Results: 4

1.
Fig. 4.

Fig. 4. From: Promyelocytic leukemia inhibits adipogenesis, and loss of promyelocytic leukemia results in fat accumulation in mice.

PML loss attenuates Sirt1-PPARγ interaction and inhibits Sirt1-nuclear corepressor(NCoR)-silencing mediator for retinoic and thyroid hormone receptor (SMRT) recruitment to the pparγ promoter during adipogenesis in 3T3-L1 cells. A: pGL3-pparγ-luc reporter plasmid was cotransfected into 293T cells with expression vectors for human PML4 and PPARγ together with renilla luciferase vector. Results are expressed as firefly luciferase activity normalized to renilla luciferase activity. B: coimmunoprecipitation of Sirt1 with PPARγ. Cell lysates of ctrl shRNA and PML shRNA-3T3-L1 cells were obtained during 0–6 days of adipogenesis and subjected to immunoprecipitation using anti-PPARγ (Santa Cruz Biotechnology) antibody. The immunoprecipitated complex was immunoblotted with anti-Sirt1 (Upstate Biotechnology) antibody. C, left: chromatin immunoprecipitation (ChIP) analysis on the pparγ promoter in ctrl shRNA 3T3-L1 cells using IgG and antibodies against Sirt1 (Upstate Biotechnology), SMRT (Santa Cruz Biotechnology), NCoR (Santa Cruz Biotechnology) and PPARγ (H-100; Santa Cruz Biotechnology) at indicated days after adipogenic induction. C, right: ChIP analyses on the pparγ promoter in ctrl shRNA and PML shRNA 3T3-L1 cells on day 2 after adipogenic induction. Twenty-five to 35 cycles of PCR reaction were performed. D: ChIP analysis on the pparγ or aP2 promoter. The chromatin-associated DNA samples were obtained in PML shRNA and ctrl shRNA cells on day 6 after differentiation and immunoprecipitated with IgG or antibodies against Sirt1 and PPARγ.

Myung K. Kim, et al. Am J Physiol Endocrinol Metab. 2011 December;301(6):E1130-E1142.
2.
Fig. 2.

Fig. 2. From: Promyelocytic leukemia inhibits adipogenesis, and loss of promyelocytic leukemia results in fat accumulation in mice.

PML depletion does not affect Akt, mTOR/S6K1, or AMP-activated protein kinase (AMPK) signaling. A: Western blotting analysis of phospho-Akt (p-Akt; phospho-Ser473), p-S6 kinase 1 (p-S6K; phospho-Thr389), p-S6 (phospho-Ser240/244), and phospho-eukaryotic initiation factor 4E-binding protein-1 (p-4E-BP1; phospho-Thr37/46). White adipose tissue (WAT) and liver proteins from 2 different male HFD-fed PML+/+ and PML−/− mice (HFD for 20 wk) were loaded, electrophoresed, blotted, and probed with various antibodies. For insulin treatment, mice were fasted for 6 h, injected intraperitoneally with 0.75 U/kg insulin. Tissues were collected 10 min after injection. Some images were obtained from spliced blots. Spaces were inserted at the splice sites. B: Western blotting analysis of the primary PML+/+ and PML−/− MEFs treated with insulin (0–100 nM, 15 min). SF, serum-free condition, cells were serum-starved overnight. C: AMPK activation in vivo measured by p-AMPK (phospho-Thr172) level in WAT, liver, and muscle from female HFD-fed PML+/+ and PML−/− mice (HFD for 20 wk). Each lane represents an individual mouse. Spaces were inserted at the splice sites on the blots. D: Western blotting analysis of the primary PML+/+ and PML−/− myocyte enhancer factors (MEFs) after metformin treatment (0–3 mM, 4 h).

Myung K. Kim, et al. Am J Physiol Endocrinol Metab. 2011 December;301(6):E1130-E1142.
3.

Fig. 1. From: Promyelocytic leukemia inhibits adipogenesis, and loss of promyelocytic leukemia results in fat accumulation in mice.

