Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a RNA-seq was performed in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3), with a threshold of |FC| > 1.5 and adjusted P-value < 0.05. KEGG enrichment analysis of genes upregulated in Adipoq Cre ; Ctnnb1 flox/flox mice relative to control mice. b mRNA expression of Wnt5a and Fzd5 in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). c Immunoblotting for Wnt5a, pCaMKK2, CaMKK2, pAMPK, AMPK, Atgl, Sirt1, Pgc1α in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). d Wnt5a and Fzd5 expression in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). e Immunoblotting for Wnt5a, pCaMKII, CaMKII, pCaMKK2, CaMKK2, pAMPK, AMPK in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). f mRNA expression of triglyceride lipolysis genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 5–6). g mRNA expression of fatty acid β-oxidation genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 6). h Immunoblotting for Cpt1, Atgl in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). i Immunoblotting for Sirt1, Pgc1α, Pparγ, Pparɑ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). j mRNA expression of Pparγ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 7). k Schematic summary of β-catenin suppression results in activation of Wnt/Ca 2+ -AMPK axis, highlighting non-canonical pathways involved in the thermogenic response. The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were one-way ANOVA with Tukey’s correction for multiple comparisons or unpaired two-tailed t -tests.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin in vitro by employing adipocytes differentiated from SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), MSAB, Rosiglitazone, or MSAB plus Rosiglitazone ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to suppress β-catenin while inhibiting the Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of Rosiglitazone-differentiated adipocytes treated with vehicle (0.1% DMSO), MSAB, or MSAB plus BAPTA-AM ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots and measured maximal respiration levels across the three treatment groups (Vehicle, MSAB, MSAB and BAPTA-AM) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests or two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress Wnt5a/Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Box5, MSAB, or MSAB plus Box5 ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to supply exogenous Wnt5a recombinant protein in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Wnt5a recombinant protein, MSAB, or MSAB plus Wnt5a recombinant protein ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots, measured basal respiration and measured maximal respiration levels in cultured peri-weaning adipocytes across the six treatment groups (Vehicle, Wnt5a recombinant protein, MSAB, MSAB plus Wnt5a recombinant protein, Box5, MSAB plus Box5) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot, Recombinant, Cell Culture

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin using adipocytes differentiated from human subcutaneous lipoaspirates. b Microscopy images of human subcutaneous adipocytes following treatment with vehicle (0.1% DMSO) and MSAB after maturation ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 30 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 30 cells). c mRNA expression of adipogenic genes in ( b ) ( n = 3 biological replicates). d mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). e Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis, Wnt/β-catenin signaling pathway and thermogenic protein in ( b ) ( n = 3 biological replicates). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: Microscopy, Staining, Labeling, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: PD0325901 alleviates thrombin-inhibited osteogenic differentiation through an IL-1β-activated feedback loop between MEK-Erk1/2 and NF-κB signal pathways: insights from bioinformatics and experimental verification

doi: 10.3389/fimmu.2026.1730337

Figure Lengend Snippet: Experimental validation of bioinformatically identified inflammatory cytokines, ECM-related, and calcification-regulatory genes. (A) Heatmap illustrating genes involved in regulating osteoblast growth. (B) PPI network of calcification-regulatory genes from BP terms, highlighting hub genes such as Igf1, Tgfb3, and MMP-9. (C) PPI network of ECM-regulatory genes derived from CC terms, highlighting hub genes such as Igf1, Wnt5a, and Spp1. (D) Western blot analysis and quantitative analysis of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin ratios in thrombin- and PD03-treated osteoblasts. (E) The ratios of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin were compared among different groups. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.

Article Snippet: The following antibodies were used in this study: β-actin (AF5001, Beyotime, China), Col1α1 (ab270993, Abcam, UK), Runx2 (AF2593, Beyotime, China), OCN (AF6297, Beyotime, China), Igf1 (AF7179, Beyotime, China), Wnt5a (29793-1-AP, Proteintech, USA), Tgfb3 (AF8142, Beyotime, China), Spp1 (A5427, Bimake, USA), COX-2 (F0327, Bimake, USA), Matrix Metalloproteinase-9 (MMP-9, AF5234, Beyotime, China), Minichromosome Maintenance Complex Component 2 (MCM2, A5172, Bimake, USA), Proliferating Cell Nuclear Antigen (PCNA, SC-25280, Santa, USA), PAR-1 (AF6837, Beyotime, China), IL-1RA (AF7218, Beyotime, China), p65 (8242S, CST, USA), p-p65 (3033T, CST, USA), Erk1/2 (4695T, CST, USA), p-Erk1/2 (4370S, CST, USA), p-Stat3 (Y705) (9145S, CST, USA), p-Stat3 (S727) (9134S, CST, USA), and Stat3 (4904T, CST, USA).

Techniques: Biomarker Discovery, Derivative Assay, Western Blot