Journal: Redox Biology

Article Title: SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

doi: 10.1016/j.redox.2022.102508

Figure Lengend Snippet: Transduction with SARS-CoV-2 proteins increases apoptosis and causes cell cycle arrest in BEAS-2B cells. A) Representative immunoblots and densitometry showing upregulated expression of AIF and CDK4, 96 hours following transduction with 3 different SARS-CoV-2 proteins (vs GFP control). Only transduction with M protein downregulates CDK4. ** P < 0.01; NS, not significant; n = 3/group. B) Representative images and mean data showing that heterologous expression of each of the three selected SARS-CoV-2 proteins increase the expression of AIF relative to the mitochondrial reporter protein, TOMM20, in BEAS-2B cells at 96 hours. Cells were stained with AIF mouse monoclonal antibody (green). Cells were also stained with TOMM20 rabbit polyclonal antibody (red). Ex/Em: 556/560-580 nm *** P < 0.001; n = 5–7/group. C) Lentiviral transduction of A549 cells with SARS-CoV-2 proteins, Nsp7, Nsp9 & M increase apoptosis, defined as cells that are annexin V positive and propidium iodide (PI) negative, relative to the GFP control. *** P < 0.001; n = 3/group. D) Lentiviral transduction of A549 cells with SARS-CoV-2 M protein causes cell cycle arrest in G1/G0, relative to GFP controls and other SARS-CoV-2 proteins. *** P < 0.001; NS; not significant; n = 3/group. E) BEAS-2B cells were loaded with mPTP staining dye-AM (Abcam ab239704; flow cytometry kit). CoCl 2 was used to quench the cytosolic fluorescence, maintaining only the mitochondrial signal. Histograms demonstrate reduced mPTP activity (as the result of the mPTP opening) in M-transfected cells (light gray) versus control (dotted histogram). mPTP activity was not affected in Nsp9-transfected cells (dark gray) compared to control. The bar graph shows the fold change comparison in mPTP activity and confirms that that M-transfected cells had significantly reduced mPTP activity (open mPTP) versus control ( p = 0.019 ; parametric one-way ANOVA). mPTP activity was calculated as MFI(CoCl 2 ) – MFI (CoCl 2 +Ionomycin); MFI – median fluorescence intensity. MFI = mean fluorescent intensity. * P < 0.05; NS; not significant; n = 4/group.

Article Snippet: To confirm MHV-1 infection, the same sections were blocked with 3% BSA in PBS at 37 °C for 1 hour and an MHV A59 Nsp9 antibody (NBP-21671, Novus Biologicals, Centennial, CO, USA) was applied (1:500 for 1 hour at 37 °C).

Techniques: Transduction, Western Blot, Expressing, Control, Staining, Flow Cytometry, Fluorescence, Activity Assay, Transfection, Comparison

Journal: Redox Biology

Article Title: SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

doi: 10.1016/j.redox.2022.102508

Figure Lengend Snippet: SARS-CoV-2 proteins inhibit HPV in human PASMC. In the representative trace (left) a control hypoxic response is shown. Note that the rapid rise in [Ca 2+ ] i in human PASMC, a surrogate for HPV. Note the loss of a significant hypoxic increases in [Ca 2+ ] i (pink bar in graph) caused by transduction with M or Nsp9 SARS-CoV-2 viral proteins. Transduction with lentiviral GFP served as the control. Also note none of the SARS-CoV-2 proteins altered the KCl-induced rise in [Ca 2+ ] i (purple). * P < 0.05, ** P < 0.01, *** P < 0.001, NS; not significant; n = 6–10 cells/group.

Article Snippet: To confirm MHV-1 infection, the same sections were blocked with 3% BSA in PBS at 37 °C for 1 hour and an MHV A59 Nsp9 antibody (NBP-21671, Novus Biologicals, Centennial, CO, USA) was applied (1:500 for 1 hour at 37 °C).

