Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Culture media composition. RGZ = rosiglitazone; ISO = isoproterenol; BM = basal medium; DM = differentiation medium; MM = maintenance medium; DMEM-HG = Dulbecco’s modified Eagle’s medium-high Glucose; BCS = bovine calf serum; P/S = penicillin-streptomycin; IBMX = 3-isobutyl-1-methylxanthine; DEX = dexamethasone; DMSO = dimethyl sulfoxide.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: Modification

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Feeding volumes used at different times for different support. RGZ = rosiglitazone; ISO = isoproterenol; BM = basal medium; DM = differentiation medium; MM = maintenance medium.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Quantitative analysis of lipidic deposits. Column graphs display the fold changes of the ORO staining expressed in percentage of positive pixels (PPP) ± SEM: statistical significance of adipocytes differentiated at T3, T10, and T17 following treatment with DM and MM (WHITE), RGZ 0.1 µM and 1 µM, or ISO 0.1 µM and ISO 1 µM versus T0 (****p < 0.0001). The graph also reports the statistical analysis of RGZ 0.1 μM T3 and RGZ 1 μM T3 versus WHITE T3 (§§§§p < 0.0001); ISO 0.1 μM T10 versus WHITE T10 (###p < 0.001); RGZ 0.1 µM T17, ISO 0.1 μM T17, and ISO 1 μM T17 versus WHITE T17 (°p < 0.05; °p < 0.0001). Abbreviations: ISO, isoproterenol; RGZ, rosiglitazone; ORO, Oil Red O.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: Staining

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Analysis of UCP-1 levels used as a marker of the adipocyte phenotype at different differentiation times and following the different treatments used. (A) UCP-1 levels stratified by time (T0, T3, T10, and T17) and by treatment (RGZ 0.1 μM, RGZ 1 μM, ISO 0.1 μM, and ISO 1 μM). (B) Representation of the variation of UCP-1 levels as a function of time and stratified by treatment (RGZ 0.1 μM, RGZ 1 μM, ISO 0.1 μM, and ISO 1 μM). Graphs display the UCP-1 levels expressed in ng/mL ± SEM. Statistical significance versus WHITE T3 (#p < 0.05) in (A) . Statistical significance versus WHITE T10 (§§§p < 0.001; §§§§p < 0.0001) in (A) . Statistical significance versus WHITE T17 (****p < 0.0001) in (A) . Statistical significance versus WHITE (****p < 0.0001) in (B) . Abbreviations: ISO, isoproterenol; RGZ, rosiglitazone. Sample size (n): eight biological replicates for white T17 and four biological replicates for the other treatments. Three technical replicates per sample.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: Marker

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Analysis of the activity of the citrate synthase enzyme representing the metabolic and mitochondrial activity of adipocyte cells at different differentiation times and following the different treatments used. (A) Enzyme activity stratified by time (T0, T3, T10, and T17) and by treatment (RGZ 0.1 μM, RGZ 1 μM, ISO 0.1 μM, and ISO 1 μM). (B) Representation of the variation of enzymatic activity as a function of time and stratified by treatment (RGZ 0.1 μM, RGZ 1 μM, ISO 0.1 μM, and ISO 1 μM). Graphs display the citrate synthase activity expressed in mU/mL ± SEM. Statistical significance versus WHITE T17 (**p < 0.01; ****p < 0.0001) in (A) . Statistical significance versus WHITE (*p < 0.05; **p < 0.01) in (B) . Abbreviations: ISO, isoproterenol; RGZ, rosiglitazone. Sample size (n): six for white T3, five for white T10, eight biological replicates for white T17; five biological replicates for RGZ 0.1 μM, RGZ 1 μM, ISO 0.1 μM, and ISO 1 μM at T3 and T10; 11 biological replicates for RGZ 0.1 μM and RGZ 1 μM at T17, and five biological replicates for ISO 0.1 μM and ISO 1 μM at T17. Three technical replicates per sample.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Representative 3D confocal images of lipid droplets (ORO, red) and UCP-1 (green) expression in (A) preadipocytes; (B) 3T3-L1 cells differentiated at T17 in white adipocytes, after treatment with DM and MM for white adipocyte differentiation; (C, D) following treatment with RGZ 0.1 µM and 1 μM; (E) and (F) following treatment with ISO 0.1 µM and 1 µM. Scale bar: 20 µM. Abbreviations: DM, differentiation medium; MM, maintenance medium; ISO, isoproterenol; RGZ, rosiglitazone; ORO, Oil Red O; UCP-1, mitochondrial uncoupling protein-1.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: Representative images of ORO staining in preadipocytes (T0) and adipocytes differentiated at T3, T10, and T17 following treatment with DM and MM without or with RGZ 0.1 µM and 1 μM and ISO 0.1 µM and 1 µM. For each group and each time point, the cells contained in 10 fields acquired at ×40 magnification for image analysis of Oil Red O expression have been analyzed. Scale bar: 20 µm. Abbreviations: DM, differentiation medium; MM, maintenance medium; ISO, isoproterenol; RGZ, rosiglitazone; ORO, Oil Red O.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Development of an adipocyte differentiation protocol using 3T3-L1 cells for the investigation of the browning process: identification of the PPAR-γ agonist rosiglitazone as a browning reference drug

doi: 10.3389/fphar.2025.1546456

Figure Lengend Snippet: (A) Quantitative analysis of lipidic droplet size. Column graphs display the fold changes of the ORO-positive droplet size expressed as an average of all single areas ± SEM. Statistical significance of RGZ 0.1 μM T3 versus WHITE T3 (#p < 0.05); RGZ 0.1 µM T10, RGZ 1 μM T10, ISO 0.1 μM T10, and ISO 1 μM T10 versus WHITE T10 (§§p < 0.01; §§§§p < 0.0001); RGZ 0.1 µM T17, RGZ 1 μM T17, ISO 0.1 μM T17, and ISO 1 μM T17 versus WHITE T17 (****p < 0.0001). Abbreviations: ISO, isoproterenol; RGZ, rosiglitazone; ORO, Oil Red O. (B) Classification of lipidic droplet size. Column graphs display the fold changes of the ORO-positive droplet size expressed as a percentage of the ratio between the number of droplets with small, medium, and large size (0–120 μm 2 , 120 μm 2 –240 µm 2 , >240 μm 2 , respectively) and the total number of droplet counts for each group ± SEM. Five fields were evaluated for lipid droplet size in each group and at each time point. Statistical significance of RGZ 0.1 µM T17, RGZ 1 μM T17, ISO 0.1 μM T17 versus WHITE T17 (*p < 0.05; **p < 0.01; ***p < 0.001). Abbreviations: ISO, isoproterenol; RGZ, rosiglitazone; ORO, Oil Red O.

Article Snippet: RGZ (Merck KGaA, Germany) and ISO (Merck KGaA, Germany) at concentrations of 0.1 μM and 1 μM was added to both DM and MM to evaluate their impact on the de novo browning process.

Techniques: