Journal: bioRxiv

Article Title: Tissue-Specific Alteration of Metabolic Pathways Influences Glycemic Regulation

doi: 10.1101/790618

Figure Lengend Snippet: Related to Figure S4. (A) Protein expression levels of missense G6PC variants were determined in Huh7 cells (n=4-5) and (B) HEK293 cells (n=5) by western blot densitometric analysis of FLAG-tagged G6PC constructs relative to tubulin control, with representative blots shown. (C) Protein expression levels of PTV Q347X were determined in Huh7 cells (n=3) and (D) HEK293 cells (n=4) by western blot densitometric analysis of V5-tagged G6PC constructs relative to tubulin control, with representative blots shown. Bars in red indicate variants that are statistical drivers of the gene-based signal. (E) Cellular localisation of V5-tagged G6PC-Q347X was assessed in Huh7 cells and overlaid with markers for the ER (calreticulin) and the trans-golgi network (TGN46). White arrows point to positions of the Golgi apparatus. Scale bar indicates 10μm. (F) Glucose-6-phosphatase activity of unglycosylated WT G6PC protein obtained from tunicamycin-treated (Tuni) HEK293 microsomes (n=2), with representative western blot of microsomal protein shown. All data presented as mean ± SEM. * p=0.01-0.05; ** p=0.001-0.01; *** p<0.001.

Article Snippet: Human G6PC (NM_000151.3) and G6PC2 cDNA (NM_021176.2) within a pCMV6-Entry vector (with a C-terminal Myc-FLAG-tag) was purchased from OriGene (RC215623 and RC211146 respectively).

Techniques: Expressing, Western Blot, Construct, Activity Assay

Journal: bioRxiv

Article Title: Tissue-Specific Alteration of Metabolic Pathways Influences Glycemic Regulation

doi: 10.1101/790618

Figure Lengend Snippet: Related to Figure S5. (A) Expression levels of the glycosylated forms (upper bands only) of G6PC2 variant proteins were determined in INS-1 832/13 cells by western blot densitometric analysis of Myc-tagged G6PC2 constructs relative to tubulin control (n=5). Representative blots are shown for untreated cells together with cells treated with proteasomal inhibitor MG-132 or lysosomal inhibitor chloroquine. (B) Glucose-6-phosphatase activity of L173T and L173V variants in G6PC (proxy for I171T and I171V in G6PC2 respectively) in HEK293 against increasing glucose-6-phosphate concentrations (n=4), with mean Vmax ± SEM and Km ± SEM values shown for WT and each variant. (C) Glucose-6-phosphatase activity of F258L variant in G6PC (proxy for F256L in G6PC2) in HEK293 against increasing glucose-6-phosphate concentrations (n=3), with mean Vmax ± SEM and Km ± SEM values shown. Vmax and Km results were computed based on the Michaelis-Menten kinetic model. (D) Effect of G6PC2 WT and variant protein expression on luciferase activity driven by ER stress response elements in HEK293 cells. Relative luciferase units corrected for background activity were normalised to WT for each reporter, from n=6 across two independent experiments (except for F256L, n=3 in one experiment) using two-way ANOVA with Fisher’s LSD test comparing each variant to WT. (E) Cellular localisation of R283X in EndoC-βH1 overlaid with markers for the ER (calreticulin) and the trans-golgi network (TGN46). White arrows point to positions of the Golgi apparatus. Scale bar indicates 10μm. (F) Insulin secretion normalised to total content at basal and high glucose conditions (with and without drug treatments) following 96-120h G6PC2 knockdown in EndoC-βH1. Unpaired two-tailed Students’ t tests were used to compare G6PC2 knockdown to control for each condition, from n=16 across 4 independent experiments. Tol: tolbutamide; Diaz: diazoxide. All data presented as mean ± SEM. * p=0.01-0.05; ** p=0.001-0.01; *** p<0.001.

Article Snippet: Human G6PC (NM_000151.3) and G6PC2 cDNA (NM_021176.2) within a pCMV6-Entry vector (with a C-terminal Myc-FLAG-tag) was purchased from OriGene (RC215623 and RC211146 respectively).

