human small airway epithelial cells  (ATCC)


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    ATCC human small airway epithelial cells
    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured <t>cells</t> with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the <t>airway</t> cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, <t>human</t> <t>small</t> airway <t>epithelial</t> cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]
    Human Small Airway Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1"

    Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

    Journal: medRxiv

    doi: 10.1101/2023.01.24.23284890

    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]
    Figure Legend Snippet: a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]

    Techniques Used: Infection, Immunohistochemistry, Immunostaining, Cell Culture, Expressing, Activity Assay, Two Tailed Test, Standard Deviation, Binding Assay

    pcs 301 010 lung  (ATCC)


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    ATCC pcs 301 010 lung
    Pcs 301 010 Lung, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pcs 301 010 lung  (ATCC)


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    ATCC pcs 301 010 lung
    Pcs 301 010 Lung, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human small airway epithelial cells  (ATCC)


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    ATCC human small airway epithelial cells
    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured <t>cells</t> with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the <t>airway</t> cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, <t>human</t> <t>small</t> airway <t>epithelial</t> cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]
    Human Small Airway Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1"

    Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

    Journal: medRxiv

    doi: 10.1101/2023.01.24.23284890

    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]
    Figure Legend Snippet: a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]

    Techniques Used: Infection, Immunohistochemistry, Immunostaining, Cell Culture, Expressing, Activity Assay, Two Tailed Test, Standard Deviation, Binding Assay

    normal primary human small airway epithelial sae cells  (ATCC)


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    ATCC normal primary human small airway epithelial sae cells
    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, <t>SAE,</t> AT2 <t>and</t> <t>LBE</t> cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.
    Normal Primary Human Small Airway Epithelial Sae Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome"

    Article Title: Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac1238

    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, SAE, AT2 and LBE cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.
    Figure Legend Snippet: A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, SAE, AT2 and LBE cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.

    Techniques Used: Expressing, Cell Culture

    human airway epithelial cell line  (ATCC)


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    ATCC human airway epithelial cell line
    Human Airway Epithelial Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human retinal pigment epithelial cells  (ATCC)


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    ATCC human retinal pigment epithelial cells
    Human Retinal Pigment Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human adult pigment epithelial cells  (ATCC)


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    ATCC human adult pigment epithelial cells
    Human Adult Pigment Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    primary hsaecs  (ATCC)


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    ATCC primary hsaecs
    ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) <t>Primary</t> <t>hSAECs</t> were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.
    Primary Hsaecs, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2"

    Article Title: Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI160766

    ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) Primary hSAECs were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.
    Figure Legend Snippet: ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) Primary hSAECs were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.

    Techniques Used: Infection, Luciferase, Activity Assay, SDS Page, Western Blot, Expressing, Flow Cytometry

    pcs 301 010  (ATCC)


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    human airway epithelial cells  (ATCC)


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    ATCC human small airway epithelial cells
    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured <t>cells</t> with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the <t>airway</t> cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, <t>human</t> <t>small</t> airway <t>epithelial</t> cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]
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    ATCC pcs 301 010 lung
    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured <t>cells</t> with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the <t>airway</t> cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, <t>human</t> <t>small</t> airway <t>epithelial</t> cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]
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    ATCC normal primary human small airway epithelial sae cells
    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, <t>SAE,</t> AT2 <t>and</t> <t>LBE</t> cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.
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    ATCC human airway epithelial cell line
    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, <t>SAE,</t> AT2 <t>and</t> <t>LBE</t> cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.
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    ATCC human retinal pigment epithelial cells
    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, <t>SAE,</t> AT2 <t>and</t> <t>LBE</t> cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.
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    ATCC human adult pigment epithelial cells
    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, <t>SAE,</t> AT2 <t>and</t> <t>LBE</t> cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.
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    ATCC primary hsaecs
    ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) <t>Primary</t> <t>hSAECs</t> were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.
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    ATCC pcs 301 010
    ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) <t>Primary</t> <t>hSAECs</t> were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.
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    ATCC human airway epithelial cells
    ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) <t>Primary</t> <t>hSAECs</t> were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.
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    a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]

    Journal: medRxiv

    Article Title: SARS-CoV-2 Spike Protein Receptor Binding-ACE2 Interaction Increases Carbohydrate Sulfotransferases and Reduces N-Acetylgalactosamine-4-Sulfatase through Phospho-p38-MAPK and RB-E2F1