Techniques: Control, Transduction

Journal: Redox Biology

Article Title: SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia

doi: 10.1016/j.redox.2022.102508

Figure Lengend Snippet: MHV-1 causes a mitochondriopathy that increases apoptosis and suppresses HPV. A) MHV-1 reduces Δ Ψm in murine cells. Mitochondria in LA-4 adenoma cells are seen in red, reflecting their uptake of TMRM, a potentiometric dye. MHV-1 reduced relative TMRM fluorescence, which is interpreted as a relative depolarization of ΔΨm ; measured 96 hours after infection. *** P <0.001; n=15 cells/group. B) MHV-1 infection upregulates AIF expression in mitochondria and causes nuclear translocation of AIF in murine cells. LA-4 murine adenoma cells were infected with MHV-1(0.05 pfu/cell) for 96 hours. Cells were immunostained for AIF (green) and loaded with MitoTracker Deep Red (Invitrogen, 150 nM) for 1 hour at 37 o C. The cells were also stained with DAPI to identify the nuclei (blue). Cells were imaged at Ex/Em: 644/655–675. Note the increase in AIF, both in mitochondria and in the nucleus. * P <0.05; n=4–6 cells/group. C) MHV-1 pneumonia induces hypoxemia, inhibits HPV, and increases AEC apoptosis . i) Male and female A/J mice develop systemic oxygen desaturation with MHV-1 infection at days 4–6. (n=4 for male Ctrl, n=4 for male MHV-1, n=5 for female Ctrl, n=3 for female MHV-1) ii) Representative RV pressure traces and mean data show that HPV is suppressed in MHV-1 mice. There was no significant increase in RV systolic pressure with hypoxia in MHV-1 infected mice, in contrast to the robust rise (HPV) in uninfected mice. Note that IP treatment with Bay K8644 significantly augmented HPV (defined as the Δ RVSP in response to hypoxia) in both control and infected mice. (n=8 for Ctrl, n=7 for MHV-1) iii) Bay K8644 (1 mg/kg IP) increases O 2 saturation. (n=9 for Ctrl, n=7 for MHV-1). The higher O 2 saturations in this panel (vs panel i) reflect the animals were being mechanically ventilated with room air (versus spontaneous breathing of room air in panel i). iv) Micro-CT images acquired using a VECTor CT scanner. The CT scans show lung consolidation in MHV-1 mice (on right). The histology insets show inflammatory infiltrates in the MHV-1 lung. Histology could not be performed on the same lungs as the CT scan, because the CT scans were done with barium infusion. v) Immunofluorescent staining of MHV-Nsp9 protein and TUNEL assay show colocalization of Nsp9 and TUNEL in MHV-1 infected lungs. This illustrates that only infected AEC developed apoptosis. The bar graph quantifies MHV-1 induced apoptosis, identified by (+) TUNEL green stain within AEC nuclei (in panel iv inset) (n=9 for Ctrl, n=9 for MHV-1). * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: To confirm MHV-1 infection, the same sections were blocked with 3% BSA in PBS at 37 °C for 1 hour and an MHV A59 Nsp9 antibody (NBP-21671, Novus Biologicals, Centennial, CO, USA) was applied (1:500 for 1 hour at 37 °C).

Techniques: Fluorescence, Infection, Expressing, Translocation Assay, Staining, Control, Micro-CT, Plasmid Preparation, Computed Tomography, TUNEL Assay

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 1 RAC3 interaction with ERa is GTP and ligand dependent in vitro and in cells. (a) HIS-tagged RAC3, pulled down with Talon beads (Clontech), interacts with full-length ERa (Invitrogen) in a GTP and estradiol (E2) dependent manner. Western blot was probed with H222.2 and pan RAC antibody. (b) Quantification of western blot by LICOR software. Averaged integrated intensity of ERa bands was normalized to average integrated intensity of RAC3 bands. (c) RAC3 interacts with ERa in ligand dependent manner as measured by mammalian two- hybrid analysis of VP-16-ERa and GAL4 tagged RAC3 and RAC1 in MCF7 C4-12 cells. (d) Co-immunoprecipitation of ERa with RAC3 in MCF7 C4-12 þ Flag-ERa cells transfected with WT- RAC3. Western blot was probed with RAC and H222.2 antibodies and all bands were taken from the same blot. Empty wells were spliced out for clarity.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: In Vitro, Western Blot, Software, Immunoprecipitation, Transfection

