ad g6pc1  (Obio Technology Corp Ltd)

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: FTH Sustains Hepatic G6Pase in Response to Systemic Polymicrobial Infection (A) Volcano plot of mean RNA expression from RNA microarray screen in the liver of Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice, 48 hr after CLP (n = 4 mice per group). (B) Validation of G6pc1 mRNA expression by qRT-PCR in the liver of Mx1 Cre Fth Δ/Δ mice not subjected to CLP (n = 13), 48 hr after CLP (n = 12) versus Fth lox/lox mice not subjected to CLP (n = 11), or 48 hr after CLP (n = 11). Data pooled from three independent experiments. (C and D) Representative western blot of G6pc1 in Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice (C) and relative quantification by densitometry (D) before (Control; Ctr) or 48 hr after CLP (n = 6 per group). (E) Quantification of liver Gpt1 mRNA by qRT-PCR, same mice as (B). (F) Liver G6Pase enzymatic activity, same mice as (B). (G) Quantification of G6PC1 mRNA levels by qRT-PCR in HepG2 cells untreated (−) or treated (+) with heme and/or TNF. Mean ± SEM from eight independent experiments with similar trend. (H) G6PC1 protein levels in HepG2 cells treated as in (G). (I) Relative quantification G6PC1 protein levels by densitometry of western blot from HepG2 cells treated as in (G) and (H). (J) Relative luciferase units (RLU) in HepG2 cells transiently co-transfected with a rat G6pc1 firefly luciferase and CMV Renilla luciferase reporters. Control cells were transfected with a promoterless firefly luciferase reporter. Cells were treated, 48 hr after transfection, with heme and TNF. Data are shown as mean RLU ± SD from four independent experiments with similar trend. (K) Liver mRNA quantification by qRT-PCR in C57BL/6 mice receiving heme (+; n = 8) (i.p. 30 mg/kg BW) or not (−; n = 9). Data pooled from three independent experiments. (L) Liver mRNA quantification by qRT-PCR in Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice receiving heme (+; i.p. 15 mg/kg BW; n = 3 per genotype) or not (−; n = 6–8 per genotype). Data pooled from one to three independent experiments. (M) Relative luciferase units (RLU) in HepG2 cells transiently co-transfected with a rat G6pc1 firefly luciferase and a CMV Renilla luciferase reporters plus a human FTH expression vector. Cells transfected with a promoterless firefly luciferase reporter were used as baseline RLU. Transfected cells were treated 48 hr thereafter with heme and TNF and analyzed 12 hr thereafter. Data shown as mean RLU ± SD from five independent experiments with similar trend. Red bars in (B), (D)–(F), (J), and (K) represent mean values and doted circles indicate individual mice. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.01. See also Figure S6 .

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Infection, RNA Expression, Microarray, Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, Activity Assay, Luciferase, Transfection, Plasmid Preparation

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Liver Gluconeogenesis Is Required to Establish Disease Tolerance to Sepsis, Related to and ( a ) Western Blot of G6PC1 and p21 in HepG2 cells untreated (-) or treated (+) with heme and TNF in the presence (+) or absence (-) of Lactacystin (Lact.) or MG132. Actin was used as loading control. Data are representative of three independent experiments with the same trend. ( b ) G6Pase enzymatic activity in HepG2 cells transduced (+) or not (-) with a G6PC1 Rec.Ad. and when indicated (+) co-transduced with a FTH Rec.Ad. Transduction with a LacZ Rec. Ad. was used as a control. NT: Not transduced. Cells were treated (+) or not (-) with heme and TNF 72 hr after Rec.Ad. transduction. Data are shown as mean ± SD, pooled from 2 independent experiments with four replicates each. ( c ) Temperature and ( d ) weight of G6pc1 lox/lox (n = 12) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 12) mice subjected to CLP. Data are shown as mean ± SEM, pooled from 3 independent experiments. ( e ) Blood glucose and ( f ) clinical severity score in Control (C57BL/6; n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice subjected to CLP. Mean ± SD pooled from 2 independent experiments. ( g ) Serological markers of organ injury in C57BL/6 (n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice 6 hr mice after CLP. ( h ) H&E stained paraffin sections representative of at least four G6pc1 lox/lox and Alb Cre ER T2 G6pc1 Δ/Δ mice 9 hr after PCI. B: bronchus; CV: central vein; G: glomerulus. Magnification 400x. ( i ) Temperature and ( h ) weight and ( j ) blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 7) mice receiving heme ( i.p ; 30 mg/kg BW). Mean ± SD from 2 independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Western Blot, Control, Activity Assay, Transduction, Staining

