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KO of Candidate Host Factor Genes Reduces Coronavirus Infection (A) Quantitative reverse-transcriptase PCR (RT-qPCR) quantification of intracellular SARS-CoV-2 levels in RNP-edited A549-ACE2 cells. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 72 h post-infection (hpi). (B) RT-qPCR quantification of intracellular SARS-CoV-2 levels in Calu-3 cells lentivirally transduced with Cas9/sgRNA cassettes targeting the indicated genes. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 48 hpi. (C) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, <t>TMEM106B</t> KO, or TMEM106B KO cells with TMEM106B cDNA add-back (AB). Cells were infected using MOI = 0.1 and harvested at 24 hpi. (D) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, VAC14 KO, or VAC14 KO cells with VAC14 cDNA AB. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (E) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, SCAP KO, MBTPS2 KO, or EXOC2 KO cells. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (F) RT-qPCR quantification of intracellular OC43 and 229E RNA levels in WT and TMEM106B, VAC14 , SCAP , MBTPS2 , or EXOC2 KO Huh7.5.1 cells. Cells were infected using MOI = 0.05 (229E) and MOI = 3 (OC43) and harvested at 48 hpi. (G–I) RT-qPCR quantification of intracellular viral RNA for (G) OC43, (H) 229E, or (I) SARS-CoV-2 in WT Huh7.5.1 cells or cell lines deficient in CCZ1B , RAB7A , VPS16 , BECN1 , PIK3R4 , or UVRAG. (J–L) RT-qPCR quantification of intracellular viral RNA for (J) SARS-CoV-2, (K) OC43, or (L) 229E in WT, PIK3R4 KO, or PIK3R4 KO cells with PIK3R4 cDNA AB. (M–O) RT-qPCR quantification of intracellular viral RNA for (M) SARS-CoV-2, (N) OC43, or (O) 229E in WT, VPS16 KO, or VPS16 KO cells with VPS16 cDNA AB. For SARS-CoV-2 infection, viral N gene transcripts were normalized to cellular RNaseP. For OC43 and 229E experiments, viral RNA was normalized to 18S RNA. For all RT-qPCR experiments, results are displayed relative to infection in WT cells, and data represent means ± SEM from three biological samples.
Tmem106b, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plasmid vac14 #47418  (Addgene inc)
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Plasmid Vac14 #47418, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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KO of Candidate Host Factor Genes Reduces Coronavirus Infection (A) Quantitative reverse-transcriptase PCR (RT-qPCR) quantification of intracellular SARS-CoV-2 levels in RNP-edited A549-ACE2 cells. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 72 h post-infection (hpi). (B) RT-qPCR quantification of intracellular SARS-CoV-2 levels in Calu-3 cells lentivirally transduced with Cas9/sgRNA cassettes targeting the indicated genes. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 48 hpi. (C) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, <t>TMEM106B</t> KO, or TMEM106B KO cells with TMEM106B cDNA add-back (AB). Cells were infected using MOI = 0.1 and harvested at 24 hpi. (D) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, VAC14 KO, or VAC14 KO cells with VAC14 cDNA AB. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (E) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, SCAP KO, MBTPS2 KO, or EXOC2 KO cells. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (F) RT-qPCR quantification of intracellular OC43 and 229E RNA levels in WT and TMEM106B, VAC14 , SCAP , MBTPS2 , or EXOC2 KO Huh7.5.1 cells. Cells were infected using MOI = 0.05 (229E) and MOI = 3 (OC43) and harvested at 48 hpi. (G–I) RT-qPCR quantification of intracellular viral RNA for (G) OC43, (H) 229E, or (I) SARS-CoV-2 in WT Huh7.5.1 cells or cell lines deficient in CCZ1B , RAB7A , VPS16 , BECN1 , PIK3R4 , or UVRAG. (J–L) RT-qPCR quantification of intracellular viral RNA for (J) SARS-CoV-2, (K) OC43, or (L) 229E in WT, PIK3R4 KO, or PIK3R4 KO cells with PIK3R4 cDNA AB. (M–O) RT-qPCR quantification of intracellular viral RNA for (M) SARS-CoV-2, (N) OC43, or (O) 229E in WT, VPS16 KO, or VPS16 KO cells with VPS16 cDNA AB. For SARS-CoV-2 infection, viral N gene transcripts were normalized to cellular RNaseP. For OC43 and 229E experiments, viral RNA was normalized to 18S RNA. For all RT-qPCR experiments, results are displayed relative to infection in WT cells, and data represent means ± SEM from three biological samples.
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protein siglec-8 sc-47418  (Santa Cruz Biotechnology)
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Protein Siglec 8 Sc 47418, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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KO of Candidate Host Factor Genes Reduces Coronavirus Infection (A) Quantitative reverse-transcriptase PCR (RT-qPCR) quantification of intracellular SARS-CoV-2 levels in RNP-edited A549-ACE2 cells. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 72 h post-infection (hpi). (B) RT-qPCR quantification of intracellular SARS-CoV-2 levels in Calu-3 cells lentivirally transduced with Cas9/sgRNA cassettes targeting the indicated genes. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 48 hpi. (C) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, TMEM106B KO, or TMEM106B KO cells with TMEM106B cDNA add-back (AB). Cells were infected using MOI = 0.1 and harvested at 24 hpi. (D) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, VAC14 KO, or VAC14 KO cells with VAC14 cDNA AB. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (E) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, SCAP KO, MBTPS2 KO, or EXOC2 KO cells. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (F) RT-qPCR quantification of intracellular OC43 and 229E RNA levels in WT and TMEM106B, VAC14 , SCAP , MBTPS2 , or EXOC2 KO Huh7.5.1 cells. Cells were infected using MOI = 0.05 (229E) and MOI = 3 (OC43) and harvested at 48 hpi. (G–I) RT-qPCR quantification of intracellular viral RNA for (G) OC43, (H) 229E, or (I) SARS-CoV-2 in WT Huh7.5.1 cells or cell lines deficient in CCZ1B , RAB7A , VPS16 , BECN1 , PIK3R4 , or UVRAG. (J–L) RT-qPCR quantification of intracellular viral RNA for (J) SARS-CoV-2, (K) OC43, or (L) 229E in WT, PIK3R4 KO, or PIK3R4 KO cells with PIK3R4 cDNA AB. (M–O) RT-qPCR quantification of intracellular viral RNA for (M) SARS-CoV-2, (N) OC43, or (O) 229E in WT, VPS16 KO, or VPS16 KO cells with VPS16 cDNA AB. For SARS-CoV-2 infection, viral N gene transcripts were normalized to cellular RNaseP. For OC43 and 229E experiments, viral RNA was normalized to 18S RNA. For all RT-qPCR experiments, results are displayed relative to infection in WT cells, and data represent means ± SEM from three biological samples.