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 2 RAC3 is an ERa co-activator. (a, d) RAC3 overexpression increases E2-induced ERE luciferase activity in MCF7 cells transfected with PGL2-ERE-luciferase, RTK and indicated constructs as described in Material and methods. MCF7 cells were treated with 10 nM E2, 10 nM E2 þ 1 mM ICI and ethanol for 6–24 h as indicated. (b, c) Western blots probed with anti-Myc antibody show comparable protein expression of Myc-tagged RAC3 and RAC1 or WT, T17 and V12 RAC3 mutants from an independent transfection. (e) RAC3 overexpression increases CCND1 gene expression in MCF7 cells transfected with RAC3 or vector. Gene expression was measured by TAQMAN assay. The symbol *P-value is 0.01. (f, g) RAC3 is present (f) and increases ERa occupancy (g) at the CCND1 promoter in MCF7 C4–12 cells stably transfected with Flag-ERa and transiently transfected with RAC3 or RAC1. Occupancy was determined by ChIP analysis and measured by real-time PCR as percent input. Western blot probed with anti-Myc antibody shows comparable protein expression of Myc-tagged RAC3 and RAC1 from an independent transfection.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: Over Expression, Luciferase, Activity Assay, Transfection, Construct, Western Blot, Expressing, Gene Expression, Plasmid Preparation, TaqMan Assay, Stable Transfection, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 3 A single amino acid confers RAC3 co-activation of ERa. (a) Sequence alignment of RAC3 and RAC1. Boxed amino acids were mutated. (b, c) Mutations at S151 of RAC3 and RAC1 alter the ability of the GTPases to activate ERa-induced transcription as measured by ERE luciferase assay in MCF7 cells, which were transiently transfected with indicated constructs. (d) RAC3 S1515A mutation decreases the affinity of RAC3 for ERa in MCF7 C4-12 cells stably transfected with Flag-ERa and transiently transfected with RAC3 WT and RAC3 S151A. After a 1 h treatment with 10 nM E2, ERa was pulled down with H222.2 antibody. Western blot was probed with pan-RAC and ERa antibodies; all bands were taken from the same blot with empty wells omitted for clarity.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: Activation Assay, Sequencing, Luciferase, Transfection, Construct, Mutagenesis, Stable Transfection, Western Blot

Journal: Oncogene

Article Title: RAC3 is a pro-migratory co-activator of ERα.

doi: 10.1038/onc.2010.583

Figure Lengend Snippet: Figure 7 Clinical consequences of RAC3 expression in ERa positive human breast tumors. Gene expression data were obtained from the NKI patient database. (Chang et al., 2003; Chang et al., 2005) All analysis was done using Prism 5 for Mac OS X from GraphPad (La Jolla, CA, USA). (a, b) Increasing RAC3 expression decreases recurrence free survival shown in Kaplan–Meier curves of recurrence free survival in years. Increasing RAC1 expression had no effect. (c, d) Increasing RAC3 expression increases the proportion of patients with metastatic events. RAC1 expression levels had no statistically significant effect.

Article Snippet: 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value < 0.0001 Time (years) R ec ur re nc e F re e S ur vi va l RAC1 Expressing Tumors 0 5 10 15 20 0 50 100 150 Not Expressed Expressed Top 25% Expressed Top 10% expressed p-value = 0.6903 Time (years) R ec ur re nc e F re e S ur vi va l Oncogene Biosynth (Itasca, IL, USA).

Techniques: Expressing, Gene Expression

Journal: Journal of Virology

Article Title: Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

doi: 10.1128/jvi.02206-06

Figure Lengend Snippet: FIG. 1. Temporal expression of A6L during VACV infection. (A) Schematic representation of vA6L-V5, a recombinant VACV en- coding A6 with a C-terminal V5 epitope tag. The relevant portion of the genome of vA6L-V5 with the inserted V5 tag (filled arrow) is depicted. For comparison, the corresponding portion of the WT WR genome is depicted with the positions of the open reading frames (open boxes with arrows) indicated by the nucleotide numbers in the complete WR genomic sequence (accession no. AY243312). (B) The kinetics of A6L expression. BS-C-1 cells were mock infected or in- fected with 10 PFU per cell of vA6L-V5. AraC was added to one of the infections at 40 g/ml to inhibit DNA replication. The cells were harvested at the indicated time (hours p.i. [hpi]) and analyzed by Western blotting (WB) with a MAb to V5 epitope as described pre- viously (15). The same amount of total proteins as determined by Bradford protein assay were analyzed.

Article Snippet: At 12 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% FBS for 60 min, and stained with monoclonal antibody to V5 or polyclonal antibody to VACV (Fitzgerald) for 1 h and a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for an additional hour.

