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: Cell & Bioscience

Article Title: Aberrant methylation of Serpine1 mediates lung injury in neonatal mice prenatally exposed to intrauterine inflammation

doi: 10.1186/s13578-022-00901-8

Figure Lengend Snippet: Demethylation promoted Rela but inhibited Hdac2 binding to the Serpine1 promoter. A–C Serpine1 expression analysed using RT-qPCR and western blot in Rela-, Hif1a-, and Rest-overexpressing MLE-12 cells. D Transcriptional activity of Rela, Hif1a, and Rest at the Serpine1 promoter analysed using luciferase assay in MLE-12 cells. E The binding ability of Rela and Hdac2 to the biotin-labelled Serpine1 promoter probe analysed via DNA pull-down assay. F–H Serpine1 expression analysed using RT-qPCR and western blot in Hdac2-knockdown MLE-12 cells. I Transcriptional activity of Rela, Hif1a, and Rest at the methylated-Serpine1 promoter analysed using luciferase assay in HEK293T cells. J Transcriptional activity of Rela at the Serpine1 promoter analysed using luciferase assay in Hdac2-knockdown MLE-12 cells. K The enrichment of TFs at the Serpine1 promoter region determined using anti-Rela and anti-Hdac2 antibodies via ChIP-qPCR in 5-Aza-CdR-treated MLE-12 cells. Data show mean ± SEM; Data was analysed using unpaired t -tests

Article Snippet: MLE-12, A549, BEAS-2B and human epithelial kidney 293 T (HEK293T) cell lines were purchased from ATCC.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Pull Down Assay, Knockdown, Methylation, ChIP-qPCR

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