Journal: mBio

Article Title: A Poxvirus Decapping Enzyme Colocalizes with Mitochondria To Regulate RNA Metabolism and Translation and Promote Viral Replication

doi: 10.1128/mbio.00300-22

Figure Lengend Snippet: Loss of mitochondrial colocalization reduces D10’s gene expression shutoff capability. (A) Inactivation of D9 and D10’s decapping activities leads to highly accumulated m 7 G caps between viral factories and nuclei in VACV-infected cells. A549DKO cells were infected with WT or vD9muD10mu (MOI = 3) or mock infected. Confocal microscopy was used to visualize m 7 G cap (α-cap antibody, green) and DNA (DAPI, blue) at 16 hpi. Three zoomed-in areas were shown on the right. (B and C) Loss of mitochondrial colocalization leads to regions with highly accumulated caps in the cytoplasm of VACV-infected cells. A549DKO cells were infected with indicated viruses at an MOI of 3. Confocal microscopy was used to visualize m 7 G cap (α-cap antibody, green) and DNA (DAPI, blue) at 16 hpi. The numbers indicate the percentages of cells containing regions with accumulated caps from at least six views. (D) vΔD9 infection does not lead to highly accumulated cap regions in the cytoplasm of VACV-infected cells. A549DKO cells were infected with indicated viruses at an MOI of 3. Confocal microscopy was used to visualize m 7 G cap (α-cap antibody, green) and DNA (DAPI, blue) at 16 hpi. The numbers indicate the percentages of cells containing regions with accumulated caps in the cytoplasm from multiple randomly pictured views. Arrows indicate two cells with an increase of cap staining in the nuclei of vΔD9-infected cells. (E) Loss of D10 mitochondrial colocalization reduces its ability to shut off gene expression. Plasmid encoding a Gaussia luciferase reporter gene under a cellular EF-1α promoter was cotransfected with the indicated plasmids encoding codon-optimized D10 or D10 mutants. Gaussia luciferase activities were measured 24 h posttransfection. (F) Western blotting of D10 and D10 mutant protein levels (a representative image of three biological repeats). Error bars represent the standard deviation of at least three replicates. ****, 0.001 < P ≤ 0.0001.

Article Snippet: The Gaussia luciferase activities were measured using a luminometer using the Pierce Gaussia luciferase flash assay kit (Thermo Scientific; catalog no. 16158).

Techniques: Gene Expression, Infection, Confocal Microscopy, Staining, Plasmid Preparation, Luciferase, Western Blot, Mutagenesis, Standard Deviation

Journal: Journal of hepatology

Article Title: Serum-based tracking of de novo -initiated liver cancer progression reveals early immunoregulation and response to therapy

doi: 10.1016/j.jhep.2015.06.021

Figure Lengend Snippet: IL-18+IL-12 therapy against MET+CAT oncogene-induced liver tumors. MET (10ug)+CAT (10ug) oncogenes along with G.luc (4ug) and HSB2 (2.4ug) were hydrodynamically-transfected into C57/Bl6 mice. On days 20–24 and 28–31 mice were treated with vehicle control (VC) or IL-18+IL-12. (A) Serum Gaussia Luciferase levels vs. Day. (B) Livers were weighed on day 61. (C) DNA was isolated from livers on day 61 and MET and CAT oncogene copy number was qPCR quantitated from 100 ng of DNA. VC n=5, IL-18+12 n=10, mean ± SEM. For B and C Wilcoxon Rank Sum test was used to determine significance.

Article Snippet: Serum luciferase levels were determined using the BioLux Gaussia Luciferase Assay kit (New England BioLabs) according to manufacturer’s direction and luminescence measurements were acquired using FLUOstar Omega microplate reader (BMG LABTECH) after controlled injection of 50 μl of substrate mixture into plates containing the serum PBS mixture.

Techniques: Transfection, Luciferase, Isolation