Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir. Data represent the average of three independent experiments ± SD. (C) POC for percentage of infection of the Huh7.5 drug screen performed at 0.5 μM. 33 drugs had >60% reduction in infection with >80% cell viability. (D) Distribution of 23 validated antivirals by drug target class. (E) Dose-response analysis of the candidates with a selectivity index (SI) > 3 identified in the screen. Data represent the average of three independent experiments ± SD.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: High Throughput Screening Assay, Infection, Microscopy

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: Cell-type-specific dependencies of entry inhibitors (A) Calu-3 human lung epithelial cells were infected with SARS-CoV-2 (MOI = 0.5) and processed for microscopy 48 hpi. (B) Dose-response analysis of Calu-3 cells treated with quinolines or remdesivir. Data represent the average of four independent experiments ± SD. (C) IC 50 , CC50, and SI for Vero, Huh7.5, and Calu-3 cells treated with a panel of quinolines or remdesivir. Data represent the average of four independent experiments ± SD. (D) Dose-response analysis of Calu-3 cells treated with the cathepsin inhibitor Z-FA-FMK. Data represent the average of four independent experiments ± SD. (E) Dose response analysis of Calu-3, Vero, and Huh7.5 cells treated with camostat. Data represent the average of ≥2 independent experiments ± SD. (F) IC 50 , CC50, and SI for camostat across cell types. (G) Immunoblot of Vero, Huh7.5, and Calu-3 cells probed for Ace2 and tubulin as a loading control. Representative blot is shown. (H) qRT-PCR of Ace2 or TMPRSS2 comparing Huh7.5 and Calu-3 cells. Data represent the mean ± SEM for ≥2 independent experiments.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Infection, Microscopy, Western Blot, Quantitative RT-PCR

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: Validation of antiviral activity of nine candidates in Calu-3 cells (A) Dose-response analysis of Huh7.5 candidate antivirals in Calu-3 cells with a SI > 3. Data represent the average of ≥4 independent experiments ± SD. (B) qRT-PCR analysis of nine candidate antivirals in Calu-3 cells. Data represent the mean ± SEM for ≥2 independent experiments.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Activity Assay, Quantitative RT-PCR

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet: Cyclosporine is antiviral against SARS-CoV-2 independent of calcineurin (A) Dose-response analysis of Huh7.5 cells treated with a panel of cyclosporins and related drugs. Data represent the average of ≥2 independent experiments ± SD. (B) Dose-response analysis of Calu-3 cells treated with a panel of cyclosporins and related drugs. Data represent the average of five independent experiments ± SD. (C) Dose-response analysis of Calu3 cells treated with cyclophilin-selective drugs NIM811 and alisporivir. Data represent the average of five independent experiments ± SD. (D) Table of IC 50 s, CC50s, and SI of Huh7.5 cells and Calu-3 cells treated with the indicated drugs. (E) qRT-PCR analysis of viral replication in Huh7.5 and Calu-3 cells treated with the indicated drugs. (F) qRT-PCR analysis of viral replication in iAT2 cells treated with the indicated drugs. (G) qRT-PCR of NHBE cells treated with the indicated drug. For (E)–(G), data represent the mean ± SEM for ≥2 independent experiments.

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Quantitative RT-PCR

Journal: Cell Reports

Article Title: Drug repurposing screens reveal cell-type-specific entry pathways and FDA-approved drugs active against SARS-Cov-2

doi: 10.1016/j.celrep.2021.108959

Figure Lengend Snippet:

Article Snippet: High-throughput screening in human Huh7.5 cells to identify antivirals against SARS-CoV-2 (A) Huh7.5 cells were infected with SARS-CoV-2 (MOI = 1) and 30 hpi processed for microscopy. (B) Dose-response analysis of Huh7.5 cells treated with hydroxychloroquine or remdesivir.

Techniques: Recombinant, SYBR Green Assay, Software

Journal: Scientific Reports

Article Title: The citrus flavanone naringenin impairs dengue virus replication in human cells

doi: 10.1038/srep41864

Figure Lengend Snippet: Naringenin half maximal inhibitory concentration (IC 50 ) and selectivity index (SI) for anti-DENV activity in Huh7.5 cells.

Article Snippet: The human-derived Huh7.5 hepatoma cell line (ATCC PTA-8561) was grown in Dulbecco’s Modified Eagle Medium/nutrient mixture F-12 (D-MEM/F-12; Gibco, Grand Island, NY, USA) supplemented with 100 IU/μg/mL penicillin/streptomycin (Gibco, Grand Island, NY, USA) and 10% FBS at 37 °C in a humidified, 5% CO 2 controlled atmosphere.

