Journal: Molecules

Article Title: Effects of Chlorogenic Acid on Cellular Senescence in an In Vitro Model of 3T3-L1 Murine Adipocytes

doi: 10.3390/molecules31010167

Figure Lengend Snippet: Effects of chlorogenic acid on adipogenesis. Here, 3T3-L1 adipocytes were treated with H 2 O 2 (100 μM) on days 5, 6, and 7 of the differentiation process for three hours, followed by CGA exposure (5, 10, or 20 μM). Cells cultured with the differentiation medium containing the CGA vehicle alone (0.1% v / v DMSO) were used as controls. ( A ) Representative images of Oil Red O staining (original magnification at ×40—scale bar 50 µm) in control cells (CTR) and in cells exposed to H 2 O 2 and treated or not with CGA. ( B ) Cell lipid accumulation was expressed as relative content vs. control cells. ( C ) PPARγ protein expression was analyzed by Western blot. The densitometry results are reported as fold changes compared to control cells. The values were normalized to the corresponding β-actin value. ( D ) FASN gene expression value was expressed as 2 −ΔΔCt and normalized against control cells. 18S rRNA was used as a housekeeping gene. All results are reported as the mean ± S.D. of three independent experiments (n = 3 biological replicates). Error bars represent S.D. a p < 0.01 vs. control cells; c p < 0.01 vs. H 2 O 2 ; d p < 0.05 vs. H 2 O 2 + CGA 5 μM; e p < 0.01 vs. H 2 O 2 + CGA 5 μM; f p < 0.05 vs. H 2 O 2 + CGA 10 μM.

Article Snippet: Membranes were then blocked using 5% non-fat dry milk solution and incubated overnight at 4 °C with the following specific primary antibodies: rabbit anti-Lamin B1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) (1:1000), mouse anti-PPAR-γ monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) (1:1500), rabbit anti-PI3K p85 monoclonal antibody (Cell Signaling Technology) (1:1000), mouse anti-p21 (Waf1/Cip1) monoclonal antibody (Santa Cruz Biotechnology) (1:500), mouse anti-phospho-p53 (Ser15) monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-phospho-p38 (Thr180/Tyr182) monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-Bax monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-Bcl-2 monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-Phospho-Akt (Ser473) monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-GLUT-4 monoclonal antibody (Cell Signaling Technology) (1:1000), rabbit anti-NF-κB p65 polyclonal antibody (Invitrogen, Milan, Italy) (1:1000), mouse anti-COX-2 monoclonal antibody (Santa Cruz Biotechnology) (1:500), and rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology) (1:6000).

Techniques: Cell Culture, Staining, Control, Expressing, Western Blot, Gene Expression