Techniques: Expressing, Variant Assay, Western Blot, Construct, Activity Assay, Luciferase, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

doi: 10.1186/s13046-023-02718-w

Figure Lengend Snippet: Deregulated expression of G6pc in human and rat HCCs and association with clinicopathological features. A Venn diagram of miR-494 putative targets (Targetscan algorithm) and downregulated genes in human (TCGA-HCC) and rat (DEN-HCC model) HCCs with respect to surrounding livers. B Kaplan–Meier curves of high and low G6pc-expressing HCCs (TCGA cohort). C-E Box plot graphs of G6pc mRNA levels in HCC and surrounding livers from the TCGA ( N = 49) and Bologna ( N = 46) cohorts and DEN-HCC rats ( N = 18). Y-axes report G6pc mRNA expression. Real Time PCR was run in triplicate. F–H Correlation graphs between miR-494 and G6pc mRNA levels in HCC tissues of the TCGA ( N = 319) and Bologna ( N = 42) cohorts and tumor nodules ( N = 23) of DEN-HCC rats. Axes report 2 −ΔΔCt values corresponding to miR-494 and G6pc levels transformed in a log2 form. Real Time PCR was run in triplicate. I Box plot graphs of G6pc mRNA levels in HCCs from the TCGA ( N = 365) and Bologna ( N = 42) cohorts according to tumor grade. On the top of each graph is reported the p-value relative to ANOVA, whereas stars represent comparison between groups (Tukey’s post hoc test). Y-axes report G6pc mRNA expression. Real Time PCR was run in triplicate. J Box plot graphs of G6pc mRNA levels in HCCs of the TCGA ( N = 298) and Bologna ( N = 25) cohorts divided according to the presence or absence of microvascular invasion (MVI). Y-axes report G6pc mRNA expression. Real Time PCR was run in triplicate. K Correlation graph between G6pc mRNA levels and tumor size of HCC patients ( N = 42) from the Bologna cohort. Axes report 2 −ΔΔ . Ct values corresponding to G6pc mRNA levels and tumor size (cm). GAPDH was used has housekeeping gene. Real Time PCR was run in triplicate. ANOVA, two-tailed unpaired Student's t-test and Pearson’s correlation were used. * P ≤ 0.05; ** P ≤ 0.01

Article Snippet: G6PC (Myc-DDK-tagged) overexpression vector (ID: RC215623) and control vector pCMV6 were from Origene.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Comparison, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

doi: 10.1186/s13046-023-02718-w

Figure Lengend Snippet: G6pc is a target of miR-494 in HCC. A Real Time PCR and WB analyses of G6pc expression following miR-494 overexpression in transfected HCC cells and infected Huh-7 cells (miR-494 Huh-7). NC: negative control precursor miRNA. PMXs: control vector. Y-axis reports 2 −ΔΔ . Ct values corresponding to G6pc mRNA levels normalized to controls (NC or pMXs). Mean ± SD values are displayed. Beta-actin was used as housekeeping gene for Real Time and WB experiments. Real Time PCR analysis was performed in two independent experiments in triplicate; WB analysis was performed in two independent experiments. B Dual-luciferase activity assay of wild type and mutant (mut) G6pc-3UTR vectors (pGL3-G6pc) co-transfected with miR-494 or anti-miR-494 (AM-494) in HepG2 cells. NC: negative control precursor miRNA. NCi: negative inhibitor control miRNA. Y-axes report the firefly/renilla ratio normalized to controls (NC or NCi). Mean ± SD values are displayed. Analysis was performed in two independent experiments in triplicate. C Real Time PCR and WB analyses of G6pc expression in tumor masses (N = 16) of xenograft mice obtained following subcutaneous injection of miR-494-overexpressing and control (pMXs) Huh-7 cells. Y-axis reports G6pc mRNA and protein levels normalized to control. Mean ± SD values are displayed. Beta-actin was used as housekeeping gene for Real Time and WB experiments. Real Time PCR was run in triplicate. D Representative images (4X magnification) of miR-494-overexpressing and control (pMXs) Huh-7 spheroids at 24 h. Data were obtained by measuring feret’s diameter (µm) of thirty randomly selected spheroids in two independent experiments. Mean ± SD values are displayed. Scale bars, 750 μm. E Representative confocal images of control (pMXs) and miR-494-overexpressing Huh-7 spheroids expressing GFP (green signal) and stained with the live-cell oxygen sensor BTP (red signal), which fluorescence emission is quenched by molecular oxygen. Traces represent red and green fluorescence intensity along the linear regions of interest traced in the merged images to visualize signal distribution. At least ten spheroids for condition were analyzed in two independent experiments. Scale bars, 25 μm. F WB analysis of G6pc and HIF-1 in Huh-7 spheroids and ( G ) miR-494-overexpressing Huh-7 cells following AM-494 transfection. NCi: negative inhibitor miRNA control. PMXs: control vector. Beta-actin was used as housekeeping gene. WB analysis was performed in two independent experiments. H Representative images (20X magnification) of PAS staining in control (pMXs) and miR-494-overexpressing Huh-7 cells. Y-axis reports the percent of PAS positive cell area normalized to control. Mean ± SD values are displayed. Five randomly selected fields were analyzed from three independent experiments. Scale bars, 20 μm. I , J Growth curves of control (pMXs) and miR-494-overexpressing Huh-7 cells in standard and starved culture conditions. Growth curves were normalized to T0. Mean ± SD values are reported. Two independent experiments were performed in quadruplicate. K Growth curves of miR-494-overexpressing Huh-7 cells transfected with G6pc overexpressing (G6pc over) or control (pCMV6) vector and grown in starved (medium without FBS) culture conditions. Growth curves were normalized to T0. Mean ± SD values are reported. Two independent experiments were performed in quadruplicate. Statistical significance was determined by two-tailed unpaired Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

