Journal: Journal of Virology

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Nonstructural Membrane Protein pK15 Recruits the Class II Phosphatidylinositol 3-Kinase PI3K-C2α To Activate Productive Viral Replication

doi: 10.1128/JVI.00544-18

Figure Lengend Snippet: Depletion of PI3K-C2α from infected endothelial cells leads to reduced KSHV lytic replication. HuARLT2-rKSHV cells were microporated with either a control siRNA (Scr), four individual siRNAs or a pool of these siRNAs against PI3K-C2α, or a single siRNA against K15, and the KSHV lytic cycle was induced 24 h later using a cocktail of RTA and SB. (A to C) Forty-eight hours after lytic induction, images for GFP and RFP expression were acquired (A), and the number of RFP-positive cells from five or more fields was quantified; cells were then lysed, and the expression level of the indicated viral proteins was assessed by Western blotting (C). Results are representative of data from two independent experiments. The scatter plot (B) represents the means ± standard deviations (SD) for five or more fields. Ordinary one-way analysis of variance (ANOVA) was used to determine P values. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D and E) Primary lymphatic endothelial cells infected with KSHV for 2 weeks (LEC-rKSHV) were transfected with either the pooled PI3K-C2α siRNA or a scrambled control. After 72 h, cells and the culture supernatant were collected. The expression levels of the indicated proteins were analyzed by Western blotting (D), and the amount of released infectious virus was assayed by titrating the culture supernatant on HEK-293 cells (E). A Mann-Whitney test was used to determine P values. **, P < 0.01.

Article Snippet: Primary juvenile foreskin lymphatic endothelial cells (LECs; Lonza) were provided by Päivi M. Ojala (University of Helsinki, Helsinki, Finland) and were grown in EGM-2MV medium (Lonza, Walkersville, MD, USA).

Techniques: Infection, Control, Expressing, Western Blot, Transfection, Virus, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Aging/aged hLECs displayed increased accumulation of ROS, which was associated with progressive decline in Prdx6, Cat and Nrf2 expression. ( A ) Excessive accumulation of ROS in aging/aged hLECs. Primary hLECs isolated from lenses of different ages were divided into six groups: 16–21 y (n = 6); 24–26 y (n = 6); 34–36 y (n = 4); 52–58 y (n = 6); 62–68 y (n = 12); 75 y (n = 4). Cells were cultured in 96 well plate (5000/well), and ROS were quantified using H2-DCF-DA dye assay as shown. Data represent the mean ± S.D. of two independent experiments. 16–21 y vs 24–26 y, 34–36 y, 52–58 y, 62–68 y and 75 y (aging samples); *p < 0.001. ( B – D ) Aging/aged hLECs showing a significant loss of Prdx6, Cat and Nrf2. Total RNA was isolated from hLECs and human lenses of different ages as indicated and was processed for real-time PCR analysis. # LECs directly detached from lenses and were used for assays to avoid cell culture effects. The data represent the mean ± S.D. from three independent experiments. p values were determined for younger vs aging samples. * p < 0.001.

Article Snippet: Primary rat LECs (rLECs) were isolated from 6-week-old Sprague-Dawley albino rats (n = 8) as described previously .

Techniques: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Rat LECs treated with SFN displayed increased levels of antioxidant genes/proteins, Prdx6, Cat, and phase II protein GST π . (A) Determination of noncytotoxic concentration of SFN in primary culture of rLECs. Primary cultures of rLECs were treated with different concentrations of SFN as indicated for 24 h. Cells were subjected to MTS assay to measure viability. Histogram reflects values; nontoxic concentrations were 2.4 μM and 4.8 μM. DMSO vs SFN treated samples; *p < 0.001, **p < 0.05. ( B and C ) rLECs treated with SFN showed enhanced expression of Prdx6 mRNA and protein. Cells were treated with 2.4 μM or 4.8 μM of SFN or DMSO as indicated. Total RNA and protein were isolated. Real-time PCR and Western analysis with Prdx6 specific probes revealed a concentration-dependent increased expression of Prdx6 mRNA ( B ) and protein ( C ). ( D and E ) Expression assays showing SFN enhanced expression of Cat in rLECs. Experiments and parameters were similar to those noted above ( B and C ). RNA and protein extract were processed for real-time PCR and Western analyses using Cat specific primers and antibody, respectively. ( F and G ) SFN-treated rLECs displayed significantly increased levels of GST π mRNA and protein in time- and concentration-dependent fashion. mRNA and cellular extracts were isolated from SFN-treated primary rLECs, and were processed for real-time PCR ( F ) and Western analysis ( G ) assays. ( C,E and G ); Upper panel, representative Immunoblot. Lower panel, densitometric analysis of protein band level; levels were normalized to corresponding β-actin levels and values are presented as histograms (C and G, dotted line shows marking of boundary of bands). ( B–G) , Data represent means ± S.D. of three independent experiments. Open vs gray and black bars; gray vs black bar; * p < 0.001, ** p < 0.05.