Journal: Cell

Article Title: Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

doi: 10.1016/j.cell.2020.12.004

Figure Lengend Snippet: KO of Candidate Host Factor Genes Reduces Coronavirus Infection (A) Quantitative reverse-transcriptase PCR (RT-qPCR) quantification of intracellular SARS-CoV-2 levels in RNP-edited A549-ACE2 cells. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 72 h post-infection (hpi). (B) RT-qPCR quantification of intracellular SARS-CoV-2 levels in Calu-3 cells lentivirally transduced with Cas9/sgRNA cassettes targeting the indicated genes. A non-targeting sgRNA was used as control. Cells were infected using MOI = 0.1 and harvested at 48 hpi. (C) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, TMEM106B KO, or TMEM106B KO cells with TMEM106B cDNA add-back (AB). Cells were infected using MOI = 0.1 and harvested at 24 hpi. (D) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, VAC14 KO, or VAC14 KO cells with VAC14 cDNA AB. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (E) RT-qPCR quantification of intracellular SARS-CoV-2 levels in WT Huh7.5.1, SCAP KO, MBTPS2 KO, or EXOC2 KO cells. Cells were infected using MOI = 0.1 and harvested at 24 hpi. (F) RT-qPCR quantification of intracellular OC43 and 229E RNA levels in WT and TMEM106B, VAC14 , SCAP , MBTPS2 , or EXOC2 KO Huh7.5.1 cells. Cells were infected using MOI = 0.05 (229E) and MOI = 3 (OC43) and harvested at 48 hpi. (G–I) RT-qPCR quantification of intracellular viral RNA for (G) OC43, (H) 229E, or (I) SARS-CoV-2 in WT Huh7.5.1 cells or cell lines deficient in CCZ1B , RAB7A , VPS16 , BECN1 , PIK3R4 , or UVRAG. (J–L) RT-qPCR quantification of intracellular viral RNA for (J) SARS-CoV-2, (K) OC43, or (L) 229E in WT, PIK3R4 KO, or PIK3R4 KO cells with PIK3R4 cDNA AB. (M–O) RT-qPCR quantification of intracellular viral RNA for (M) SARS-CoV-2, (N) OC43, or (O) 229E in WT, VPS16 KO, or VPS16 KO cells with VPS16 cDNA AB. For SARS-CoV-2 infection, viral N gene transcripts were normalized to cellular RNaseP. For OC43 and 229E experiments, viral RNA was normalized to 18S RNA. For all RT-qPCR experiments, results are displayed relative to infection in WT cells, and data represent means ± SEM from three biological samples.