Article Snippet: G6PC (Myc-DDK-tagged) overexpression vector (ID: RC215623) and control vector pCMV6 were from Origene.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Over Expression, Transfection, Infection, Negative Control, Control, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis, Injection, Staining, Fluorescence, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

doi: 10.1186/s13046-023-02718-w

Figure Lengend Snippet: MiR-494 regulates cellular metabolism in HCC cells. A Oxygen consumption rate in control (pMXs) and miR-494-overexpressing Huh-7 cells measured in standard medium (endogenous respiration); in the presence of oligomycin A (Oligo) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Mean ± SD values are displayed. Three independent experiments were performed. B Respiratory control ratio of control (pMXS) and miR-494-overexpressing Huh-7 cells. Three independent experiments were performed. C Representative confocal images of control (pMXs) and miR-494-overexpressing Huh-7 cells after staining with TMRM (red) and Mitotracker Green (MTG, green). Inset panels show magnification of selected areas and the violin plot refers to the quantification of the mean length of mitochondrial branches (µM). Data were obtained by measuring at least thirty randomly selected cells in two independent experiments. D Quantification of ΔΨm in control (pMXs) and miR-494-overexpressing Huh-7 cells. Data were obtained by measuring five randomly selected fields in three independent experiments and are expressed as TMRM signal intensity normalized to control. Mean ± SD values are displayed. E Enzymatic activity of succinate dehydrogenase (SDH) and citrate synthase (CS) in control (pMXs) and miR-494-overexpressing Huh-7 cells. The Y-axis reports the enzymatic activity (µmol*min −1 *mg −1 ) normalized to control. Mean ± SD values are displayed. Three independent experiments were analyzed in duplicate. F Real Time PCR analysis of succinate dehydrogenase (SDH) and citrate synthase (CS) expression in control (pMXs) and miR-494 overexpressing Huh-7 cells. Y-axis reports 2 −ΔΔCt values corresponding to mRNA levels normalized to control. Mean ± SD values are displayed. Beta-actin was used as housekeeping gene. Real Time PCR analysis was performed in two independent experiments in triplicate. G Extracellular lactate quantification by HPLC in control (pMXs) and miR-494 overexpressing Huh-7 cells. Mean ± SD values are displayed. Three independent experiments were analyzed in duplicate. H Growth curves of control (pMXs) and miR-494 overexpressing Huh-7 cells in the presence of Antimycin A. Growth curves were normalized to T0. Mean ± SD values are reported. I Growth curves of miR-494 overexpressing Huh-7 cells transfected with G6pc overexpressing or empty (pCMV6) vector in the presence of Antimycin A. Growth curves were normalized to T0. Mean ± SD values are reported. H , I Live imaging curves were performed in two independent experiments in quadruplicate. Statistical significance was determined by two-tailed unpaired Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. PMXs: empty vector

Article Snippet: G6PC (Myc-DDK-tagged) overexpression vector (ID: RC215623) and control vector pCMV6 were from Origene.