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Liver Gluconeogenesis Is Critical to Establish Disease Tolerance to Sepsis (A) G6pc1 protein expression in the liver of control ( G6pc1 lox/lox ; Ctr) and Alb Cre ER T2 G6pc1 Δ/Δ ( G6pc1 Δ/Δ ) mice. (B) Blood glucose levels of G6pc1 lox/lox (n = 12) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 12) mice subjected to CLP. Data are shown as mean ± SEM from three independent experiments. (C) Survival of control ( G6pc1 lox/lox ; n = 10) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 10) mice subjected to CLP. Data were pooled from two independent experiments with similar trend. (D) Blood glucose of control (C57BL/6; n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice subjected to PCI. Data are shown as mean ± SD from two independent experiments with the same trend. (E) Survival of control (C57BL/6; n = 10) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 8) subjected to PCI. Data pooled from three independent experiments with similar trend. (F) CFU for aerobic (Ae) and anaerobic (An) bacteria, 6 hr after PCI. Red bars represent mean values and dotted circles represent individual mice. PC, peritoneal cavity. (G) Blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 7) mice receiving heme (i.p. 30 mg/kg BW). Mean ± SD from two independent experiments. (H) Survival of same mice as (G). (I) Blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 9) mice receiving LPS (i.p. 5 mg/kg BW). Mean ± SD from two independent experiments. (J) Survival of same mice as (I). (K) Blood glucose levels in G6pc1 lox/lox (n = 9) and Alb Cre ER T2 Gpc1 Δ/Δ (n = 10) mice receiving poly(I:C) (intra-retro orbital [i.r.o.] 30 mg/kg BW). Mean ± SD from two independent experiments. (L) Survival of same mice as (K). Numbers in gray (B, D, G, I, and K) are live/total mice at each time point. ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S6 .

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Expressing, Control, Bacteria

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: FTH Overexpression or Ferritin Administration Induce Disease Tolerance to Sepsis (A) Survival of C57BL/6 mice transduced 48 hr before severe CLP with FTH Rec.Ad. (n = 11), ferroxidase-deficient FTH Rec.Ad. (FTH m ; n = 11), LacZ Rec.Ad. (n = 12), or receiving vehicle (PBS; n = 19). Data pooled from six independent experiments with similar trend. (B) CFU for aerobic (Ae) and anaerobic (An) bacteria, 12 hr after CLP in mice treated as in (A). (C) Representative western blot in HepG2 cells transduced with LacZ, FTH, or mutated FTH (FTH m ) Rec.Ad. (D and E) Densitometry of G6PC1 (D) and FTH protein (E) expression normalized to α-tubulin (n = 4 per group). (F) Expression of G6pc1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (−) to heme, TNF, and deferoxamine (DFO). Mean ± SEM from three independent experiments done in duplicates. (G) Survival of C57BL/6 mice receiving apoferritin (n = 16), ferritin (n = 10), BSA (n = 12), or saline (n = 12), 24 hr before severe CLP. Data pooled from five independent experiments. (H) CFU for aerobic (Ae) and anaerobic (An) bacteria 12 hr after severe CLP in mice treated as in (G). Data from three independent experiments. (I) Survival of C57BL/6 mice receiving apoferritin (n = 22), BSA (n = 20), or saline (n = 16) 6 hr after severe CLP. Data pooled from seven independent experiments. Red bars represent mean values and dotted circles represent individual mice (B and G). PC, peritoneal cavity; NS, non-significant. ∗ p < 0.05, ∗∗ p < 0.01. See also Figure S7 .

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Over Expression, Bacteria, Western Blot, Transduction, Expressing, Quantitative RT-PCR, Saline

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Antioxidants Bypass the Requirement of FTH in the Establishment of Normoglycemia and Disease Tolerance to Sepsis (A–C) Relative weight (A), blood glucose (B), and survival (C) of Mx1 Cre Fth Δ/Δ mice subjected to CLP and receiving NAC (n = 7; 15 mg/kg) or vehicle (n = 6). Data pooled from two independent experiments. (D) Colony forming units (CFU) for aerobic (Ae) and anaerobic (An) bacteria, 48 hr after CLP. PC, peritoneal cavity. Red bars represent mean values and doted circles represent individual mice. Pooled from two independent experiments. (E) Expression of G6PC1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (−) to heme, TNF, and/or NAC. Data shown as mean ± SD from four independent experiments. (F and G) Blood glucose (F) and survival (G) of Mx1 Cre Fth Δ/Δ mice subjected to CLP and receiving BHA (n = 11; 50 mg/kg) or vehicle (n = 9). Data shown as mean ± SD. Pooled from three independent experiments. (H–J) Relative weight (H), blood glucose (I), and survival (J) of Alb Cre ER T2 G6pc1 Δ/Δ mice subjected to CLP and receiving NAC (n = 5) or vehicle (n = 5). Mean ± SD from one experiment. NS, non-significant. ∗ p < 0.05, ∗∗ p < 0.01. Numbers in gray (A, B, F, I, and J) indicate live/total mice.

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Bacteria, Expressing, Quantitative RT-PCR

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet:

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Virus, Recombinant, cDNA Synthesis, Luciferase, SYBR Green Assay, Labeling, Enzyme-linked Immunosorbent Assay, Bioassay, Kinase Assay, Gene Expression, Software, Combined Bisulfite Restriction Analysis Assay, Stripping Membranes, Control