Journal: International Journal of Molecular Sciences

Article Title: Phenolic Compounds with Antioxidant Properties from Canola Meal Extracts Inhibit Adipogenesis

doi: 10.3390/ijms21010001

Figure Lengend Snippet: ( A ) Immunofluorescence staining of PPARγ in CME treated mesenchymal stem cells (CH310T1/2) visualized at 10 µm: ( UN ) undifferentiated cells—negative control; ( DC ) differentiated cells—positive control. Differentiated cells treated with CMEs: Acetone extract ( AE ); methanol extract ( ME ); butanol extract ( BE ); ethanol extract ( EE ); hexane extract ( HE ); chloroform extract ( CE ); and water extract ( WE ). Captured images with no number labels are cells stained with fluorescein isothiocyanate (FITC). Images labeled with “1” show cells stained with 4’,6-diamidino-2-phenylindole (DAPI); images labeled with “2” show the normal view of cells, and images labeled with “3” are merged images of FITC stained cells, DAPI stained cells, and normal images, respectively. ( B ) Quantitative analysis of PPARγ gene expression in C3H10T1/2 cells treated with CMEs. Results are expressed as means ± standard deviation ( n = 3). Bars with different letters have mean values that are significantly different ( p < 0.05).

Article Snippet: The cells were then incubated with anti-PPARγ (81B8) rabbit monoclonal antibody (1:50) Cell Signalling Technology ( Danvers , MA, USA ) for one hour at ambient temperature, then washed gently with PBS and incubated in the dark with anti-rabbit IgG (Fab 2)–Alexa Fluor ® 488 (1:100; Cell Signalling Technology) for one hour.

Techniques: Immunofluorescence, Staining, Negative Control, Positive Control, Labeling, Gene Expression, Standard Deviation

Journal: Endocrinology

Article Title: SUMO-1 regulates body weight and adipogenesis via PPARγ in male and female mice.

doi: 10.1210/en.2012-1846

Figure Lengend Snippet: Figure 6. Expression of PPAR target genes in SUMO-1-less cells. A and B, Embryonic fibroblasts from WT and Sumo1-null (KO) mice were differentiated into adipocytes for 14 days. Plin (A) and Fsp27 mRNA (B) levels were measured by qRT-PCR and normalized to Gapdh mRNA levels. C, PPAR protein level was measured by immunoblotting with anti-PPAR antibody. The level of -actin was used as loading control. D, Immortalized WT and KO MEFs were transfected with the PPRE-Luc reporter construct together with PPAR and -galactosidase expression plasmids. Luciferase activities were normalized to -galactosidase activities. E, WT and SUMO-1-depleted (Sumo1 shRNA) 3T3-L1 preadipocytes were differentiated into adipocytes for 7 days. Loading of PPAR onto the aP2 promoter was determined by chromatin immunoprecipitation using anti-PPAR antibody or normal rabbit IgG. qRT-PCR was performed using primers for the aP2 promoter, and the data are expressed relative to IgG. F, PPAR protein levels were measured by immunoblotting with anti-PPAR-antibody; -actin was used as loading control. Results are shown as mean SEM. Significance of differences was calculated with Student’s t test. AC, Differentiated adipocytes, -gal, -galactosidase; Luc, luciferase.

Article Snippet: Immunoblotting was performed using rabbit polyclonal PPAR antibody (81B8; Cell Signaling Technology, Danvers, MA), mouse monoclonal SUMO-1 antibody (sc-5308; Santa Cruz Biotechnology, Santa Cruz, CA), -actin antibody (sc-47778; Santa Cruz), or -tubulin antibody (sc-5286; Santa Cruz).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Transfection, Construct, Luciferase, shRNA, Chromatin Immunoprecipitation