Article Snippet: The following cDNA sequence containing plasmids were obtained: hACE2 (Addgene, #1786, gift from Hyeryun Choe) , TMPRSS2 (Addgene, #53887, gift from Roger Reeves) , TMEM106B (Genscript, OHu17671), VAC14 (Addgene, #47418, gift from Peter McPherson) , PIK3R4 (Addgene, #23488, gift from William Hahn & David Root) ( ) and VPS16 (Addgene, #67023, gift from Noboru Mizushima) ( ).

Techniques: Infection, Reverse Transcription, Quantitative RT-PCR, Control, Transduction

Characterization of Gene-Edited Cells, Related to <xref ref-type=Figure 3 (A) Genotyping of clonal Huh7.5.1. Targeted loci were PCR-amplified, Sanger-sequenced and aligned to WT reference sequence. Frameshifts are highlighted in blue. (B) Western blot analysis of WT, KO and KO cells with respective cDNA add-backs (AB) for TMEM106B, VAC14 and PIK3R4. Lysates to probe for TMEM106B were prepared under non-reducing conditions and bands appear as dimers. GAPDH was used as loading control. (C) Cell viability measurement of 229E infected WT and KO Huh7.5.1 cells. Cells were infected with 229E (moi = 0.05) and viability was determined 8 dpi using Cell Titer Glo. Values are displayed as means ± SD from three biological samples. (D) Cell viability measurement of OC43 infected WT and KO Huh7.5.1 cells. Cells were infected with OC43 (moi = 3) and viability was determined 8 dpi using Cell Titer Glo. Values are displayed as means ± SD from two biological samples. (E) Analysis of cell proliferation of RNP-edited A549-ACE2 cells. Cells were plated in 96-wells and confluency was measured daily using an automated microscope. Values are displayed as means ± SD from four separate wells per cell line. (F) Analysis of cell proliferation of WT and clonal KO Huh7.5.1 cells. Cells were plated in 96-wells and cell proliferation was measured daily using Cell Titer Glo. Values are displayed as means ± SD from three separate wells per cell line per time point. " width="100%" height="100%">

Journal: Cell

Article Title: Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

doi: 10.1016/j.cell.2020.12.004

Figure Lengend Snippet: Characterization of Gene-Edited Cells, Related to Figure 3 (A) Genotyping of clonal Huh7.5.1. Targeted loci were PCR-amplified, Sanger-sequenced and aligned to WT reference sequence. Frameshifts are highlighted in blue. (B) Western blot analysis of WT, KO and KO cells with respective cDNA add-backs (AB) for TMEM106B, VAC14 and PIK3R4. Lysates to probe for TMEM106B were prepared under non-reducing conditions and bands appear as dimers. GAPDH was used as loading control. (C) Cell viability measurement of 229E infected WT and KO Huh7.5.1 cells. Cells were infected with 229E (moi = 0.05) and viability was determined 8 dpi using Cell Titer Glo. Values are displayed as means ± SD from three biological samples. (D) Cell viability measurement of OC43 infected WT and KO Huh7.5.1 cells. Cells were infected with OC43 (moi = 3) and viability was determined 8 dpi using Cell Titer Glo. Values are displayed as means ± SD from two biological samples. (E) Analysis of cell proliferation of RNP-edited A549-ACE2 cells. Cells were plated in 96-wells and confluency was measured daily using an automated microscope. Values are displayed as means ± SD from four separate wells per cell line. (F) Analysis of cell proliferation of WT and clonal KO Huh7.5.1 cells. Cells were plated in 96-wells and cell proliferation was measured daily using Cell Titer Glo. Values are displayed as means ± SD from three separate wells per cell line per time point.

Article Snippet: The following cDNA sequence containing plasmids were obtained: hACE2 (Addgene, #1786, gift from Hyeryun Choe) , TMPRSS2 (Addgene, #53887, gift from Roger Reeves) , TMEM106B (Genscript, OHu17671), VAC14 (Addgene, #47418, gift from Peter McPherson) , PIK3R4 (Addgene, #23488, gift from William Hahn & David Root) ( ) and VPS16 (Addgene, #67023, gift from Noboru Mizushima) ( ).