Article Snippet: Primary rat LECs (rLECs) were isolated from 6-week-old Sprague-Dawley albino rats (n = 8) as described previously .

Techniques: Concentration Assay, MTS Assay, Expressing, Isolation, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: SFN induced Nrf2 expression and enhanced nuclear accumulation in both SRA-hLECs and rLECs. ( A ) Effect of SFN concentration(s) on expression of Nrf2 mRNA in SRA-hLECs. Total RNA was isolated and real-time PCR was performed using specific primers. mRNA expression of Nrf2 was adjusted/normalized to the mRNA copies of β-actin. Histogram represents mean ± S.D. obtained from three independent experiments. Open vs gray and black bars; gray vs black bar; * p < 0.001. ( B ) SFN-mediated induction of Nrf2 expression and nuclear localization. Cultured SRA-hLECs were treated with different concentrations of SFN for 6 h. Cytosol and nuclear extract were immunoblotted with anti-Nrf2 antibody. β-actin was used as loading control. Upper panel, An apparent increased nuclear translocalization of Nrf2 was observed. Lower panel, Histogram showing relative density of protein bands (Nrf2/β-actin). ( C ) Nrf2 activation by SFN accompanied accumulation of Nrf2 in whole cell lysates and led to nuclear accumulation in time- and concentration-dependent fashion in rLECs. Primary culture of rLECs treated with different concentrations of SFN were processed for extraction of total cell extract as well as cytosolic and nuclear fractions at predefined time intervals as indicated. Cellular extract ( C , a) or nuclear fraction ( C , b) containing equal amounts of protein were immunoblotted with anti-Nrf2 antibody. β-actin was used as loading control. A significant accumulation of Nrf2 in nucleus was observed when examined at 2 h and onwards compared to basal levels (untreated control).

Article Snippet: Primary rat LECs (rLECs) were isolated from 6-week-old Sprague-Dawley albino rats (n = 8) as described previously .

Techniques: Expressing, Concentration Assay, Isolation, Real-time Polymerase Chain Reaction, Cell Culture, Control, Activation Assay, Extraction

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: In vivo DNA binding assay revealed that SFN reinforced binding activity of Nrf2 in SRA-hLECs and aging/aged primary hLECs. ( A ) Schematic representation of the regulatory region of proximal promoter of human Prdx6 gene-containing ARE binding sites showing primer location and sequences used in ChIP assay. (B) SFN induced increase in DNA binding activity of Nrf2 to Prdx6 gene promoter containing ARE site in SRA-hLECs. ChIP experiment was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis Kit. Chromatin samples prepared from SRA-hLECs treated with varying concentrations (0, 3 µM and 6 µM) of SFN for 24 h were subjected to ChIP assay with a ChIP grade antibody, anti-Nrf2 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primers designed to amplify −400 to −305 region of the human Prdx6 promoter bearing Nrf2/ARE sites as shown. Histogram shows the amplified DNA band visualized with real-time PCR analysis. DMSO (0) vs 3 µM and 6 µM SFN and 3 µM vs 6 µM SFN treatment; *p < 0.001. ( C ) SFN reinforced the enrichment of Nrf2 to its responsive element, ARE, present in Prdx6 gene promoter in aging/aged primary hLECs. ChIP assay was conducted using anti-Nrf2 antibody. Immunoprecipitated DNA fragments were purified and processed for qPCR analysis using primers indicated above and in the Methods section, but in primary hLECs of variable ages treated with different concentrations (0, 3 µM, 6 μM) of SFN. Histograms represent the concentration dependence of SFN-induced enrichment of Nrf2 at ARE sites in Prdx6 gene promoter. Open vs gray and black bars and gray vs black bar; * p < 0.001. Data revealed a significant augmentation of Nrf2 binding to ARE by SFN in all ages of LECs, but in aged cells there was a loss in Nrf2 binding to ARE. ( D ) SFN failed to activate mutant Prdx6 promoter disrupted at Nrf2/ARE site. Upper panel, diagram of 5′-regulatory region of Prdx6 promoter spanning from −918/+30 bp containing ARE site and its mutant plasmid linked to CAT reporter gene used for CAT activity. Lower panel, CAT activity of the wild-type (WT) Prdx6 promoter and its mutant (Mut) at ARE site and empty CAT vector in SRA-hLECs treated with SFN or DMSO (control). Wild-type or its mutant Prdx6 promoter construct along with pGFP-Vector were cotransfected into SRA-hLECs and CAT activity was measured. CAT activity (lower panel) was normalized to GFP readings (O.D.). Histogram represents the mean ± S.D. obtained from three independent experiments. WT vs Mut and DMSO vs SFN treated samples; *p < 0.001. ( E ) SFN reinforced Prdx6 transcription in aging/aged primary human LECs. As described above, hLECs of variable ages were transfected with the same wild-type Prdx6 promoter ARE site (upper panel). Lower panel, relative CAT activity of the wild-type promoter in SFN-treated aging/aged hLECs. All data are presented as mean ± S.D. values derived from three independent experiments. DMSO vs SFN treated samples; younger (21 y old) vs aging samples; *p < 0.001.