Techniques: Expressing, Infection, Recombinant, Comparison, Sequencing, Western Blot, Bradford Protein Assay

Journal: Journal of Virology

Article Title: Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

doi: 10.1128/jvi.02206-06

Figure Lengend Snippet: FIG. 4. A6 protein expressed by vA6L-mut2 has a reduced stability at 40°C. (A) The A6 protein level in cells infected by vA6L-mut2 or vA6L-V5. BS-C-1 cells were infected with 10 PFU per cell of vA6L- mut2 or vA6L-V5 and maintained at 31 or 40°C. The cells were har- vested at 9 or 12 h p.i. and analyzed with SDS-PAGE and Western blotting (WB) with antibodies to the V5 epitope and VACV late protein D8. (B) Pulse-chase analysis of A6 protein. BS-C-1 cells were infected with vA6L-mut2 or vA6L-V5 at 40°C. At 12 h p.i., the cells were metabolically labeled with [35S]methionine-cysteine for 30 min. The cells were either harvested immediately (P) or replenished with normal medium and harvested 4 h later (C). The cell lysates as well as proteins precipitated from the cell lysates with anti-V5 antibody were analyzed with SDS-PAGE and autoradiography. IP, immunoprecipi- tate.

Article Snippet: At 12 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% FBS for 60 min, and stained with monoclonal antibody to V5 or polyclonal antibody to VACV (Fitzgerald) for 1 h and a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for an additional hour.

Techniques: Infection, SDS Page, Western Blot, Pulse Chase, Metabolic Labelling, Labeling, Autoradiography

Journal: Journal of Virology

Article Title: Vaccinia Virus A6L Encodes a Virion Core Protein Required for Formation of Mature Virion

doi: 10.1128/jvi.02206-06

Figure Lengend Snippet: FIG. 6. vA6L-mut2 is not defective for forming viral DNA-containing factories. (A). Localization of A6 protein in infected cells. HeLa cells grown on coverslips were infected with 0.1 PFU/cell of vA6L-V5 at 37°C. At 12 h p.i., the cells were fixed, permeabilized, blocked, and stained with a monoclonal antibody to V5 and a Cy2-conjugated secondary antibody. The DNA was stained with Hoechst dye. The images were acquired with an AX-70 Olympus fluorescence microscope. (B). BS-C-1 cells grown on coverslips were infected with 0.1 PFU/cell of vA6L-V5 or vA6L-mut2 at 40°C. At 12 h p.i., the cells were fixed, permeabilized, blocked, and stained with polyclonal antibody to VACV. The DNA was stained with Hoechst dye. The arrows point to typical DNA-containing viral factories.

Article Snippet: At 12 h p.i., the cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 10% FBS for 60 min, and stained with monoclonal antibody to V5 or polyclonal antibody to VACV (Fitzgerald) for 1 h and a Cy2-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for an additional hour.

Techniques: Infection, Staining, Microscopy

Journal: Scientific Reports

Article Title: A screen for novel hepatitis C virus RdRp inhibitor identifies a broad-spectrum antiviral compound

doi: 10.1038/s41598-017-04449-3

Figure Lengend Snippet: Assays with HCV genotype 3a replicon. ( A ) G418 resistant HCV-3a replicon expressing Huh7.5 cells were treated with indicated compounds for 48 h and the firefly luciferase activity was plotted as relative luciferase units (RLU). DMSO treated HCV-3a replicon expressing Huh 7.5 cells was taken as 100%. ( B ) The toxicity of these compounds in the replicon expressing cells was measured using WST-1 assay reagent. The values are depicted as percentages with the DMSO treated cells taken as 100%. ( C ) The replicon expressing cells were treated with varying concentrations of 66E2 and relative luciferase unit is plotted against the concentration of 66E2. EC 50 is the compound concentration that inhibits 50% of viral replication (RLU). ( D ) Huh7.5 cells were treated with indicated concentrations of 66E2 and cytotoxicity determined using WST-1 assay reagent. The values are plotted as percentages with the DMSO treated cells taken as 100%. CC 50 is the compound concentration that produces 50% of cytotoxicity. ( E ) Western blot analysis. Replicon expressing Huh7.5 cells were treated with different concentrations of 66E2 and CMC for 48 hours and expression of NS5A monitored using anti NS5A antibody. Expression of GAPDH was determined for loading control. ( F ) Reverse-transcriptase quantitative PCR analysis was performed to determine the levels of positive and negative sense HCV-3a RNA upon treatment with 66E2 and CMC at 5 μM for 48 h. The % mean is shown above the bars and the error bars are standard deviations. All the assays in the figure were performed in triplicates and results presented are representative of at least three independent assays.

Article Snippet: Antibodies against HCV NS5A (ab13833) and DENV (ab26837 and ab9202) were from Abcam and anti GAPDH was purchased from Santa Cruz Biotechnology (USA).

Techniques: Expressing, Luciferase, Activity Assay, WST-1 Assay, Concentration Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction