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{"CAPTION FIG1.png": "'\\nFig. 1: **Dynamin 2 negatively regulates viral gene expression.****A.** E1A expression at 2 h pi as measured after virus infection alone (V), or infection following a pericentamen (Buf+ V) at 250 nM or 500 nM, and in the solvent control (DMSO+ V). **B.** Effect on E1A mRNA expression of pericentamen with cytochalasin D prior to virus infection (Cyto D+ V) at 250 nM or 2 pM; solvent control (DMSO+ V), virus alone (V); 2 h pi (DMSO+ V vs. Cyto D+ V 2 nM; \\\\(\\\\text{ip}<0.05\\\\), ANNOVA). **C.** Effect on E1A mRNA expression of pretreatment with nocodazole prior to viral infection (Noc + V) at 10 \u03bcM or 30 \u03bcM, virus alone (V), and DMSO control (DMSO+ V). **D.** Confocal microscopy representation of viral entry at 2 h pi in the presence of solvent control (panel 1, DMSO+ V) or 30 \u03bcM nocodazole (panel 2, Noc + V). **C.** Cy3 labeled HAdV-D37 (red), anti- mtDNA staining (green). **E.** Western blot showing protein expression levels in keratocytes after dynamic (DMSO+ knockdown: mock transfected and virus infected (V), scRNA transfected and virus infected (cRNA+ V), cNDM2 transfection prior to viral infection (sIDN2+ V). Western blot for a-AP2A1, and adaptor protein shown as negative control for specificity of DNM2 knockdown. Actin blot shows load control. Dynamic 2 mRNA expression levels by qRT-PCR are represented in bar graph just below. **F.** Early gene expression (E1A) as measured by qRT-PCR from same RNA pool as in E: virus alone (V), scrambled RNA (cRNA+ V), dynamic 2 knockdown, virus infected cells (sIDN2M2 + V) (\u201cp \\\\(<0.001\\\\), ANOVA). **G.** Western blot shows expression of CDmetry in empty vector prior to virus infection (EV+ V) and mCherry-dynamin 2 (arrow) prior to virus infection (OE-DNM2+ V). The bar graph just below shows mRNA expression of dynamic 2. **H.** E1A mRNA expression as measured in cells after dynamic 2 vector pretreatment, performed on same RNA pool from G (\u201cp = 0.01, Students _\u03b5_-test). Error bars represent standard deviation of the mean. Each experiment was repeated at least 5 times with similar results.\\n\\n'", "CAPTION FIG3.png": "'\\n\\n## References\\n\\nFig. 3: **Dynamin 2 knockdown increases nuclear targeting of adenovirus.****A.** Acetylated tubulin (red) after infection with EdU labeled virus (green) at 1 and 2 h, pH, after empty vector transfection prior to virus infection (EV), dynamin 2 vector transfection prior to infection (OE-DNM2), pretreatment with scRNA or dynamin 2 siRNA (siRNA (siRNA). DAPI staining (blue) of nuclei utilized to outline blue-free images (a-1 through h-1), in order to better visualize virions in cell nuclei. **B.** Quantification of green fluorescence (pixel units) from **A** (2h pH) within ovals outlines (nuclei) as measured using ImageJ and shown graphically in box plot (\u201dr \\\\(<\\\\) 0.0001, Kruskal-Wallis). Data was obtained from 20 cells per experimental group in three experimental replicates. **C.** PCR for HAdV-D37 E1A, hexon and penton base genomic DNA measured to show relative levels of viral entry at 30 min pi. **D.** Quantitative real-time PCR for E1A DNA under the same experimental conditions. Upper graph shows the standard curve for E1A DNA. Lower graph demonstrates no statistical difference between experimental groups Each experiment was repeated at least 3 times with similar results.\\n\\n'", "CAPTION FIG4.png": "'\\n\\n**Fig. 4.** Dynamln **2** knockdown increases virions in the cytosol relative to the endosome. A. Medshad schematic of $LD assay (Susomalainen et al., 2013). In this assay, SLO permeabilizes only cell membranes and not endosomal membranes, so that Cy3-labeled virions within endosome appear red, while virions in the cytosol appear yellow upon binding to acid-Cy3 antibody with a green chromophore. B. Sreptolyan O (SLO)-penetration assay performed 1 h p. Panel 1 shows the no-SLO control, where there is no staining for GM-130, labeling absence of antibody penetration. Panels 2-5 show equivalent staining for GM130 (green), indicating that the transfection did not affect pore formation by SLO. **C.