Techniques: Concentration Assay, Activity Assay

Journal: Cell host & microbe

Article Title: Induction of Selenoprotein P mRNA during Hepatitis C Virus Infection Inhibits RIG-I-Mediated Antiviral Immunity.

doi: 10.1016/j.chom.2019.02.015

Figure Lengend Snippet: Figure 4. The RIG-I-Mediated Signaling Pathway, but not the MDA5- or TLR3-Mediated Signaling Pathway, Is Involved in the SEPP1 mRNA Knockdown Stimulation of the Type I IFN Response (A) SEPP1 mRNA knockdown-augmented IFN-b1 induction was lost by the suppression of RIG-I in PHHs. SEPP1 mRNA knockdown PHHs were prepared by lentivirus-mediated gene silencing. At 3 days after lentivirus infection, the indicated siRNA or its control were transfected into the cells. At 3 days after siRNA transfection, the cells were stimulated with HCV-RNA (left) or Poly(I:C) (right). (B) SEPP1 mRNA knockdown-augmented IFN-b1 induction is lost in RIG-I KO cells. RIG-I or MDA5 KO HepG2 cells and their controls were infected with lentivirus shSEPP1 (L-shSEPP1#1 and #2). These cells were stimulated with HCV-RNA (left) or Poly(I:C) (right). (C and D) Replication of influenza virus and EMCV in SEPP1 mRNA knockdown hepatocytes. SEPP1 mRNA knockdown HepG2 cells were prepared by infection with lentivirus shSEPP1 (L-shSEPP1#1 and #2). At 6 days after gene silencing, the cells were infected with influenza virus (C, left) or EMCV (C, right). Influenza virus and EMCV replication levels were evaluated by the expression of nucleoprotein (NP) (D, left) and EMCV RNA (D, right), respectively. Data are expressed as mean ± SEM. n = 3. Data were combined from at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays Cell Counting Kit-8 Dojindo Molecular Technologies Cat# 347-07621 High Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat# 4368814 7900HT Real-Time PCR System Applied Biosystems VeriKine Human Interferon Beta ELISA Kit PBL Assay Science Cat# 41410-1 ViewRNA ISH Cell Assay Kit Affymetrix Cat# TFA-QVC0001 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript SP6 Transcription Kit Invitrogen Cat# AM1330 RIP Assay Kit MBL Cat# RN1001 Pierce Magnetic RNA-Protein Pull-Down Kit Thermo Fischer Scientific Cat# 20164 Experimental Models: Cell Lines HepG2 ATCC N/A Huh7.5 Professor C. M. Rice N/A KH This paper N/A Primary Human Hepatocyte (PHH) Phoenix Bio N/A Primary Mouse Hepatocyte (PMH) This paper N/A Bone-marrow-derived dendritic cells (BM-DCs) This paper N/A Experimental Models: Organisms/Strains Sepp1-knockout mice Lab stored Hill et al., 2003 Oligonucleotides See Table S3 N/A N/A Recombinant DNA See Table S4 N/A N/A Software and Algorithms GraphPad Prism GraphPad https://www.graphpad.com/ scientificsoftware/prism/ ImageJ ImageJ https://imagej.nih.gov/ij/

Techniques: Knockdown, Infection, Control, Transfection, Virus, Expressing

Journal: Cell host & microbe

Article Title: Induction of Selenoprotein P mRNA during Hepatitis C Virus Infection Inhibits RIG-I-Mediated Antiviral Immunity.