Loss of promyelocytic luekemia (PML) results in fat accumulation after high-fat diet (HFD) in mice. A: body weight (BW) and body fat index of female HFD-fed PML+/+ and PML−/− mice (n = 10–15/genotype). Body fat index was calculated by dividing the fat mass by total BW. B: BW and body fat index of male HFD-fed PML+/+ and PML−/− mice (n = 8–10/genotype). C: weights of epididymal (Epi) and retroperitoneal (Retro) fat normalized by BW. The weight of subcutaneous (sc) fat was estimated roughly by subtracting the sum of Epi and Retro fat from the total body fat and normalized by BW. Fat pads were obtained from HFD-fed male PML+/+ and PML−/− mice (HFD for 16 wk; n = 3–4/genotype). D: skin sections of the male HFD-fed PML+/+ and PML−/− mice (HFD for 24 wk) were stained with hematoxylin and eosin (H & E). Two different individual mice/genotype were used. E: histological sections of sc fat pads of male HFD-fed PML+/+ and PML−/− mice (HFD for 24 wk; left). E, right: adipocyte cell size in sc fat [arbitrary units (AU)]. For fat cell size histogram, digital images of H & E-stained adipose tissue slides were analyzed using image scope software (Aperio). Three different individual mice/genotype were used. F: food intake per mouse per day measured over 14 days (n = 5–6/genotype). Female HFD-fed PML+/+ and PML−/− mice were used (HFD for 4 and 16 wk). G: locomoter activity as measured by beam breaks/24 h using female HFD-fed PML+/+ and PML−/− mice (HFD for 16 wk; n = 8/genotype). X total indicates X total laser beam interruptions. H: glucose (GTT) and insulin tolerance tests (ITT) of female HFD-fed PML+/+ and PML−/− mice (HFD for 16–18 wk). For GTT, mice were fasted for 16 h, and 1 mg/g glucose was injected intraperitoneally. Blood glucose was measured at indicated times after injection. For the ITT, mice were fasted for 4 h before intraperitoneal injection with 0.4 U/kg of human insulin (Sigma) (n = 8–12/genotype). I: blood insulin (left) and glucose levels (right) in female HFD-fed PML+/+ and PML−/− mice (HFD for 16–18 wk; n = 8–12 of each group). Mice were either fed ad libitum or fasted (16 h). J: blood insulin (left) and glucose levels (right) in male HFD-fed PML+/+ and PML−/− mice (HFD for 40 wk; n = 8–10 of each group). Mice were either fed ad libitum or fasted (16 h). Weeks indicate weeks on HFD. *P < 0.05; **P < 0.01; ***P < 0.001.

Myung K. Kim, et al. Am J Physiol Endocrinol Metab. 2011 December;301(6):E1130-E1142.
4.

Fig. 3. From: Promyelocytic leukemia inhibits adipogenesis, and loss of promyelocytic leukemia results in fat accumulation in mice.

PML regulates adipogenesis in culture. A: PML protein in WAT, skeletal muscle (Mus), and liver from male BD-fed PML+/+ mice (BD for 12 wk). B: decrease in PML expression during adipogenesis (0–5 days) in 3T3-L1 cells shown by Western blotting analysis (upper panel). RT-PCR analyses of PML, CCAAT/enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor-γ (PPARγ) during adipogenesis (0–9 days) in 3T3-L1 cells (bottom). C: PML staining and Oil Red O staining. Top: confocal microscopy of 3T3-L1 cells on day 5 after adipogenic induction (+adipo). Bottom: representative pictures of the control 3T3-L1 cells cultured in the absence of adipogenic stimuli (0–7 days) (−adipo). Scale bars, 10 μm. D: %lipid accumulation in ctrl shRNA and PML shRNA 3T3-L1 cells on day 5 after adipogenic induction (left) and a picture of the Oil Red O staining of control (ctrl) shRNA and PML shRNA 3T3-L1 cells cultured in the presence (+adipo) or absence (−adipo) of the adipogenic stimuli for 5 days (right). E. %lipid accumulation in PML+/+ and PML−/− MEFs on day 5 after adipogenic induction. F: expressions of the C/EBP proteins and PPARγ during adipogenesis in ctrl shRNA and PML shRNA cells (left) and in PML+/+ and PML−/− MEFs (right). G: mRNA expression levels (RT-PCR) of various genes on day 5 after adipogenic induction in ctrl shRNA and PML shRNA-3T3-L1 cells. H: Western blotting analysis of the aP2 protein level in ctrl shRNA and PML shRNA-3T3-L1 cells (on day 5 after adipogenic induction). I: mRNA expression levels (RT-PCR) of PPARγ, C/EBPα, and sirtuin 1 (Sirt1) in subcutaneous fat of male HFD-fed PML+/+ and PML−/− mice (HFD for 16 wk); n = 3. J: RT-PCR and Western blotting analyses of the PML expression level in WAT from fed or fasted male BD-fed PML+/+ mice. Mice were either fed ad libitum or fasted (24 h). Each lane in Western blot represents an individual mouse. K: circulating free fatty acid (FFA) levels in PML+/+ and PML−/− mice under fed or fasted conditions. Male BD-fed PML+/+ and PML−/− mice were used (BD for 14–16 wk; n = 5–6 of each group). L: FFA release in the culture medium in the absence (basal) or presence of 10 μM isoproterenol (ISO; 2 h) analyzed using epididymal white adipose samples obtained from female HFD-fed PML+/+ and PML−/− mice (HFD for 4–6 wk; n = 3 of each group). **P < 0.01.

Myung K. Kim, et al. Am J Physiol Endocrinol Metab. 2011 December;301(6):E1130-E1142.

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