Endogenous FGF2 and HIF-1α expression in ischemic skin extracts.

<p>A. Representative Western blot analyses of FGF2 and HIF-1α at the indicated post-operative time. The molecular weights of FGF2 mouse isoforms are indicated. B. Levels of endogenous FGF2 mRNA determined by RT-qPCR analysis. Results are expressed relative to the level from control mice and represent mean±SE (n = 5 mice per post-operative time). *P<0.05 vs. control. C. Quantification of total FGF2 detected by Western blot analysis after normalization to GAPDH and to endogenous mRNA level. **P<0.001 vs. control.</p

Ischemia induced by a dorsal skin flap model in IRES FGF2-Luc transgenic mice.

<p>A. Representation of FGF2 mRNA and mouse protein isoforms (left panel) and of the bicistronic mRNA expressed by the RFL12 transgenic mice (right panel). This bicistronic cassette expresses, under control of the CMV (Cytomegalovirus) promoter, LucR and LucF reporter genes in a cap- or FGF2 IRES-dependent manner, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003078#pone.0003078-Creancier1" target="_blank">[18]</a>. B. Ischemia induction using a skin flap model modified from Ceradini <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003078#pone.0003078-Ceradini1" target="_blank">[26]</a> and representative Laser Doppler analysis performed 18, 48 and 72 hours after surgery. A U-shaped peninsular skin incision was created on the dorsal surface of 8-week old female RFL12 mice. The two vascular pedicles arising from the lateral thoracic arteries were sectioned. To avoid necrotic tissues, the study of gene expression was performed on the proximal part of the skin flap indicated by the white square. The color scale illustrates blood flow variations from maximal (red) to minimal perfusion (dark blue). C. Quantification of laser Doppler analysis. Ctr corresponds to non-operated mice. Results represent mean±SE on at least 3 mice per post-operative time.</p

Crosstalk between FGF2 and HIF-1α.

<p>A. FGF2 and HIF-1α gene silencing using targeted-siRNA. 911 cells were transfected with FGF2 (left) or HIF-1α (right) targeted siRNA (siFGF2 or siHIF-1α, respectively). Protein contents were measured in normoxic (FGF2) and hypoxic (HIF-1α) cells 48 h after transfection. SiC corresponds to a control scrambled siRNA. B. Effect of FGF2 gene silencing on HIF-1α accumulation in 911 cells cultivated in normoxic (N) and hypoxic conditions. C. Effect of HIF-1α gene silencing on FGF2 accumulation in 911 cells cultivated in normoxic (N) and hypoxic conditions.</p

DataSheet_1_Membrane estrogen receptor-α contributes to female protection against high-fat diet-induced metabolic disorders.pdf

BackgroundEstrogen Receptor α (ERα) is a significant modulator of energy balance and lipid/glucose metabolisms. Beyond the classical nuclear actions of the receptor, rapid activation of intracellular signaling pathways is mediated by a sub-fraction of ERα localized to the plasma membrane, known as Membrane Initiated Steroid Signaling (MISS). However, whether membrane ERα is involved in the protective metabolic actions of endogenous estrogens in conditions of nutritional challenge, and thus contributes to sex differences in the susceptibility to metabolic diseases, remains to be clarified.MethodsMale and female C451A-ERα mice, harboring a point mutation which results in the abolition of membrane localization and MISS-related effects of the receptor, and their wild-type littermates (WT-ERα) were maintained on a normal chow diet (NCD) or fed a high-fat diet (HFD). Body weight gain, body composition and glucose tolerance were monitored. Insulin sensitivity and energy balance regulation were further investigated in HFD-fed female mice.ResultsC451A-ERα genotype had no influence on body weight gain, adipose tissue accumulation and glucose tolerance in NCD-fed mice of both sexes followed up to 7 months of age, nor male mice fed a HFD for 12 weeks. In contrast, compared to WT-ERα littermates, HFD-fed C451A-ERα female mice exhibited: 1) accelerated fat mass accumulation, liver steatosis and impaired glucose tolerance; 2) whole-body insulin resistance, assessed by hyperinsulinemic-euglycemic clamps, and altered insulin-induced signaling in skeletal muscle and liver; 3) significant decrease in energy expenditure associated with histological and functional abnormalities of brown adipose tissue and a defect in thermogenesis regulation in response to cold exposure.ConclusionBesides the well-characterized role of ERα nuclear actions, membrane-initiated ERα extra-nuclear signaling contributes to female, but not to male, protection against HFD-induced obesity and associated metabolic disorders in mouse.</p

Additional file 1: Figure S1. of The AF-1-deficient estrogen receptor ERα46 isoform is frequently expressed in human breast tumors

Clinical parameters of the breast tumor samples. IDC invasive ductal carcinoma, ILC invasive lobular carcinoma. Figure S2. Expression of Flag-ERα36 in transiently transfected MDA-MB231 as detected by immunocytochemistry using an anti-Flag antibody. Figure S3. Analysis of potential correlation between the clinical parameters of the breast tumor samples and the expression of ERα46 isoform. A significant P value (indicated in red) was only found between ERα46 expression and HER-2 positive breast tumors. IDC invasive ductal carcinoma, ILC invasive Lobular Carcinoma. Figure S4. Results of the proteomic analysis of the ERα46 protein detected in tumor samples. A) Western blot with the SP1 antibody obtained after immunoprecipitation of ERα with HC20 antibody in two human tumors overexpressing the putative ERα46 isoform. B and C) Sequence coverage obtained from the peptides identified by proteomic analysis shown in bold red on ERα66 and on ERα46 isoforms M (methionine): putative translational start sites generating the ERα46 isoform. Figure S5. The stress-induced increase in LucF activity is reproducible in MCF7 cells (A) and is not due to the generation of mono-cistronic LucF transcripts via an internal promoter or cryptic splicing as observed in MDA-Lenti-AB exposed to two siRNAs-lucR (B and C). Figure S6. Modulation profiles of the interaction of ERα46 (red) and ERα66 (blue) with coregulators in A) Apo proteins and B) in response to E2 binding. C) Profile of EC50 values of 4-OH-tamoxifen- (red) and fulvestrant- (blue) induced modulation of ERα46 and ERα66 coregulators interaction when use in antagonists mode with 6.3 nM E2. Figure S7. List of primers used in the expression profiling of target genes. Figure S8. Fold-changes (FC) in gene expression± SEM in MDA-ERα46 and MDA-ERα66 cells in response to 4-h treatment with E2 (10–8 M). P values were determined by Mann-Whitney test. Significant P values are indicated in red. (PPTX 939 kb