-sided t-test. ∗P < 0.05.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Control, Injection, Immunofluorescence, Staining