Journal: Cell metabolism

Article Title: Cytosolic Aspartate Availability Determines Cell Survival When Glutamine Is Limiting

doi: 10.1016/j.cmet.2018.07.021

Figure Lengend Snippet: Key Resources Table

Article Snippet: pLentiCRISPRv2 (lentiviral Cas9 expressing vector) , Laboratory of Dr. Feng Zhang , Addgene Cat #52961.

Techniques: Recombinant, shRNA, Expressing, Plasmid Preparation, Software, Flow Cytometry

Journal: PLOS Pathogens

Article Title: The PRMT5/WDR77 complex restricts hepatitis E virus replication

doi: 10.1371/journal.ppat.1011434

Figure Lengend Snippet: (A) Western blot analysis of lysates from HepG2C3A cells transduced with PRMT5, WDR77 or non-targeting control plentiCRISPRv2 sgRNA lentivirus. Tubulin was used as the loading control. (B) HepG2C3A cells were infected with HEV Kernow C1/p6 virus. Immunofluorescence of ORF2 was performed at day 3 post infection with ORF2 monoclonal antibody (2G8) . (C) Flow cytometry analysis of HEV infection by ORF2 staining using 2G8 antibody was performed at day 3 post infection. (D) RT-qPCR analysis of HEV genomic RNAs at day 3 post infection. (E) Intracellular viral titer of Kernow C1/p6 was determined at day 7 post transfection in S10-3 cells. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; ***, P < 0.001 by one-way ANOVA. All data are representative of three independent experiments.

Article Snippet: plentiCRISPRv2 vector (78852, Addgene) was digested by BsaI restriction enzyme (R3733, NEB) to prepare backbone, then annealed sgRNA oligo was ligated with backbone by T4 DNA ligase (M0202, NEB) to construct knockout sgRNA. sgRNA target sequences are listed in .

Techniques: Western Blot, Transduction, Control, Infection, Virus, Immunofluorescence, Flow Cytometry, Staining, Quantitative RT-PCR, Transfection

Journal: PLOS Pathogens

Article Title: The PRMT5/WDR77 complex restricts hepatitis E virus replication

doi: 10.1371/journal.ppat.1011434

Figure Lengend Snippet: (A) Western blot analysis of lysates from S10-3 cells transduced with PRMT5, WDR77 or non-targeting control plentiCRISPRv2 sgRNA lentivirus. Actin was used as the loading control. (B-D) S10-3 cells were transfected with HEV replicon RNAs of Kernow C1/p6 Gluc replicon (B), Sar55 Gluc replicon (C) and pSHEV3 Gluc replicon (D). Supernatants were collected and Gluc activity was measured at day 2 post transfection. Data are normalized with non-targeting control sgRNA. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA. All data are representative of three independent experiments.

Article Snippet: plentiCRISPRv2 vector (78852, Addgene) was digested by BsaI restriction enzyme (R3733, NEB) to prepare backbone, then annealed sgRNA oligo was ligated with backbone by T4 DNA ligase (M0202, NEB) to construct knockout sgRNA. sgRNA target sequences are listed in .

Techniques: Western Blot, Transduction, Control, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

doi: 10.1101/2021.03.01.433431

Figure Lengend Snippet: A, Normalized RNA-seq reads were aligned to the GRCh38 and Vervet-African green monkey genomes and quantified with Salmon (v1.3.0). The read counts for ACE2 , TMPRSS2 , FURIN , CTSB , CTSL , and NRP1 are given for the indicated cell lines. See also and . B, qRT-PCR for ACE2 and TMPRSS2 expression normalized to 1μg input RNA for each cell line. Cercopithecus aethiops specific primers against TMPRSS2 were used for the Vero E6 samples. Each bar represents mean, error bars indicate SEM (n=3). C, Immunoblot showing ACE2 expression across 10 lung and upper airway cancer cell lines and Vero E6 cells (representative of n=3). ACE2 expression was quantified using Licor Image Studio software in which ACE2 levels were normalized to β-ACTIN, set relative to Vero E6, and are indicated below the immunoblots. D, qRT-PCR for cell-associated SARS-CoV-2 RNA at 4 and 72 hpi at MOI=0.015 or 0.15. MOIs were determined by titration on Vero E6 cells. Error bars represent SEM (n=3). * indicates p<0.05 where significance was determined using two-way ANOVA and the Šidák correction for multiple comparisons. E, qRT-PCR for cell-associated SARS-CoV-2 RNA in H522 cells across various time points and MOIs. Error bars represent SEM (n=2). F, qRT-PCR for SARS-CoV-2 RNA in the supernatant of H522 cells across various time points and MOIs. Error bars represent SEM (n=2). G, Percent of SARS-CoV-2 infected H522 and Vero E6 cells determined by FACS for Nucleocapsid positive cells across various time points and MOIs. Error bars represent SEM (n=2). H, Plaque assays on H522 and Vero cells using two viral dilutions (10 −2 and 10 −1 ). Data are representative of three independent experiments. I , Representative images of H522 cells infected with SARS-CoV-2 at MOI=1. H522 cells were fixed and stained for SARS-CoV-2 RNA (green) by RNAScope reagents and Nucleocapsid (N) protein (red) at 4 and 96hpi and imaged by confocal microscopy (representative of n=2). See also . J , Representative images using transmission electron microscopy (TEM) on Vero E6 and H522 cells infected with SARS-CoV-2 (MOI=0.1 pfu/cell and 24 hpi for Vero, MOI: 1 pfu/cell and 96 hpi for H522).

Article Snippet: H522 cells were transduced with a pLentiCRISPRv2 derived vector targeting ACE2 (Genscript).

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Software, Titration, Infection, Staining, RNAscope, Confocal Microscopy, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

doi: 10.1101/2021.03.01.433431

Figure Lengend Snippet: A, Representative immunoblot showing ACE2 expression and Vinculin as the loading control in Vero E6, H522, H522-ACE2, basal HBEC, and basal HBEC-ACE2 cells. B , Viruses were pre-treated with increasing concentrations of S neutralizing antibody for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of the S neutralizing antibody. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). *** indicates p<0.001 where significance was determined using two-way ANOVA and the Dunnett correction for multiple comparisons. C, SARS-CoV-2 viruses were pre-treated with increasing amounts of soluble ACE2-Fc for 1 h and then cells were infected with SARS-CoV-2 at MOI=0.1 in the presence of ACE2-Fc. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 24 hpi and was normalized to mock treated (n=3). ** indicates p<0.01 and *** indicates p<0.001 where significance was determined using two-way ANOVA and the Dunnett correction for multiple comparisons. D, Representative images of cells infected with VSV-SARS-CoV-2-S Δ21 at 0 and 8hpi using an Incucyte® S3 Live Cell Analysis System (n=3). Percent GFP positive cells seeded in triplicate were quantified over time with the shaded grey region indicating standard deviation.

Article Snippet: H522 cells were transduced with a pLentiCRISPRv2 derived vector targeting ACE2 (Genscript).

Techniques: Western Blot, Expressing, Control, Infection, Quantitative RT-PCR, Cell Analysis, Standard Deviation

Journal: bioRxiv

Article Title: Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

doi: 10.1101/2021.03.01.433431

Figure Lengend Snippet: A , Cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR at 72 hpi (n=3). *** indicates p<0.001 where significance was determined using two-way ANOVA and the Dunnett correction for multiple comparisons. B , Polyclonal populations of H522 and Calu-3 (ACE2 +/+ and ACE2 −/− ) cells were infected with SARS-CoV-2 virus and cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 4 and 72 hpi (n=8). Error bars indicate the SEM. *** indicates p<0.001 where significance was determined using two-way ANOVA and the Tukey correction for multiple comparisons. See also . C , Polyclonal populations of H522 and Calu-3 (ACE2 +/+ and ACE2 −/− ) cells were pre-treated with 20 μg/ml of the indicated blocking antibodies for 1 h and then infected with SARS-CoV-2 at MOI=0.1 in the presence of the blocking antibodies. Cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 72 hpi (n=3). Error bars indicate the SEM. *** indicates p<0.001 where significance was determined using two-way ANOVA and the Tukey correction for multiple comparisons. See also . D , Monoclonal populations from H522 ACE2 +/+ (6 clones), ACE2 −/− (2 clones), and ACE2 +/− (1 clone) were infected with SARS-CoV-2 at MOI=0.1 and cell-associated SARS-CoV-2 RNA was detected by qRT-PCR 4 and 72 hpi (n≥3). Error bars indicate the SEM. See also .

Article Snippet: H522 cells were transduced with a pLentiCRISPRv2 derived vector targeting ACE2 (Genscript).

Techniques: Blocking Assay, Infection, Quantitative RT-PCR, Virus, Clone Assay