Journal: PLoS Pathogens

Article Title: Divergent Effects of Human Cytomegalovirus and Herpes Simplex Virus-1 on Cellular Metabolism

doi: 10.1371/journal.ppat.1002124

Figure Lengend Snippet: HSV-1 replication is inhibited by reducing flux from glucose toward pyrimidine nucleotide synthesis. ( A ) Schematic diagram of glucose flux to pyrimidine nucleotide biosynthesis. Red lines mark siRNA-targeted reactions catalyzed by pyruvate carboxylase (PC) and aspartate transaminase 2 (GOT2). (OAA: oxaloacetate, AKG: oxoglutarate, Gln: glutamine). ( B ) RNA interference knockdown of pyruvate carboxylase (marked by arrow) in MRC5 cells. Cells were transfected with non-targeting siRNAs (NT) or siRNAs targeting pyruvate carboxylase (PC) and harvested at indicated time points after transfection. Pyruvate carboxylase levels in the cells were detected by western blot using specific antibodies. Beta-actin was employed as a loading control. ( C ) Buildup of 13 C 3 -labeled malate after switching MRC5 cells to uniformly 13 C-labeled glucose medium for 2 hours at 10 hpi of HSV-1 (F) infection. The cells have been transfected with a universal non-targeting siRNA (NT) or an siRNA targeting pyruvate carboxylase (PC) 120 h prior to infection (significance: p = 0.007). Symbols indicate experimental data points ±1 s.d.; n = 3; values are given in arbitrary units. ( D ) Production of infectious HSV-1 (F) and HCMV (AD169) virions in cells transfected with siRNAs against pyruvate carboxylase (PC), aspartate transaminase 2 (GOT2), or a universal negative control (NT). The transfection and infection of MRC5 cells were performed as described in Materials and Methods . Values are expressed relative to non-targeting control (±1 s.d.; n = 3). Conditions resulting in significantly altered virus production (p≤0.05) compared to treatment with the universal negative control are marked with a star.

Article Snippet: siRNA transfection Double stranded siRNA molecules directed against pyruvate carboxylase ( 5′-GACUGUACGCGGCCUUCGATT ), aspartate transaminase 2 ( 5′-CUAUUGAGAGCUUCACACATT ), and a Universal Negative Control (SIC001) were purchased from Sigma.

Techniques: Transfection, Western Blot, Labeling, Infection, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Paracrine Fibroblast Growth Factor 1 Functions as Potent Therapeutic Agent for Intrahepatic Cholestasis by Downregulating Synthesis of Bile Acid

doi: 10.3389/fphar.2019.01515

Figure Lengend Snippet: The mRNA and protein levels of FGF1 in liver were downregulated in the ANIT-induced intrahepatic cholestasis mouse model. Eight-week-old C57BL/6J mice were orally administrated with olive oil (control group) or 75 mg/kg ANIT (cholestasis mouse model) for once and then liver tissues were collected and analyzed after 48 h. (A , B) Evaluation of liver function as determined by serum levels of ALT and AST. (C , D) Serum BA concentrations (C) and hepatic BA pool (D) of each group were measured. (E) The levels of hepatic FGF1 gene expression of each group were examined by real-time polymerase chain reaction (RT-PCR); (F) Representative western blot analysis (up panel) and densitometric quantification (down panel) of FGF1 protein expression in liver tissues from ANIT-induce intrahepatic cholestasis mouse model and control group; data are normalized to GAPDH. Data are presented as mean ± SEM; ****p

Article Snippet: Alanine transaminase (ALT) and aspartate transaminase (AST) Assay Kits were purchased from Sigma Aldrich (St. Louis, MO, USA) and performed in accordance with the protocols provided with manufacturers.

Techniques: Mouse Assay, AST Assay, Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot