Journal: PLoS Genetics

Article Title: Acceleration of the Glycolytic Flux by Steroid Receptor Coactivator-2 Is Essential for Endometrial Decidualization

doi: 10.1371/journal.pgen.1003900

Figure Lengend Snippet: (A) hESC decidualization following six days of culture in EPC cocktail with or without 2-DG (50 mM concentration). Top panels show the morphological changes of hESCs following the indicated treatments. Histograms at bottom display relative transcript levels of IGFBP-1 and PRL following indicated treatments. (B) Glycolytic flux assay of hESCs previously transfected with control or SRC-2 siRNA and cultured for 24 hrs in EPC (see section). (C) Heat map displays metabolites that are significantly altered with SRC-2 knockdown as assessed by LC-MS analysis. Prior to LC-MS analysis, hESC lines derived from three subjects were transfected with either control or SRC-2 siRNA in triplicate and cultured in EPC media. Inset shows the significant percentage decrease in intracellular levels of fructose 2, 6-bisphosphate with SRC-2 knockdown. (D) Schematic shows the position of PFKFB3 and fructose 2, 6-bisphosphate in the glycolytic pathway.

Article Snippet: Polyclonal anti-rabbit SRC-2 (Bethyl laboratories, Montgomery, TX), Polyclonal anti-rabbit PFKFB3 (Cell Signaling, Danvers, MA) and a monoclonal anti-mouse β-actin (Sigma-Aldrich) antibodies were used for detecting SRC-2, PFKFB3 and β-actin proteins respectively.

Techniques: Concentration Assay, Flux Assay, Transfection, Control, Cell Culture, Knockdown, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: PLoS Genetics

Article Title: Acceleration of the Glycolytic Flux by Steroid Receptor Coactivator-2 Is Essential for Endometrial Decidualization

doi: 10.1371/journal.pgen.1003900

Figure Lengend Snippet: (A) Real-time PCR shows the rapid induction profile of PFKFB3 in hESCs cultured in the presence of EPC cocktail for the indicated time points. (B) Relative mRNA levels of PFKFB3 and SRC-2 in hESCs transfected with control or SRC-2 siRNA and cultured in the presence of EPC cocktail for the indicated time points. (C) Schematic depicts the reported position of a PR binding site in PFKFB3 promoter (region 1 ); region 2 denotes a non-binding region (negative control) for PR (TSS indicates transcriptional start site). Histogram below shows the real-time PCR result following ChIP of PR or SRC-2 on region 1 and 2. (D) Morphology of hESCs transfected with control or PFKFB3 siRNA following six days of culture in the presence of the EPC cocktail. (E) Transcript levels of IGFBP-1, PRL and PFKFB-3 in hESCs transfected with control or PFKFB3 siRNA and cultured in the presence of EPC cocktail at indicated time points. (F) Crystal violet stained control siRNA or PFKFB3 siRNA transfected hESCs after 10 days of culture in EPC cocktail (left panels). Quantitation of retained crystal violet stain from control siRNA or PFKFB3 siRNA transfected hESCs is displayed by histogram. (G) A cell viability assay (MTT) of hESCs transfected with control and PFKFB3 siRNA and cultured for the indicated time points. **** P <0.0001.

Article Snippet: Polyclonal anti-rabbit SRC-2 (Bethyl laboratories, Montgomery, TX), Polyclonal anti-rabbit PFKFB3 (Cell Signaling, Danvers, MA) and a monoclonal anti-mouse β-actin (Sigma-Aldrich) antibodies were used for detecting SRC-2, PFKFB3 and β-actin proteins respectively.

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Transfection, Control, Binding Assay, Negative Control, Staining, Quantitation Assay, Viability Assay