Journal: Cell

Article Title: TMEM41B Is a Pan-flavivirus Host Factor

doi: 10.1016/j.cell.2020.12.005

Figure Lengend Snippet:

Article Snippet: Human: HEK293T/17 (embryonic kidney epithelial) , ATCC , Cat.#CRL-11268 ; RRID: CVCL_1926.

Techniques: Virus, Subcloning, Western Blot, Recombinant, Infection, Transfection, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, SYBR Green Assay, cDNA Synthesis, Sequencing, Derivative Assay, Plasmid Preparation, Software

Journal: Journal of Lipid Research

Article Title: Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake

doi: 10.1194/jlr.M041335

Figure Lengend Snippet: miR-185 downregulates SREBP-2 mRNA and protein level by binding to MREs within SREBP-2 mRNA 3′UTR. A: Luciferase activity was quantitated in 293T cells transfected with a control luciferase reporter plasmid (Con Luc), SREBP-2 3′UTR containing reporter plasmid (3′UTR), SREBP-2 3′UTR mutant (3′UTR MUT), and pre-miR-185 (miR185) or control pre-miR (Con miR). Luciferase activity was measured in 293T cells as described in the Materials and Methods section. Bar graphs represent mean ± SEM from three independent experiments. B: The fold change in SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185, Antagamer-miR-185 (Antagomer) (100 nM), or control miR (Con miR) transfected HepG2 cells. Bar graphs represent mean ± SEM from three independent experiments. C: SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185 or control miR transfected THLE-2 cells. Values were normalized to the level of GAPDH. Bar graphs represent mean ± SEM from three independent experiments. D: SREBP-2 protein level was determined using whole cell lysates by Western analysis in miR-185 overexpressing and control miR transfected HepG2 cells. β-Actin was used as a loading control. FL, full-length SREBP-2; M, mature form of SREBP-2.

Article Snippet: THLE-2, HepG2, and 293T cells were obtained from ATCC.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot

Journal:

Article Title: The Long Terminal Repeat of Jaagsiekte Sheep Retrovirus Is Preferentially Active in Differentiated Epithelial Cells of the Lungs

doi:

Figure Lengend Snippet: Cell lines used in this study

Article Snippet: H441 and H358 cells were grown in RPMI 1640 medium (Gibco BRL) adjusted to contain 1.5 g of sodium bicarbonate per liter, 4.5 g of glucose per liter, and 10 mM HEPES with 10% FBS. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Cell line Origin Tissue or cell type ATCC no. or reference A549 Human Lung carcinoma CCL-185 H441 Human BAC HTB-174 H358 Human BAC CRL-5807 293T Human Embryonic kidney Lebkowsky et al. ( 33 ) OA1 Sheep Brain fibroblast CRL-6538 OAT Sheep Sheep testis CRL-6546 FLL Sheep Primary fetal lamb MRI a JS7 Sheep BAC Jassim ( 30 ) CP-MRI Sheep Choroid plexus MRI a CP-ATCC Sheep Choroid plexus CRL-1700 mtCC1-2 Mouse Clara cell Magdaleno et al. ( 34 ) BV2 Mouse Microglia A. Tenner a F9 Mouse Testicular carcinoma CRL-1720 FOP Mouse Mammary carcinoma J. Hassel a IC-21 Mouse Peritoneal macrophage TIB-186 MHS Mouse Alveolar macrophage CRL-2019 C2C12 Mouse Myoblast CRL-1772 ABI-2 Mouse Hybridoma HB-33 MLE-12 Mouse Lung epithelium CRL-2110 TCMK Mouse Mouse kidney CCL-139 NIH 3T3 Mouse Mouse embryo CCL-92 MLE-15 Mouse Type II pneumocyte Wikenheiser et al. ( 59 ) ST3 Mouse Thymus stroma Brightman et al. ( 10 ) Open in a separate window a Cells were provided directly by an investigator or institution with no reference available.

