Journal: Advanced Science

Article Title: Caspase‐6 Controls Lipid and Energy Metabolism in Diet‐Induced Obesity

doi: 10.1002/advs.202514784

Figure Lengend Snippet: Caspase‐6 cleaves PPARγ and SP1 to control ATGL expression in adipocytes. (A, B) Immunoblot of PPARγ and ATGL proteins in eWAT (A) and iWAT (B) of WT and C6KO mice fed 60% HFD for 12 weeks (n = 4). Two tailed unpaired Student's t ‐test. (C) In vitro cleavage of recombinant PPARγ by active caspase‐6. Immunoblot of PPARγ. (D) Immunoblot of lysates from 3T3‐L1 adipocytes treated with cycloheximide (5 µg/ml) and TNFα (25 ng/ml) for indicated time with or without pretreatment of Emricasan (50 µg/ml) for 1 hr. (E, F) Immunoblot SP1 protein in eWAT (E) and iWAT (F) of WT and C6KO mice fed HFD for 12 weeks (n = 3). Two tailed unpaired Student's t ‐test. (G) In vitro cleavage of recombinant SP1 by active caspase‐6. Coomassie blue staining of gel. (H) Immunoblot of lysates from 3T3‐L1 adipocytes treated with cycloheximide (CHX, 5 µg/ml) and TNFα (25 ng/ml) for indicated time with or without pretreatment of Emricasan (50 µg/ml) for 1 hr. (I) Immunoblot of lysates from ex vivo cultured eWAT from WT or C6KO, treated with CHX (15 µg/ml) and TNFα (75 ng/ml) for indicated time. (J) Pnpla2 expression in 3T3‐L1 adipocytes treated with Rosiglitazone (5µM) or T0070907 (100 nM) in the presence or absence of Mithramycin (10 µM) for 6 h (n = 3). Two tailed unpaired Student's t ‐test. (K) In vitro cleavage of WT and mutant (D69E) PPARγ2 expressed in HEK293T by recombinant active caspase‐6. (L) In vitro cleavage of WT and mutant (D185E) SP1 expressed in HEK293T by recombinant active caspase‐6. (M) PPARγ activity reporter assay in HEK293T cell expressing PPRE‐H2B‐eGFP along with WT PPARγ or PPARγ D69E mutant in the absence or presence of CHX‐TNFα treatment (n = 8). Two tailed unpaired Student's t ‐test. (N‐V) Pearson correlation between CASP6 and PPARγ target genes in human adipose tissues ( GSE245948 ): Correlation matrix (N), AQP7 (O), CPT1B (P), FADS2 (Q), ME3 (R), PLIN4 (S), PCK2 (T), SLC27A1 (U), and SLC27A4 (V). (n = 76). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant active caspase‐6 was obtained from Enzo Life Sciences (#BML‐SE170), recombinant PPARγ from Sino Biological (#12019‐H20B), and recombinant SP1 from Active Motif (#81181).

Techniques: Control, Expressing, Western Blot, Two Tailed Test, In Vitro, Recombinant, Staining, Ex Vivo, Cell Culture, Mutagenesis, Activity Assay, Reporter Assay

Journal: Advanced Science

Article Title: Caspase‐6 Controls Lipid and Energy Metabolism in Diet‐Induced Obesity

doi: 10.1002/advs.202514784

Figure Lengend Snippet: Adipocyte‐specific caspase‐6 knockout protects against diet‐induced obesity and insulin resistance. Flox and adipocyte‐specific caspase‐6 knockout (ACKO) mice fed 60% HFD for 12 weeks. (A) Schematic diagram of generating Casp6 flox (Flox) mice and adipocyte‐specific Casp6 knockout (ACKO) mice. (B) Immunoblot of caspase‐6 protein in adipose tissues and livers. (C) Body weight (n = 9‐11). (D‐F) Tissue weights (n = 9‐11): eWAT (D), iWAT (E), BAT (F). Two tailed unpaired Student's t ‐test. (G–I) Indirect calorimetry (n = 6): oxygen consumption rate (G), carbon dioxide production (H), and energy expenditure (I). ANCOVA analysis with body weight as a covariate. (J, K) GTT (J, n = 9‐10) and ITT (K, n = 8‐9) with AUC quantification. GTT/ITT: two‐way ANOVA followed by Šídák's‐corrected post hoc test. (L) Immunoblots for the expression levels of PPARγ, SP1 and ATGL in eWAT and iWAT of Flox and ACKO mice fed HFD for 12 weeks. (M) Graphical summary. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant active caspase‐6 was obtained from Enzo Life Sciences (#BML‐SE170), recombinant PPARγ from Sino Biological (#12019‐H20B), and recombinant SP1 from Active Motif (#81181).

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

Journal: Frontiers in Pharmacology

Article Title: Acetylshikonin mitigates diet-induced MASLD by targeting PPARγ-mediated metabolic dysfunction

doi: 10.3389/fphar.2026.1735481

Figure Lengend Snippet: AS attenuates steatosis via the multi-target and PPARγ pathways. (A) Molecular docking models showing AS binding to key targets including PPARγ, PPARα, mTOR, FASN, HIF1A, and ESR1, generated using AutoDock Vina. (B) Thermal shift assay showing increased thermal stability of recombinant PPARγ protein upon AS binding at various temperatures. (C) qPCR analysis showing reduced hepatic Pparγ mRNA levels in AS-treated HFHC-fed mice (n = 6). (D) Western blot analysis demonstrating dose-dependent downregulation of the PPARγ protein in PA/OA-stimulated Hepa1-6 cells treated with AS (n = 3); representative blots and quantification are shown. (E,F) Lipid droplet accumulation assessed using BODIPY staining in Hepa1-6 cells co-treated with AS and the PPARγ antagonist GW9662; fluorescence quantification confirms the synergistic effect; scale bar: 10 μm. All quantitative data are shown as the mean ± SD; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To block non-specific binding, the membranes were incubated in 5% skimmed milk powder for 2 h. The membranes were incubated overnight at 4 °C with primary antibodies against PPARγ protein (16643-1-AP, 1:1000, Proteintech) and GAPDH (60004-1-Ig, 1:10,000, Proteintech).

Techniques: Binding Assay, Generated, Thermal Shift Assay, Recombinant, Western Blot, Staining, Fluorescence