doi: 10.1016/j.chom.2019.02.015

Figure Lengend Snippet: Figure 5. SEPP1 mRNA Binds Directly to RIG-I (A) Co-localization analysis of RIG-I protein and SEPP1 mRNA in HepG2 cells by confocal microscopy. HepG2 cells were transfected with a FLAG-tagged RIG-I WT expression vector. At 72 h after transfection, SEPP1 mRNA (green) was visualized by FISH, and FLAG-tagged RIG-I (red) was identified by immunofluo- rescence staining (upper). Co-localization was estimated using Pearson’s correlation coefficient and Manders’ co-localization coefficients M1 (red per green) and M2 (green per red) (lower) (n = 30). (B) Specific binding of SEPP1 mRNA to RIG-I, but not MDA5, in HepG2 cells. RIP assay with HepG2 cell lysates using an anti-RIG-I antibody (left-upper), anti- MDA5 antibody (right-upper), or negative control (anti-IgG antibody). RIG-I (left-lower) or MDA5 (right-lower) protein in the input or post-immunoprecipitation (IP) samples was detected by western blot analysis. Immunoprecipitated GPx1 or SEPP1 mRNA was quantitated by RTD-PCR. Data were normalized by GPx1 or SEPP1 mRNA in the input before IP. (C) Influence of an RNA-binding domain mutation of RIG-I for the binding of SEPP1 mRNA. RIP assay with HepG2 cell lysates expressing FLAG-tagged RIG-I WT using an anti-FLAG antibody or negative control (anti-IgG antibody) (left). Immunoprecipitated GPx1 or SEPP1 mRNA was quantitated by RTD-PCR. Data were normalized by GPx1 or SEPP1 mRNA in the input before IP (left-upper). FLAG-tagged RIG-I WT protein in the input or post-IP samples was detected by western blot analysis (left-lower). RIP assay with HepG2 cell lysates expressing FLAG-tagged RIG-I WT, K888E mutant, or its backbone vector (pSF-EF1a) using an anti- FLAG antibody (right-upper). FLAG-tagged RIG-I WT or K888E mutant protein in the input or post-IP samples was detected by western blot analysis (right-lower). (D) In vitro binding of synthesized capped SEPP1 mRNA and purified RIG-I. RNA pull-down assay of recombinant RIG-I WT or K888E mutant using SEPP1 or GPx1 mRNA. Proteins pulled down by mRNA were detected by western blot analysis with an anti-RIG-I antibody. (E) ATPase activity assay. Purified RIG-I (200 nM) was incubated with ATP in the presence or absence of 100 nM HCV-RNA, Poly(I:C), and SEPP1 mRNA, as indicated. The amount of ADP produced in a reaction was quantified using a luciferase-luciferin reaction (described in the STAR Methods). (F) RIG-I expression in the cytoplasmic and mitochondrial fractions. Cytoplasmic and mitochondrial proteins from SEPP1 mRNA knockdown and control HepG2 cells were fractionated as described in the STAR Methods. RIG-I expression was detected by western blot analysis. LDH, cytoplasmic marker; COX IV, mitochondrial marker. Data are expressed as mean ± SEM. n = 3. Data were combined from at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays Cell Counting Kit-8 Dojindo Molecular Technologies Cat# 347-07621 High Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat# 4368814 7900HT Real-Time PCR System Applied Biosystems VeriKine Human Interferon Beta ELISA Kit PBL Assay Science Cat# 41410-1 ViewRNA ISH Cell Assay Kit Affymetrix Cat# TFA-QVC0001 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript SP6 Transcription Kit Invitrogen Cat# AM1330 RIP Assay Kit MBL Cat# RN1001 Pierce Magnetic RNA-Protein Pull-Down Kit Thermo Fischer Scientific Cat# 20164 Experimental Models: Cell Lines HepG2 ATCC N/A Huh7.5 Professor C. M. Rice N/A KH This paper N/A Primary Human Hepatocyte (PHH) Phoenix Bio N/A Primary Mouse Hepatocyte (PMH) This paper N/A Bone-marrow-derived dendritic cells (BM-DCs) This paper N/A Experimental Models: Organisms/Strains Sepp1-knockout mice Lab stored Hill et al., 2003 Oligonucleotides See Table S3 N/A N/A Recombinant DNA See Table S4 N/A N/A Software and Algorithms GraphPad Prism GraphPad https://www.graphpad.com/ scientificsoftware/prism/ ImageJ ImageJ https://imagej.nih.gov/ij/

Techniques: Confocal Microscopy, Transfection, Expressing, Plasmid Preparation, Staining, Binding Assay, Negative Control, Immunoprecipitation, Western Blot, RNA Binding Assay, Mutagenesis, In Vitro, Synthesized, Pull Down Assay, Recombinant, Activity Assay, Incubation, Produced, Luciferase, Knockdown, Control, Marker

Journal: Cell host & microbe

Article Title: Induction of Selenoprotein P mRNA during Hepatitis C Virus Infection Inhibits RIG-I-Mediated Antiviral Immunity.