Techniques:

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: VACV Protein C6 Downregulates HDAC5 (A) HDAC5, but not HDAC1, is proteasomally degraded during VACV infection. Bar charts and statistics were generated as described in Figure 5 . (B) Viral class centroid profiles compared to an inverted profile of HDAC5, which had additionally been scaled from 0 to 1. (C) Profile of HDAC5 scaled as in (B), and Tp2-class VACV proteins with known roles in regulation of IFN or ISGs. (D) C6 targets HDAC5. HFFF-TERTs were infected in biological triplicate with WT VACV or vΔC6 (lacking gene C6L ) ( Unterholzner et al., 2011 ) (MOI = 5 and 12 h). The scatterplot shows all proteins quantified. A Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. (E) Representative immunoblot demonstrating that C6 specifically targets HDAC5. By comparison, HDAC1 was unmodified (MOI = 5). Quantitation of all three replicate immunoblots are shown (right panel). Data are represented as mean ± SEM, ∗ p < 0.05 from a two-tailed t test. (F) Representative immunoblot demonstrating rescue of HDAC5 expression by inhibition of the proteasome (MOI = 5). MG132 was added 2 h after VACV infection. Quantitation of all three replicate immunoblots is shown (right panel). Data are represented as mean ± SEM, ∗∗ p < 0.00005 from a two-tailed t test. (G) Immunoblot demonstrating that inducible expression of C6 is sufficient for HDAC5 degradation. HEK293T cells inducibly expressing C6 with an N-terminal Myc or C-terminal TAP tag were either treated or untreated with 100 ng/ml doxycycline overnight then lysates immunoblotted.

Article Snippet: HEK293T (human embryo kidney epithelial cell line) , ATCC , ATCC: CRL-11268.

Techniques: Infection, Generated, Two Tailed Test, Western Blot, Comparison, Quantitation Assay, Expressing, Inhibition

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: HDAC5 Restricts VACV and HSV-1 Infection (A) Diminished replication of VACV and HSV-1 in an U2OS cell line inducibly expressing HDAC5-FLAG. Infections were performed at MOI = 0.001, for 2 days with VACV and 3 days with HSV-1, after induction with 100 ng/ml doxycycline overnight. Data are represented as mean ± SEM, p values were calculated using a two-tailed t test (n = 3). ∗ p < 0.05 and ∗∗ p < 0.01; ns: not significant. (B) Immunoblot of HDAC5-FLAG expression in U2OS cell lines. (C) Enhanced replication of VACV and HSV-1 in HeLa CRISPR/Cas9 HDAC5 −/− knockout clones compared to parental cell lines. Infections were performed as detailed in (A). Data are represented as mean ± SEM, p values were calculated using a two-tailed t test (n = 3). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (D) Immunoblot confirmed knockout of HDAC5. Sequencing of genomic DNA from clones H5KO1 and H5KO2 confirmed frameshift mutations in both alleles ( A). (E) Reintroduction of HDAC5 in HDAC5 −/− cells restored restriction of VACV and HSV-1 replication. H5KO1 cells were transduced with either empty vector (EV) or HDAC5-FLAG. Infections were performed as detailed in (A). Data are represented as mean ± SEM, p values were calculated using a two-tailed t test (n = 3). ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001. (F) Immunoblot analysis of HDAC5-FLAG expression in H5KO1 cell lines. (G) Enhanced replication of VACV and HSV-1 in HEK293T CRISPR/Cas9 HDAC5 −/− knockout clones compared to parental cell lines. Infections were performed as detailed in (A). Data are represented as mean ± SEM, p values were calculated using a two-tailed t test (n = 3). ∗ p < 0.05 and ∗∗ p < 0.01. (H) Immunoblot confirming knockout of HDAC5. Sequencing of genomic DNA from clones H5KO3 and H5KO4 confirmed frameshift mutations in both alleles ( A).

Article Snippet: HEK293T (human embryo kidney epithelial cell line) , ATCC , ATCC: CRL-11268.

Techniques: Infection, Expressing, Two Tailed Test, Western Blot, CRISPR, Knock-Out, Clone Assay, Sequencing, Transduction, Plasmid Preparation

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet:

Article Snippet: HEK293T (human embryo kidney epithelial cell line) , ATCC , ATCC: CRL-11268.

Techniques: Virus, Western Blot, Derivative Assay, Subcloning, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, TA Cloning, Sequencing, Mass Spectrometry, CRISPR, Disruption, Plasmid Preparation, Software, Quantitative Proteomics, Imaging, Flow Cytometry, Fluorescence, Microscopy