Journal: Journal of Virology

Article Title: Selective 4-Thiouracil Labeling of RNA Transcripts within Latently Infected Cells after Infection with Human Cytomegalovirus Expressing Functional Uracil Phosphoribosyltransferase

doi: 10.1128/JVI.00880-18

Figure Lengend Snippet: TB40/E GATA::UPRT IE2-2AeGFP displays wild-type growth kinetics in fibroblasts. (A) Schematic representation of the recombinant virus. BAC-derived TB40/Ewt-mCherry (WT) was used to generate TB40/E GATA::UPRT IE2-2AeGFP using standard recombineering techniques. Black arrows indicate unaltered, flanking ORFs. The stop codon of UL122 (white arrow) was replaced in the first recombination event with galK, which was subsequently replaced with a 2A self-cleaving element in frame with eGFP. A second recombineering event in the United States region of the genome inserted a double-stranded DNA fragment containing the GATA2 promoter and codon-optimized UPRT downstream of US34A. Striped boxes denote conserved homology arms. TRL and TRS, long and short terminal repeats, respectively; UL and US, unique long and short regions, respectively, IRL and IRS, long and short internal repeats, respectively. (B) Fibroblasts (MRC-5) were infected at an MOI = 1 PFU/cell with either TB40/Ewt-eGFP (WT) or TB40/E GATA::UPRT IE2-2AeGFP (UPRT). Cell-free virus was collected over 8 d and quantified by TCID50 (n = 3). Inoc, inoculum.

Article Snippet: Primary neonatal human foreskin fibroblasts (NuFF-1; GlobalStem) or primary human embryonic lung fibroblasts (MRC5; ATCC) were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, and 100 U/ml each penicillin and streptomycin.

Techniques: Recombinant, Virus, Derivative Assay, Infection

Journal: Journal of Virology

Article Title: Selective 4-Thiouracil Labeling of RNA Transcripts within Latently Infected Cells after Infection with Human Cytomegalovirus Expressing Functional Uracil Phosphoribosyltransferase

doi: 10.1128/JVI.00880-18

Figure Lengend Snippet: Humanized T. gondii UPRT is expressed by the recombinant HCMV and allows for viral transcript enrichment of thiol-RNAs. (A) Fibroblasts were infected with TB40/E GATA::UPRT IE2-2AeGFP (MOI = 0.1 PFU/cell), and UPRT expression was assessed by RT-qPCR over the indicated times. All samples were analyzed in triplicate and normalized to GAPDH. AU, arbitrary units. (B and C) Fibroblasts were infected with either TB40/Ewt-eGFP (WT) or TB40/E GATA::UPRT IE2-2AeGFP (UPRT) at an MOI = 1 PFU/cell. At 36 hpi, media containing DMSO, 100 ∝M 4-thiouracil (4tU), or 200 ∝M 4-thiouridine (4sU) was added for the respective labeling duration (DMSO, 6 h; 4tU, 6 h; 4sU, 2 h). Total RNA was isolated, and 50 μg was used for thiol-specific biotinylation. (B) Thiol-specific biotinylated RNA (1 μg) was separated on a denaturing urea gel, transferred to nitrocellulose, and detected by probing with streptavidin-HRP specific antibody. A representative Northern blot is shown. Migration of the ribosomal 28S and 18S RNA species is indicated on the right. (C) Biotinylated RNA (25 μg) was incubated with streptavidin microbeads and column purified. Equal volumes of eluted RNA were used to generate cDNA, and UL122 expression was assessed by RT-qPCR. Samples were analyzed in triplicate and normalized to total biotinylated RNA reserved prior to streptavidin purification.

Article Snippet: Primary neonatal human foreskin fibroblasts (NuFF-1; GlobalStem) or primary human embryonic lung fibroblasts (MRC5; ATCC) were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, and 100 U/ml each penicillin and streptomycin.

Techniques: Recombinant, Infection, Expressing, Quantitative RT-PCR, Labeling, Isolation, Northern Blot, Migration, Incubation, Purification

Journal: Redox Biology

Article Title: The Nrf2-inducers tanshinone I and dihydrotanshinone protect human skin cells and reconstructed human skin against solar simulated UV ☆

doi: 10.1016/j.redox.2013.10.004

Figure Lengend Snippet: Tanshinones, phenanthrenequinone-based natural products from the medicinal plant Salvia miltiorrhiza , cause Nrf2 transcriptional activation in human dermal fibroblasts. (A) Chemical structure of four major tanshinones [tanshinone I (T-I), dihydrotanshinone (DHT), tanshinone IIA (T-II-A), and cryptotanshinone (CT)]. (B) After transfection using an expression vector encoding an mGST-ARE firefly luciferase (F) reporter gene, human dermal Hs27 fibroblasts were exposed to test compounds (T-I, DHT, T-IIA, CT, SF; 5 µM; 16 h) or remained untreated. A plasmid encoding renilla luciferase (R) driven by the herpes simplex virus thymidine kinase promoter was included in all transfections to normalize transfection efficiency. Dual luciferase activities (R/F) were measured, and potency of fold induction is expressed as relative luminescence units (R/F) versus untreated control transfectants (mean±SD, n =3; ⁎ p <0.05).

