Journal: The Journal of Biological Chemistry

Article Title: Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue

doi: 10.1074/jbc.M117.811794

Figure Lengend Snippet: PDC-E2 interacts with STAT5A in GH-stimulated murine and human adipocytes. Fully differentiated 3T3-L1 adipocytes (A) or human adipocytes (B) were treated with 5 nm mGH (A) or hGH (B) for 15–20 min. Control (CTL) cells were untreated. A, monolayers were collected and subjected to subcellular fractionation that was optimized to separate cytoplasmic (cyto) and nuclear (nuc) compartments. The “Nuc +” fraction was free from cytoplasmic contamination and enriched for nuclei. Immunoprecipitation (IP) reactions utilized anti-STAT5A or anti-PDC-E2 antibodies and contained 300 μg of total protein per reaction. Interacting PDC-E2 (70 kDa) and STAT5A (95 kDa) proteins were detected by WB. Mock samples contained IP antibody but no extract. In the bottom two panels, the presence of STAT5A and PDC-E2 in the extracts used for immunoprecipitation was examined directly by Western blotting using 50 μg of total protein per lane. Experiments using 3T3-L1 adipocytes have been repeated more than three times on independent batches of cells. B, fully differentiated human adipocytes, derived from preadipocytes isolated from the visceral omental adipose depot of obese individuals, were purchased from Zenbio. Monolayers were collected, and whole-cell lysates were prepared. IP reactions and Western blotting were performed as described for A, except that 75 μg of total protein was used for each IP. The middle panel in B demonstrates the presence of activated STAT5 phosphorylated at tyrosine 694/699 (STAT5pY). The experiment with human adipocytes was performed one time.

Article Snippet: Human adipocytes were maintained as instructed by Zen-Bio in Omental Adipocyte Medium Human (catalog no. OM-AM) upon receipt in our laboratory until use within 3 weeks.

Techniques: Control, Fractionation, Immunoprecipitation, Western Blot, Derivative Assay, Isolation

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-29a in Osteoblasts Represses High-Fat Diet-Mediated Osteoporosis and Body Adiposis through Targeting Leptin

doi: 10.3390/ijms22179135

Figure Lengend Snippet: Effects of miR-29a mimics and antisense oligonucleotides on osteogenic differentiation and adipocyte formation of bone marrow mesenchymal stem cells. Forced miR-29a expression promoted mineralized matrix formation ( a ) (von Kossa staining; scale bar, 40 µm) together with increased Runx2 and Ocn expression ( b ). miR-29a mimetics repressed fluorescence adipocyte formation ( c ) (fluorescence Nile red staining; scale bar, 8 µm) and reduced aP2 and LPL expression ( d ). miR-29a mimetics promoted Ucp-1, Pgc-1α ( e ), P2rx5, and Pat2 ( f ) expression in mesenchymal stem cells incubated in brown adipogenic medium, whereas Tcf21 and Hoxc9 ( g ) expression were repressed in cell cultures grown in white adipogenic medium. miR-29a antisense oligonucleotide (miR-29a-AS) inhibited osteogenesis and brown adipocyte marker expression but upregulated adipocyte formation together with upregulated white adipocyte marker expression. Mean ± S.E. is calculated from 3 experiments. * p < 0.05 upon ANOVA test and Bonferroni post hoc test. Scramble, scrambled control; miR-29a, miR-29a mimics; miR-29a-AS, miR-29a antisense oligonucleotides.

Article Snippet: In some experiments, a total of 10 5 cells/well (24-well plates) were incubated in a differentiation medium for brown adipocytes (BADTM, Cosmo Bio Co., Ltd., Tokyo, Japan) and white adipocytes (WATDM, Cosmo Bio Co., Ltd., Tokyo, Japan) for 15 days, respectively, according to the maker’s instructions.

Techniques: Expressing, Staining, Fluorescence, Incubation, Marker, Control

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-29a in Osteoblasts Represses High-Fat Diet-Mediated Osteoporosis and Body Adiposis through Targeting Leptin

doi: 10.3390/ijms22179135

Figure Lengend Snippet: Effects of forced miR-29a expression or knockdown on leptin expression in mesenchymal stem cells and HFD-fed bone tissue. Bioinformatics prediction showing miR-29a targeted genes, which involved lipid or adipocyte metabolism ( a ). Sequences of 3′-UTR in human and murine leptin targeted by miR-29a ( b ). Gain of miR-29a function inhibited leptin expression, whereas miR-29a knockdown promoted the expression ( c ). miR-29a overexpression attenuated HFD-induced leptin expression in bone tissue ( d ). Mean ± S.E. of in vitro and in vivo models are calculated from 3 experiments and 4–5 mice, respectively. * p < 0.05 upon ANOVA test and Bonferroni post hoc test. Scramble, scrambled control; miR-29a, miR-29a mimics; miR-29a-AS, miR-29a antisense oligonucleotide; ND, standard rodent chow; HFD, high-fat diet.

Article Snippet: In some experiments, a total of 10 5 cells/well (24-well plates) were incubated in a differentiation medium for brown adipocytes (BADTM, Cosmo Bio Co., Ltd., Tokyo, Japan) and white adipocytes (WATDM, Cosmo Bio Co., Ltd., Tokyo, Japan) for 15 days, respectively, according to the maker’s instructions.

Techniques: Expressing, Knockdown, Over Expression, In Vitro, In Vivo, Control