doi: 10.1016/j.chom.2019.02.015

Figure Lengend Snippet: Figure 6. Functional Domains for the Regulation of RIG-I Activity in SEPP1 mRNA (A) Schematic diagram of GPx1 mRNA and selenocysteine incorporation mediated by the SECISs (upper). Schematic diagram of the deletion mutants of SEPP1 mRNA (lower). (B) Effect of SEPP1 mRNA deletion mutants on IFN-b1 mRNA induction in HepG2 cells. HepG2 cells were co-transfected with HCV-RNA (left) or Poly(I:C) (right) and the indicated SEPP1 mRNA deletion mutants (25 pM). (C) RNA pull-down assay using SEPP1 mRNA deletion mutants. HepG2 cells expressing FLAG-tagged RIG-I were lysed and FLAG-tagged RIG-I protein was pulled down by control GPx1 mRNA or SEPP1 mRNA deletion mutants. (D) EMSA of ORF-1 mRNA with RIG-I. Increasing amounts of purified RIG-I WT or K888E protein were incubated with 10 ng ORF-1 mRNA and subjected to electrophoresis on a 0.5% agarose gel (upper). For the competition assay (below), the indicated amounts of non-labeled HCV-RNA or Poly(I:C) were incubated with 500 ng purified RIG-I WT, and magnetic beads bound with full-length SEPP1 or ORF-1 mRNA were added. The magnetic beads were collected and pulled- down RIG-I was detected by western blot analysis. (E) Dose-dependent inhibition of RIG-I activity by SEPP1 mRNA. Different amounts (20–100 pM) of full-length SEPP1, ORF-1, and ORF-2 mRNA were co-transfected with HCV-RNA or Poly(I:C) into SEPP1 mRNA knockdown HepG2 cells (upper part of the graph). Different amounts (20–100 pM) of full-length SEPP1 mRNA were co-transfected with HCV-RNA or Poly(I:C) into SeP KO HepG2 cells overexpressing mutant RIG-I (F539A) (lower part of the graph). IFN-b1 mRNA was detected by RTD-PCR. (F) Association of SEPP1 mRNA and each component of RIG-I. Schematic representation of purified GST fused with each component of RIG-I (upper). Asso- ciation of full-length SEPP1 or ORF-1 mRNA with CARDs, helicase domain, and CTD of RIG-I (middle). For the competition assay (lower left), the indicated amounts of non-labeled HCV-RNA and magnetic beads bound with full-length SEPP1 or ORF-1 mRNA were incubated with purified RIG-I CTD. The magnetic beads were collected and pulled-down RIG-I was detected by western blot analysis. Restriction of CARD release from full-length SEPP1 mRNA (lower right). A mixture of purified GST-DCARD-His and GST-CARD was bound to full-length SEPP1 mRNA, ORF-1 mRNA, and HCV-RNA, respectively. CARDs associated with the helicase domain were pulled down using the His tag. Data are expressed as mean ± SEM. n = 3–5. Data were combined from at least 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Critical Commercial Assays Cell Counting Kit-8 Dojindo Molecular Technologies Cat# 347-07621 High Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat# 4368814 7900HT Real-Time PCR System Applied Biosystems VeriKine Human Interferon Beta ELISA Kit PBL Assay Science Cat# 41410-1 ViewRNA ISH Cell Assay Kit Affymetrix Cat# TFA-QVC0001 Dual-Luciferase Reporter Assay System Promega Cat# E1910 MEGAscript SP6 Transcription Kit Invitrogen Cat# AM1330 RIP Assay Kit MBL Cat# RN1001 Pierce Magnetic RNA-Protein Pull-Down Kit Thermo Fischer Scientific Cat# 20164 Experimental Models: Cell Lines HepG2 ATCC N/A Huh7.5 Professor C. M. Rice N/A KH This paper N/A Primary Human Hepatocyte (PHH) Phoenix Bio N/A Primary Mouse Hepatocyte (PMH) This paper N/A Bone-marrow-derived dendritic cells (BM-DCs) This paper N/A Experimental Models: Organisms/Strains Sepp1-knockout mice Lab stored Hill et al., 2003 Oligonucleotides See Table S3 N/A N/A Recombinant DNA See Table S4 N/A N/A Software and Algorithms GraphPad Prism GraphPad https://www.graphpad.com/ scientificsoftware/prism/ ImageJ ImageJ https://imagej.nih.gov/ij/

Techniques: Functional Assay, Activity Assay, Transfection, Pull Down Assay, Expressing, Control, Incubation, Electrophoresis, Agarose Gel Electrophoresis, Competitive Binding Assay, Labeling, Magnetic Beads, Western Blot, Inhibition, Knockdown, Mutagenesis