Techniques: Control, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Imaging, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

doi: 10.1186/s13046-023-02718-w

Figure Lengend Snippet: MiR-494 regulates lipid metabolism in HCC cells. A Representative confocal images of control (pMXs) and miR-494-overexpressing Huh-7 cells stained with Nile Red to visualize lipid droplets (LDs) accumulation. The cells were cultured for 24 h under standard conditions (RPMI), in medium without glucose, and in the presence of 2-deoxy glucose (2-DG). The Y-axis shows the quantification of LDs number per cell in each condition normalized to pMXs cells cultured in standard conditions. Mean ± SD values are reported. Two independent experiments were performed. Scale bar = 20 µm. B Representative confocal images of miR-494-overexpressing Huh-7 cells following transfection with G6pc overexpressing (G6pc over) or control (pCMV6) vectors for 24 h and stained with Nile Red. The column bar graph below shows the quantification of LDs number per cell respect normalized to control. Two independent experiments were performed. Scale bar, 20 µm. C Growth curves of control (pMXs) and miR-494-overexpressing Huh-7 cells cultured in no-glucose conditions or ( D ) in the presence of 2-DG. Growth curves were normalized to T0. Mean ± SD values are reported. The experiments were performed in two independent experiments in quadruplicate. E Growth curves of miR-494-overexpressing Huh-7 cells transfected with G6p-overexpressing (G6pc over) or control (pCMV6) vector and cultured in no glucose conditions or ( F ) in the presence of 2-DG. Growth curves were normalized to T0. Mean ± SD values are reported. The experiments were performed in two independent experiments in quadruplicate. G Real time PCR analysis of metabolic genes involved in lipid metabolism and pentose phosphate pathway in control (pMXs) and miR-494-overexpressing Huh-7 cells grown for 24 h in no glucose medium or ( H ) in the presence of 2-DG. Y-axis reports 2 −ΔΔ . Ct values corresponding to mRNA levels. Mean ± SD values are displayed. Beta-actin was used as housekeeping gene. Real Time PCR analysis was performed in two independent experiments in triplicate. The statistical analysis was performed using two-tailed unpaired Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

Article Snippet: G6PC (Myc-DDK-tagged) overexpression vector (ID: RC215623) and control vector pCMV6 were from Origene.

Techniques: Control, Staining, Cell Culture, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

doi: 10.1186/s13046-023-02718-w

Figure Lengend Snippet: MiR-494 is a circulating biomarker and a therapeutic target in HCC. A Correlation graphs between tissue and serum miR-494 levels in DEN-HCC rats (N = 10). Axes report 2 −ΔΔCt values corresponding to tissue and serum miR-494 levels transformed in a log2 form. U6RNA and cel-miR-39 were used as control genes for tissue and circulating miRNAs, respectively. Real Time PCR analysis was run in triplicate. B Box plot graph of serum miR-494 levels in xenograft mice ( N = 8) control (pMXs) and miR-494-overexpressing Huh-7 cells. Y-axis reports 2 −ΔΔCt values corresponding to serum miR-494 levels. Cel-miR-39 was used as control gene. Real Time PCR analysis was run in triplicate. C Box plot graphs of G6pc and ( D ) ETFDH tissue levels in HCC patients from the Bologna cohort expressing high ( N = 10) and low ( N = 12) miR-494 serum levels. Y-axes report 2 −ΔΔCt values corresponding to mRNA levels. GAPDH was used as housekeeping gene. Real Time PCR analysis was run in triplicate. E Box plot graph of baseline miR-494 serum levels in responder (R; N = 43) and non-responder (NR; N = 23) sorafenib-treated patients from the Bologna cohort. Y-axis reports 2 −ΔΔCt values corresponding to circulating miR-494 levels. Cel-miR-39 was used as control gene. Real Time PCR analysis was run in triplicate. F Correlation graph between extracellular miR-494 levels and sorafenib resistance in HCC cell lines ( N = 8). Axes report 2 −ΔΔCt values corresponding to extracellular miR-494 levels transformed in a log2 form and sorafenib resistance expressed as the ratio (percent value) of cell viability between treated and untreated cells. Real Time PCR analysis was run in triplicate. G Correlation graph between miR-494 serum levels and tumor size in sorafenib-treated DEN-HCC rats ( N = 12). Axes report 2 −ΔΔCt values corresponding to circulating miR-494 levels and tumor size (volume) of HCC nodules (mm 3 ). Tumor volume was calculated with the formula V = (D1*D2*D3)/2. All the values were transformed in a log2 form. Cel-miR-39 was used as control gene. Real Time PCR analysis was run in triplicate. H Box plot graph of G6pc tissue levels in responder (R; N = 7) and non-responder (NR; N = 7) HCC nodules from sorafenib-treated rats. Y-axis reports 2 −ΔΔCt values corresponding to G6pc mRNA levels. Beta-actin was used as housekeeping gene. Real Time PCR analysis was run in triplicate. I Correlation graph between G6pc tissue levels and miR-494 serum levels in sorafenib-treated DEN-HCC rats ( N = 11). Axes report 2 −ΔΔCt values corresponding to G6pC mRNA levels and circulating miR-494 levels transformed in a log2 form. Beta-actin and cel-miR-39 were used as control genes for tissue mRNAs and circulating miRNAs, respectively. Real Time PCR analysis was run in triplicate. J Correlation graph between G6pc tissue levels and tumor size of HCC nodules ( N = 14) from sorafenib-treated DEN-HCC rats. Axes report 2 −ΔΔCt values corresponding to G6pC mRNA levels and tumor size (volume, mm 3 ) of HCC nodules transformed in a log2 form. Beta-actin was used as housekeeping gene. Real Time PCR analysis was run in triplicate. K Growth curves of miR-494-overexpressing Huh-7 cells transfected with antimiR-494 (AM-494) and negative control (NCi) and cultured in serum-deprived medium or ( L ) in the presence of 2-DG or ( M ) sorafenib. Growth curves were normalized to T0. Mean ± SD values are reported. The curves were performed in two independent experiments in quadruplicate. The statistical analysis was performed using two-tailed unpaired Student's t-test and Pearson’s correlation. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001