Article Snippet: Human dermal neonatal foreskin Hs27 fibroblasts from ATCC and human immortalized HaCaT keratinocytes were cultured in DMEM containing 10% fetal bovine serum.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Virus, Control

Journal: Redox Biology

Article Title: The Nrf2-inducers tanshinone I and dihydrotanshinone protect human skin cells and reconstructed human skin against solar simulated UV ☆

doi: 10.1016/j.redox.2013.10.004

Figure Lengend Snippet: Tanshinone I and dihydrotanshinone upregulate protein levels of Nrf2 and Nrf2 targets in human dermal fibroblasts. (A) Dose–response relationship of tanshinone-induced the upregulation of Nrf2 protein levels. Hs27 fibroblasts were exposed to four types of tanshinones (T-I, DHT, T-IIA, CT; 5 µM, each; 4 h) followed by immunoblot analysis. SF (5 μM; 4 h) treatment was included as a positive control. (B) Time course of tanshinone-induced the upregulation of Nrf2, NQO1, and γ -GCS. Hs27 fibroblasts were exposed to tanshinones (T-I, DHT; 5 µM, 2–24 h) and total cell lysates were subjected to immunoblot analysis. Detection of GAPDH served as a loading control. (C) Quantitative analysis of triplicate gels as presented in panel A. (D) Quantitative analysis of triplicate gels as presented in panel B. Relative intensity of protein bands was determined using gel documentation software as described in “Materials and methods” (mean±SD, n =3; ⁎ p <0.05).

Article Snippet: Human dermal neonatal foreskin Hs27 fibroblasts from ATCC and human immortalized HaCaT keratinocytes were cultured in DMEM containing 10% fetal bovine serum.

Techniques: Western Blot, Positive Control, Control, Software

Journal: Redox Biology

Article Title: The Nrf2-inducers tanshinone I and dihydrotanshinone protect human skin cells and reconstructed human skin against solar simulated UV ☆

doi: 10.1016/j.redox.2013.10.004

Figure Lengend Snippet: Tanshinones increase Nrf2 half-life via interference with ubiquitination, elevate intracellular glutathione, and protect dermal fibroblasts against UV-induced cytotoxicity. (A) Tanshinone-modulation of Nrf2 and Nrf2 target gene expression. Hs27 Fibroblasts were treated with T-I and DHT (5 μM; 16 h) and mRNA was extracted. The relative mRNA levels of Nrf2 and its downstream genes ( NQO1 , GCLM ) were determined by quantitative real-time RT-PCR with triplicate samples in each experiment. The experiment was conducted in triplicate and expressed as mean±SD (* p <0.05). (B) Tanshinone-modulation of Nrf2-ubiquitination. Hs27 cells were co-transfected with plasmids encoding the indicated proteins (Nrf2, HA-Ub). Cells were then treated with SF, T-I or DHT (5 μM; 4 h) along with MG132 (10 μM; 4 h) before cell lysates were collected for the ubiquitination assay. For the detection of ubiquitin-conjugated Nrf2, anti-Nrf2 immunoprecipitates were analyzed by immunoblotting using an anti-HA antibody. (C) Tanshinone-modulation of Nrf2 protein half-life. Hs27 cells were left untreated or treated with T-I or DHT (5 μM; 4 h). Cycloheximide (50 μM) was added and cells were harvested at the indicated time points (0–45 min). Cell lysates were subjected to immunoblot analysis using anti-Nrf2 and anti-GAPDH antibodies. The intensity of the bands was quantified using Quantity One software and plotted against the time after cycloheximide treatment. (D) Intracellular total glutathione levels relative to untreated control were determined in fibroblasts exposed to tanshinones (T-I, DHT) or SF (5 μM, each; 24 h) using the QuantiChrom glutathione assay kit as detailed in Materials and Methods. (E and F) To assess cytoprotective effects of tanshinone treatment on UV-induced cytotoxicity, fibroblasts were first preincubated in the presence of T-I or DHT (5 µM; 24 h) and then exposed to full spectrum solar simulated UV [solar simulated light (SSL); panel E] or solar simulated UVA delivered in the presence of the photosensitizer riboflavin [5 μM (UVA+riboflavin); panel F], followed by assessment of cell viability at 24 h post-irradiation. Data are expressed as means±SD ( ⁎ p <0.05; n =3).

Article Snippet: Human dermal neonatal foreskin Hs27 fibroblasts from ATCC and human immortalized HaCaT keratinocytes were cultured in DMEM containing 10% fetal bovine serum.

Techniques: Ubiquitin Proteomics, Targeted Gene Expression, Quantitative RT-PCR, Transfection, Western Blot, Software, Control, Glutathione Assay, Irradiation