Article Snippet: Primary rat LECs (rLECs) were isolated from 6-week-old Sprague-Dawley albino rats (n = 8) as described previously .

Techniques: In Vivo, DNA Binding Assay, Binding Assay, Activity Assay, Control, Amplification, Real-time Polymerase Chain Reaction, Immunoprecipitation, Purification, Concentration Assay, Mutagenesis, Plasmid Preparation, Construct, Transfection, Derivative Assay

Journal: Scientific Reports

Article Title: Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress

doi: 10.1038/s41598-017-14520-8

Figure Lengend Snippet: Prdx6 knockdown experiments revealed that SFN exerted its cytoprotective activity against UVB-induced cell injuries through Prdx6 regulation. (A) Survival experiment showing increased susceptibility of As-Prdx6 transfected cells to UVB-induced oxidative stress. SRA-hLECs were transfected with As-Prdx6 (4 μg), and the effect of As-Prdx6 was confirmed through immunoblotting with anti-Prdx6 antibody (data not shown). The transfectants were divided into different groups as shown and equal numbers of cells were cultured for assay to avoid transfection effect. Survival assay (MTS assay) showed a significant reduction in viability of As-Prdx6 (lined bar) transfectants compared to empty-vector transfectants (gray and black bars) against UVB stress. ( B ) H2-DCF-DA assay showing ROS levels after UVB stress as indicated. Result are presented as Fluorescent Unit. ( A and B ), Open vs gray bar, gray vs black bar and black vs lined bar; *p < 0.001. ( C and D ) SFN protected primary rLECs against UVB exposure. ( C) rLECs were pretreated with 2.4 µM and 4.8 μM of SFN or DMSO (vehicle control) and then exposed to UVB stress. Effects on viability were determined after 24 h by MTS assay. ( D ) Effect of SFN on lowering ROS expression. rLECs were treated with DMSO, 2.4 µM or 4.8 μM of SFN and were exposed to UVB stress as indicated. ROS expression was quantified. All histograms are presented as the mean ± S.D. values derived from two independent experiments. C and D, open vs gray bar and gray vs black bar; *p < 0.001. ( E and F ) SFN rescued primary aging hLECs from UVB stress. ( E ) SFN augmented viability of aging hLECs undergoing UVB stress. Cultured hLECs of variable ages were exposed to UVB as indicated, and effects on cell viability were determined after 24 h by MTS assay. ( F ) Effect of SFN on lowering ROS expression. hLECs were treated with SFN as in (E). ROS levels were measured with H2-DCF-DA. Histogram represents the data mean ± S.D. obtained from two independent experiments. 21 y vs 56 y and 62 y, 56 y vs 62 y, Open vs gray bar and gray vs black bar; ** p < 0.05, * p < 0.001.

Article Snippet: Primary rat LECs (rLECs) were isolated from 6-week-old Sprague-Dawley albino rats (n = 8) as described previously .

Techniques: Knockdown, Activity Assay, Transfection, Western Blot, Cell Culture, Clonogenic Cell Survival Assay, MTS Assay, Plasmid Preparation, Control, Expressing, Derivative Assay