Techniques: Amplification, Sequencing, Western Blot, Control, Infection, Microscopy

Cholesterol Is Required for S-Mediated Entry of SARS-CoV-2 (A) Western blot of ACE2 and TMEM106B levels from Huh7.5.1- ACE2/TMPRSS2 cells edited with non-targeting (NT) or TMEM106B -targeting RNPs. Lysates were prepared under non-reducing conditions and TMEM106B appears as dimer. GAPDH was used as loading control. Molecular weight markers are indicated on the left. (B) VSV-SARS-CoV-2-S infection of clonal Huh7.5.1- ACE2/TMPRSS2 cells edited with RNPs targeting the specified genes. A NT sgRNA was used as control. Cells were harvested at 8 hpi and analyzed for GFP+ cells using flow cytometry. Values represent five biological replicates and are displayed as means ± SD (C) VSV-SARS-CoV-2-S infection of PF-429242-treated cells. Huh7.5.1- ACE2/TMPRSS2 cells were pretreated with different concentrations of PF-429242 for 2 h and then infected with virus. Cells were analyzed by flow cytometry at 14 hpi and analyzed for GFP+ cells using flow cytometry. Values represent two biological replicates at each concentration and are displayed as means ± SD (D) VSV-SARS-CoV-2-S infection of 25-HC-treated cells. Huh7.5.1- ACE2/TMPRSS2 cells were pretreated with different concentrations of 25-HC for 2 h and then infected with virus. Cells were analyzed by flow cytometry at 14 hpi. Values represent two biological replicates at each concentration and are displayed as means ± SD

Journal: Cell

Article Title: Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

doi: 10.1016/j.cell.2020.12.004

Figure Lengend Snippet: Cholesterol Is Required for S-Mediated Entry of SARS-CoV-2 (A) Western blot of ACE2 and TMEM106B levels from Huh7.5.1- ACE2/TMPRSS2 cells edited with non-targeting (NT) or TMEM106B -targeting RNPs. Lysates were prepared under non-reducing conditions and TMEM106B appears as dimer. GAPDH was used as loading control. Molecular weight markers are indicated on the left. (B) VSV-SARS-CoV-2-S infection of clonal Huh7.5.1- ACE2/TMPRSS2 cells edited with RNPs targeting the specified genes. A NT sgRNA was used as control. Cells were harvested at 8 hpi and analyzed for GFP+ cells using flow cytometry. Values represent five biological replicates and are displayed as means ± SD (C) VSV-SARS-CoV-2-S infection of PF-429242-treated cells. Huh7.5.1- ACE2/TMPRSS2 cells were pretreated with different concentrations of PF-429242 for 2 h and then infected with virus. Cells were analyzed by flow cytometry at 14 hpi and analyzed for GFP+ cells using flow cytometry. Values represent two biological replicates at each concentration and are displayed as means ± SD (D) VSV-SARS-CoV-2-S infection of 25-HC-treated cells. Huh7.5.1- ACE2/TMPRSS2 cells were pretreated with different concentrations of 25-HC for 2 h and then infected with virus. Cells were analyzed by flow cytometry at 14 hpi. Values represent two biological replicates at each concentration and are displayed as means ± SD

Article Snippet: The following cDNA sequence containing plasmids were obtained: hACE2 (Addgene, #1786, gift from Hyeryun Choe) , TMPRSS2 (Addgene, #53887, gift from Roger Reeves) , TMEM106B (Genscript, OHu17671), VAC14 (Addgene, #47418, gift from Peter McPherson) , PIK3R4 (Addgene, #23488, gift from William Hahn & David Root) ( ) and VPS16 (Addgene, #67023, gift from Noboru Mizushima) ( ).

Techniques: Western Blot, Control, Molecular Weight, Infection, Flow Cytometry, Virus, Concentration Assay

Journal: Cell

Article Title: Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

doi: 10.1016/j.cell.2020.12.004

Figure Lengend Snippet:

Article Snippet: The following cDNA sequence containing plasmids were obtained: hACE2 (Addgene, #1786, gift from Hyeryun Choe) , TMPRSS2 (Addgene, #53887, gift from Roger Reeves) , TMEM106B (Genscript, OHu17671), VAC14 (Addgene, #47418, gift from Peter McPherson) , PIK3R4 (Addgene, #23488, gift from William Hahn & David Root) ( ) and VPS16 (Addgene, #67023, gift from Noboru Mizushima) ( ).

Techniques: Virus, Recombinant, Purification, Gel Extraction, One Step RT-PCR, Sequencing, CRISPR, Library Amplification, Cloning, Software, Imaging