    doi: 10.1101/2023.01.24.23284890

    Figure Lengend Snippet: a , b . In lung tissue obtained post-mortem from patients who died due to SARS-CoV-2 infection, immunohistochemistry showed increased total chondroitin sulfate compared to normal control lung tissue. c , d . In contrast, intensity and distribution of ARSB were markedly reduced in the Covid-19 lung tissue. Marked decline is evident in the membrane immunostaining, compared to the normal control. e , f . Carbohydrate sulfotransferase (CHST) 15 which is the sulfotransferase that adds a 6-sulfate group to chondroitin 4-sulfate to form chondroitin-4,6-sulfate (chondroitin sulfate E), has regions of marked intensity of immunostaining in both the Covid-19 lung and the normal lung. g , h . In vascular smooth muscle tissue of the Covid-19 lung, the CHST15 immunostaining is much more intense and less diffuse than in the normal lung vascular tissue. i . Treatment of the cultured cells with Interferon (IFN)β amplifies the impact of SPRBD by increasing the expression of ACE2 more than four times the control level (p<0.1×10 −5 , n=6). j . Corresponding to the findings in the infected lung tissue, total chondroitin sulfate (CS) increased by ∼2 ug/g protein (p=0.001, n=6) and total sulfated glycosaminoglycans increased by over 3 ug/g protein (sGAG) (p=0.0005, n=6) in the AEC following exposure to the SPRBD. Increases are more following combined treatment with SPRBD and IFNβ (>5 µg/g protein for CS, n=6; 7.7 µg/g protein for sGAG, n=6). k . Consistent with the observed increases in chondroitin sulfate in Covid-19 lung tissue and in the airway cells, sulfotransferase activity increased by 63% following SPRBD and by over 200% following the combination of SPRBD and IFNβ (n=6, n=6) l . Arylsulfatase B (ARSB) activity declined in the AEC following exposure to the SPRBD (p=0.005, n=6), and declined over 50% by the combined exposure to SPRBD and IFNβ. m . The mRNA expression of ARSB also declined (p=2.6×10 −5 ; n=6) following SPRBD, and declined further following exposure to the combination of SPRBD and IFNβ. n . Expression of both CHST15 and of CHST11, which transfers 4-sulfate groups to N-acetylgalactosamine residues to create chondroitin 4-sulfate, is significantly upregulated following exposure to the SPRBD and the combination of SPRBD and IFNβ. All of the p-values were determined using unpaired t-test, two-tailed, with unequal variance, and error bars represent one standard deviation. [ACE2=angiotensin converting enzyme 2 receptor; AEC=normal, human small airway epithelial cells; ARSB=arylsulfatase B=N-acetylgalactosamine-4-sulfatase; SPRBD=SARS CoV-2 spike protein receptor binding domain]

    Article Snippet: Normal, primary, human small airway epithelial cells (PCS-301-010, ATCC, Manassas, VA) were grown in Airway Epithelial Cell Basal Medium (PCS-300-030, ATCC) supplemented with Bronchial Epithelial Cell Growth Kit (PCS-300-040) and Penicillin-Streptomycin (penicillin 10 U/ml; streptomycin 10 µg/ml), per recommendations.

    Techniques: Infection, Immunohistochemistry, Immunostaining, Cell Culture, Expressing, Activity Assay, Two Tailed Test, Standard Deviation, Binding Assay

    A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, SAE, AT2 and LBE cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.

    Journal: Nucleic Acids Research

    Article Title: Cellular APOBEC3A deaminase drives mutations in the SARS-CoV-2 genome

    doi: 10.1093/nar/gkac1238

    Figure Lengend Snippet: A3A mRNA expression in human airway and lung cells. ( A ) The A3A mRNA level in each cell line was quantified by RT–ddPCR 18 h after treatment without (control) or with IFN-ß and/or TNF-α under normoxic conditions (N). A549, Calu-3, SAE, AT2 and LBE cells and HNEpCs were also cultured under hypoxic conditions (H). The mean mRNA copy numbers of A3A relative to those of the housekeeping gene RPP40 are shown ( n = 3). ( B ) The CD11b and CD68 mRNA levels in each untreated cell line were quantified by RT–ddPCR. Human MDMs were used as positive controls. The mean mRNA copy numbers of CD11b and CD68 relative to those of RPP40 are shown ( n = 3). The error bars indicate the + SD values.

    Article Snippet: Normal primary human small airway epithelial (SAE) cells and normal primary human lobar bronchial epithelial (LBE) cells were obtained from ATCC.

    Techniques: Expressing, Cell Culture

    ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) Primary hSAECs were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.

    Journal: The Journal of Clinical Investigation

    Article Title: Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

    doi: 10.1172/JCI160766

    Figure Lengend Snippet: ( A ) A549-ACE2 cells were treated with or without type I IFN (250 U/mL) for 18 hours and then with 20 μM rapamycin (Rap), everolimus (Eve), temsirolimus (Tem), ridaforolimus (Rid), or an equivalent volume of DMSO (D) for 4 hours. HIV-CoV-2 (100 ng p24 equivalent) was added to cells, and infection was measured by luciferase activity 48 hours after infection. Luciferase units were normalized to 100 in the DMSO condition in the absence of IFN. ( B ) A549-ACE2 cells from A were subjected to SDS-PAGE and Western blot analysis. Immunoblotting was performed with anti–IFITM2/-3, anti-ACE2, and anti-actin (in that order) on the same nitrocellulose membrane. The numbers and tick marks indicate the size (kDa) and position of protein standards in ladders. ( C ) Primary hSAECs were treated with 20 μM rapamycin, everolimus, temsirolimus, ridaforolimus, or an equivalent volume of DMSO for 4 hours. VSV-CoV-2 (50 μL) was added to cells, and infection was measured by GFP expression 24 hours after infection using flow cytometry. ( D ) A549-ACE2 cells were treated with varying concentrations of everolimus or DMSO (equivalent to 30 μM everolimus) for 4 hours. SARS-CoV-2 (nCoV-WA1-2020; MN985325.1) was added to the cells at an MOI of 0.1, and infectious titers were measured in VeroE6 cells by calculating the TCID 50 per milliliter of supernatants recovered 24 hours after infection. TCID 50 (PFU/mL) values are shown. Means and the standard error were calculated from 3–4 experiments. * P < 0.05 and ** P < 0.01, by 1-way ANOVA versus DMSO.

    Article Snippet: Primary hSAECs were purchased from ATCC (PCS-301-010).

    Techniques: Infection, Luciferase, Activity Assay, SDS Page, Western Blot, Expressing, Flow Cytometry