Article Snippet: G6PC (Myc-DDK-tagged) overexpression vector (ID: RC215623) and control vector pCMV6 were from Origene.

Techniques: Biomarker Discovery, Transformation Assay, Control, Real-time Polymerase Chain Reaction, Expressing, Transfection, Negative Control, Cell Culture, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

doi: 10.1186/s13046-023-02718-w

Figure Lengend Snippet: Schematic picture of miR-494/G6pc axis involvement in metabolic plasticity of HCC cells and therapeutic potential of combined antimiR-494-based strategies. Metabolic alterations occurring in tumor cells are schematized in the central part of the picture. Circulating miR-494 levels in responder and non-responder HCC patients undergoing sorafenib treatment are presented in the right part of the picture. Treatment combinations with antimiR-494 (AM-494) are proposed for non-responder patients

Article Snippet: G6PC (Myc-DDK-tagged) overexpression vector (ID: RC215623) and control vector pCMV6 were from Origene.

Techniques:

Journal: PLoS Genetics

Article Title: The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

doi: 10.1371/journal.pgen.1002761

Figure Lengend Snippet: (A) HepG2 cells were treated with DMSO, NOG (1 mM), or DMOG (0.1 mM) for 12 hr. (B) shRNA-mediated screen. Each bar represents a single shRNA construct. Data were presented as fold relative to the scramble control. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Left, gene expression data were presented as fold relative to the scramble control from three experiments. Full names of individual genes are listed in . Right, levels of PEPCK and G6Pase protein were determined. (D) Jhdm1a knockdown or scramble HepG2 cells were treated with NOG (1 mM) for 12 hr. (E) Jhdm1a knockdown or scramble HepG2 cells were treated with a combination of dibutyryl cyclic-AMP (cAMP, 0.5 mM) and dexamethasone (Dex, 1 µM) in DMEM medium for 6 hr. Data were from two experiments. (F) Lentiviral knockdown of Jhdm1a in mouse hepatoma HepA1-6 cells. Data were from two experiments. (G) Adenoviral knockdown of Jhdm1a in mouse primary hepatocytes. Experiments were repeated three times with similar results.

Article Snippet: 26 μg protein extracts were separated by SDS-PAGE and probed with antibody against C/EBPα (Santa Cruz, sc-61), PEPCK (ABcam, ab28455) or G6Pase (Santa Cruz, sc-25840).