** SLO assay pretreated in cells transfected prior to viral infection with empty vector (EV), dynamic 2 overexpression centerset (OEPNRH2), scRNA, or siRNA (elDMM2). In SLO treated groups ( + SLO), viruses in endosomes appear red, while virions in cytosol appear yellow. As a control, left panel shows same groups treated with triton X100 without SLO (-SLO), and thus viruses in both cytosol and endosomes appear yellow. Each experiment was repeated at least 3 times with similar results.\\n\\n'", "CAPTION FIG5.png": "'\\n\\n**Fig. 5. Cytastolic plus is associated with reduced cytidine expression. A. HCF percented with EV, GE-DRM2, scRNA and siRNA2 were infected for at a MOI of 100, and ultrastructural images taken to locate vitrion distribution. Scale bar = 500 nm. Electron microscopy was performed three times with similar results. B. Cytokine arrays were performed on 500 ml of culture supernatants collected from each green after infection at MOI of 10 for 2.5 h. Reference spots (RS) were used as decaturmetric controls to quantify the mean spot pixel density. Directed cytokines included CC12 (11), CC15 (2), CXCL1 (3), CXCL10 (4), CXCL11 (5), IL-1ra (6), IL-6 (7), CXCL8 (8), MIF (9) and Seppa B1 (10). C. Bar graph showing collated results from 3 separate assays. The mouse of densitometry measurements from three independent experiments were plotted, normalizing to reference spots. Error bars represent standard deviations of the masses. \" indicates those cytokines for which expression was reduced significantly by dynamic 2 siRNA, as compared to control scRNA (\\\\({}^{\\\\circ}\\\\)P \\\\(\\\\leq\\\\) 0.05, Students t-test).\\n\\n'", "CAPTION FIG6.png": "'\\nFig. 6: **Dynamin 2 regulates MIOK localization.****A.** Confocal microscopy of cells pretreated with empty vector (eV), dynamic 2 expressing vector (QE-DNM2), \\\\(\\\\propto\\\\)RNA, or dynamic 2 siRNA (sIDNM2) and infected with Cy3-labeled HA4V-D37 (red) for 1 h, and stained with anti-perferentiin (green), and DAPI (blue). **B.** Distance from green signals (arrows) to the closest edge of adjacent nuclei (100 cells per experimental group in three experiments) was measured using Leica application suite X (LAS X, Leica Microsystems, Wetzlar, Germany), and graphed in boxplots (\u2018p \\\\(<\\\\) 0.0001, Kruskal-Wallis). **C.** Transmission electron microscopy of infected keratocytes from same pretreatment groups, with 3 distinct images shown per group shown. NU: nucleus. Scale bar = 500 nm. **D.** Measurements taken from each microtubule organizing center to nearest nuclear membrane were performed on 10 cells per group by a masked observer. Data shown reflects means and standard deviation for each group (\u2018p \\\\(<\\\\) 0.05, Students-t-act-act). **E.** Confocal microscopy showing co-localization (yellow) of nuclear pore complex protein Nwq5S (green), and Cy3-labeled HA4V-D37 (red), 2 h pl. Each experiment was performed three times with similar results.\\n\\n'", "CAPTION FIG7.png": "'Fig. 7. Effect of dynamic 2 mutations on viral gene expression. A schematic presentations of dynamic 2 wild type and mutant clones. B Western Blot for wild type (Wt), 4551-553, and 746-786, with alpha tubulin load control. C qRT-PCR for E1A mRNA expression with the same treatment groups as A (\\\\(\\\\text{rp}\\\\leq 0.0\\\\text{S}\\\\), ANOVA, with Tukey procedure). Experiments in both B and C were performed three times with similar results.\\n\\n'", "CAPTION FIG8.png": "'\\n\\n**Fig. 8. Schematic of the role of dynamic 2 in viral trafficking. Our experiments in primary fibroblasts indicate that apex dynamic 2 aggregates (OE-NMD2), vitamin accumulate in endosomes, and hence are called from reaching the microtubule organizing center (ATCC) and nucleus (Mue). Knockdown of dynamic 2 (slDMH2) localizes virions in the cytosol, with increased acetylation of microtubules (CMU), and movement of FITC closer to the nuclear membrane, thus enabling nuclear entry.**'"} |