Techniques: shRNA, Construct, Control, Gene Expression, Knockdown

Journal: PLoS Genetics

Article Title: The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

doi: 10.1371/journal.pgen.1002761

Figure Lengend Snippet: (A) Jhdm1a knockdown or scramble adenoviruses were transduced into the liver of wild-type male C57BL/6J mice (n = 5 per group). Mice fed ad libitum were sacrificed at Day 5 after viral infusion. (Left) mRNA levels of PEPCK, G6Pase and Jhdm1a in the liver were measured and normalized to U36b4. **, P<0.005. (Right) PEPCK and G6Pase protein. (B) Blood insulin levels at fed state were measured at Day 5. (C) Jhdm1a knockdown or scramble adenoviruses were transduced into the liver of wild-type male C57BL/6J mice (n = 10 per group). At Day 5, mice were i.p. injected with pyruvate (2 g/kg body weight) after a starvation for 16 hr and blood glucose levels were measured. *, P<0.05. (D and E) Adenoviruses expressing wild-type Jhdm1a, H212A point mutant, or GFP were transduced into the liver of male ob/ob mice (n = 5 per group). Gene expression was measured on Day 5 and blood glucose levels were measured on Day 3 after a 5-hr fasting. Changes of blood glucose level relative to Day 0 are presented. *, P<0.03; **, P<0.01. ( F ) Hepatic Jhdm1a mRNA levels in male C57BL/6J mice (n = 5 per group) fed ad libitum , or fasted for 5 hr or 20 hr. (G) Male C57BL/6J mice (n = 4) were i.p. injected with glucagon (300 µg/kg), insulin (0.75 U/Kg), or PBS. Hepatic Jhdm1a mRNA levels were examined 6 hr after injection. (H) Hepatic Jhdm1a mRNA levels in lean mice and diabetic ob/ob mice (n = 3 per group).

Article Snippet: 26 μg protein extracts were separated by SDS-PAGE and probed with antibody against C/EBPα (Santa Cruz, sc-61), PEPCK (ABcam, ab28455) or G6Pase (Santa Cruz, sc-25840).

Techniques: Knockdown, Injection, Expressing, Mutagenesis, Gene Expression

Journal: PLoS Genetics

Article Title: The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

doi: 10.1371/journal.pgen.1002761

Figure Lengend Snippet: (A) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Expression levels of known transcriptional regulators for gluconeogenesis were examined. Data are presented as fold relative to the scramble control from three experiments. ***, P<0.00005. (B) C/EBPα expression in Jhdm1a knockdown mouse primary hepatocytes. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Endogenous C/EBPα association with known binding sites on the PEPCK and G6Pase promoters was examined by ChIP assay. (D) Gene expression in HepG2 cells infected with lentiviruses expressing C/EBPα or vector. (E) Increased C/EBPα expression in the liver of wild-type C57BL/6J mice (n = 5 per group) with Jhdm1a knockdown. C/EBPα mRNA level and protein level were shown from independent groups of mice. **, P<0.02. (F) Decreased C/EBPα expression in the liver of ob/ob mice (n = 5 per group) ectopically expressing wild-type Jhdm1a, but not in the liver expressing H212A point mutant. ***, P<0.001. (G) HepG2 cells were infected with lentiviruses expressing C/EBPα shRNA and selected with puromycine. Cells were then infected with lentiviruses expressing Jhdm1a shRNA without selection. Data were shown from one representative of four experiments. Note, the low induction of PEPCK and G6Pase expression by Jhdm1a knockdown is due to the lack of selection pressure.

Article Snippet: 26 μg protein extracts were separated by SDS-PAGE and probed with antibody against C/EBPα (Santa Cruz, sc-61), PEPCK (ABcam, ab28455) or G6Pase (Santa Cruz, sc-25840).

Techniques: shRNA, Expressing, Control, Knockdown, Binding Assay, Gene Expression, Infection, Plasmid Preparation, Mutagenesis, Selection

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Maternal protein restriction induces alterations in insulin signaling and ATP sensitive potassium channel protein in hypothalami of intrauterine growth restriction fetal rats

doi: 10.3164/jcbn.12-28

Figure Lengend Snippet: Primers for real time reverse transcription polymerase chain reaction

Article Snippet: Antibodies to Kir6.2, IRα, IRS2, p85α, SUR1, phosphoenolpyruvate carboxykinase (PEPCK), and Glucose 6-phosphatase (G6Pase) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Maternal protein restriction induces alterations in insulin signaling and ATP sensitive potassium channel protein in hypothalami of intrauterine growth restriction fetal rats

doi: 10.3164/jcbn.12-28

Figure Lengend Snippet: Maternal undernutrition increased hepatic gluconeogenesis enzymes expression in fetuses. (A) Real time PCR analysis of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) mRNA level in control (white bars) and IUGR (black bars) livers. (B and C) Immunoblotting assay of PEPCK and G6Pase protein expression and representative immunoblots. The data were normalized to the control, and represent the mean values (± SE), n = 6 rats/group. * p <0.05; ** p <0.01 vs control.

Article Snippet: Antibodies to Kir6.2, IRα, IRS2, p85α, SUR1, phosphoenolpyruvate carboxykinase (PEPCK), and Glucose 6-phosphatase (G6Pase) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot