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Quantification of <t>ACE2</t> , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.
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gene exp ace2 hs01085333 m1  (Thermo Fisher)
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Quantification of <t>ACE2</t> , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.
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Quantification of <t>ACE2</t> , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.
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Quantification of <t>ACE2</t> , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.
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Quantification of <t>ACE2</t> , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.
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Humoral epitope dominance from immunization of C57BL/6 or Ig‐humanized mice with CoV‐1 or CoV‐2 Spike MVA. C57BL/6 and Ig‐humanized mice were immunized with 2 × 10 8 PFU IV of CoV‐1, CoV‐2 or control MVA on days 0 and 14 and the immune response examined on day 28. (a) Composite model of the CoV‐2 RBD (gray, PDB 7msq) showing the co‐binding of ACE2 to the Class 1/2 epitope (blue, PDB 6m0j), S309 antibody Fab to the Class 3 epitope (orange, PDB 7r6w), EY6A antibody Fab to the Class 4 epitope (purple, PDB 6zcz), and S2H97 antibody Fab to the Class 5 epitope (green, PDB 7m7w). (b) Representative flow cytometric plots of spleen B cells from a CR3022 immunoglobulin transgenic mouse expressing membrane IgM encoding the Class 4 antibody on most B cells, showing staining with CoV‐2 RBD followed by a cocktail of each of the fluorescent antibodies <t>and</t> <t>ACE2‐Fc</t> indicated on the Y and Y axis. The gating strategy applied to identify B cells that bind CoV‐2 RBD (red box) through epitopes competed by the indicated fluorescent conjugates (dashed boxes) is shown. (c) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (d) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (e) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (f) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (g) Mean Fluorescence Intensity (MFI) of serum antibody response blocking the binding of fluorescent ACE2 (Class1/2) or canonical antibodies to the Class 3, 4, or 5 epitopes to CoV‐2 conjugated to the surface of sheep red blood cells. Data points represent individual mice. Columns show mean ± SEM. A two‐way ANOVA was applied to figures A‐K and a one‐way ANOVA to figure H. If ANOVA revealed significant differences, unpaired t ‐tests were applied to compare between different groups. Welch's correction was applied if variances between groups were statistically different. ns = not significant * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data pooled from two independent experiments.
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Quantification of ACE2 , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Quantification of ACE2 , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Gene Expression, Quantitative RT-PCR, Cell Culture

Differential ACE2, TMPRSS2, NRP1, CTSL, CD147, AXL, and DPP4 protein expression. Whole-cell lysates of the indicated cells were analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone 14H4), CTSL (mAb clone 33/1), CD147 (mAb clone HIM6), AXL (polyclonal goat IgG, AF154), and DPP4 (polyclonal goat IgG, AF1180). ( a ) Representative immunoblots from 2 to 4 independent experiments. ( b ) Densitometric quantification normalized to actin, expressed as mean ± SEM from 2 to 4 independent experiments. CTSL, cathepsin L.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Differential ACE2, TMPRSS2, NRP1, CTSL, CD147, AXL, and DPP4 protein expression. Whole-cell lysates of the indicated cells were analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone 14H4), CTSL (mAb clone 33/1), CD147 (mAb clone HIM6), AXL (polyclonal goat IgG, AF154), and DPP4 (polyclonal goat IgG, AF1180). ( a ) Representative immunoblots from 2 to 4 independent experiments. ( b ) Densitometric quantification normalized to actin, expressed as mean ± SEM from 2 to 4 independent experiments. CTSL, cathepsin L.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Expressing, Western Blot

Validation of flow cytometry protocols and anti-ACE2 antibodies. ( a ) Comparison of cell detachment protocols. ( b ) Comparison of anti-ACE2 antibody staining by flow cytometry. ( c, d ) Binding of 2019-nCoV Spike Protein S1 (RBD) protein and blocking with anti-ACE2 antibody (mAb clone AC384) assessed by ( c ) flow cytometry and ( d ) immunofluorescence (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of 3 independent experiments. ( e ) Cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in A549 cells. Data are representative of 2 independent experiments.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Validation of flow cytometry protocols and anti-ACE2 antibodies. ( a ) Comparison of cell detachment protocols. ( b ) Comparison of anti-ACE2 antibody staining by flow cytometry. ( c, d ) Binding of 2019-nCoV Spike Protein S1 (RBD) protein and blocking with anti-ACE2 antibody (mAb clone AC384) assessed by ( c ) flow cytometry and ( d ) immunofluorescence (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of 3 independent experiments. ( e ) Cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in A549 cells. Data are representative of 2 independent experiments.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Biomarker Discovery, Flow Cytometry, Comparison, Staining, Binding Assay, Blocking Assay, Immunofluorescence, Expressing

Cell surface expression of ACE2, TMPRSS2, NRP1, CD147, AXL, and DPP4 receptors is associated with exclusive binding of SARS-CoV-2 Spike Protein S1 (RBD) to ACE2-expressing cells. ( a ) Flow cytometry analysis of cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in the indicated cell lines and primary skin cells. Data are representative of 2–4 independent experiments. ( b, c ) Representative immunofluorescence microscopy images showing ( b ) ACE2 expression (mAb clone Poly5036) and ( c ) binding of 2019-nCoV Spike Protein S1 (RBD) in red. Cell nuclei are stained with DAPI (white). Bar = 10 μm. The proportions of cells expressing ACE2 and binding to spike are shown. Data are representative of at least 2 independent experiments. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Cell surface expression of ACE2, TMPRSS2, NRP1, CD147, AXL, and DPP4 receptors is associated with exclusive binding of SARS-CoV-2 Spike Protein S1 (RBD) to ACE2-expressing cells. ( a ) Flow cytometry analysis of cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in the indicated cell lines and primary skin cells. Data are representative of 2–4 independent experiments. ( b, c ) Representative immunofluorescence microscopy images showing ( b ) ACE2 expression (mAb clone Poly5036) and ( c ) binding of 2019-nCoV Spike Protein S1 (RBD) in red. Cell nuclei are stained with DAPI (white). Bar = 10 μm. The proportions of cells expressing ACE2 and binding to spike are shown. Data are representative of at least 2 independent experiments. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Expressing, Binding Assay, Flow Cytometry, Immunofluorescence, Microscopy, Staining

Differentiation of human keratinocytes moderately upregulates ACE2. NHEKs and N/TERT-2G cells were cultured as undifferentiated cells (denoted as undiff.) or differentiated cells (denoted as Diff.). ( a ) ACE2 and K RT 10 mRNA levels relative to control (2 -ΔΔCT ), quantified by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 2 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) ACE2, CTSL, and K10 protein expression analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), CTSL (mAb clone 33/1), and K10 (rabbit polyclonal Poly19054) in ( b ) NHEK and ( c ) N/TERT-2G cells. Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. CTSL, cathepsin L; K, keratin; NHEK, normal human epidermal keratinocyte.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Differentiation of human keratinocytes moderately upregulates ACE2. NHEKs and N/TERT-2G cells were cultured as undifferentiated cells (denoted as undiff.) or differentiated cells (denoted as Diff.). ( a ) ACE2 and K RT 10 mRNA levels relative to control (2 -ΔΔCT ), quantified by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 2 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) ACE2, CTSL, and K10 protein expression analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), CTSL (mAb clone 33/1), and K10 (rabbit polyclonal Poly19054) in ( b ) NHEK and ( c ) N/TERT-2G cells. Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. CTSL, cathepsin L; K, keratin; NHEK, normal human epidermal keratinocyte.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Cell Culture, Control, Quantitative RT-PCR, Expressing, Western Blot

TLR3-mediated activation upregulates ACE2 expression in human epidermal keratinocytes. NHEK and N/TERT-2G cells were stimulated with Poly (I:C) and IFN-α + β for 24 hours. ( a ) ACE2 and CTSL mRNA levels relative to control (2 -ΔΔCT ), measured by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 3 independent experiments. Data were analyzed using the Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) Western blot analysis of ACE2 (mAb clone AC384) and CTSL (mAb clone 33/1). Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. ( d ) Western blot analysis of TMPRSS2 (mAb clone S20014A) expression. Shown is immunoblot representative of 2 independent experiments. ( e ) Representative immunofluorescence microscopy images showing ACE2 expression (mAb clone Poly5036) (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of at least 2 independent experiments. CTSL, cathepsin L; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; TLR3, toll-like receptor 3.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: TLR3-mediated activation upregulates ACE2 expression in human epidermal keratinocytes. NHEK and N/TERT-2G cells were stimulated with Poly (I:C) and IFN-α + β for 24 hours. ( a ) ACE2 and CTSL mRNA levels relative to control (2 -ΔΔCT ), measured by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 3 independent experiments. Data were analyzed using the Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) Western blot analysis of ACE2 (mAb clone AC384) and CTSL (mAb clone 33/1). Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. ( d ) Western blot analysis of TMPRSS2 (mAb clone S20014A) expression. Shown is immunoblot representative of 2 independent experiments. ( e ) Representative immunofluorescence microscopy images showing ACE2 expression (mAb clone Poly5036) (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of at least 2 independent experiments. CTSL, cathepsin L; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; TLR3, toll-like receptor 3.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Activation Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Microscopy, Staining

SARS-CoV-2 Delta variant (B.1.617.2) binds to human epidermal keratinocytes but does not replicate. ( a ) Experimental design of SARS-CoV-2 Delta variant (B.1.617.2) infection. ( b, c ) A549-hACE2, ( b ) NHEK, and ( c ) N/TERT-2G cells unstimulated or stimulated with Poly (I:C) were noninfected (denoted as NI) or infected for 48 hours at the indicated MOI with SARS-CoV-2 Delta variant (B.1.617.2). SARS-CoV-2 E gene RNA levels normalized to GAPDH (2 -ΔCT ) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition with the noninfected control (denoted as NI). Only significant P -values ( P < .05) are indicated above the infected conditions in the figure. ∗∗∗∗ P < .0001, ∗∗∗ P < .001, and ∗∗ P < .01. ( d ) A549-hACE2, NHEK, and N/TERT-2G cells were noninfected (denoted as NI) or infected for 72 hours with SARS-CoV-2 Delta variant (B.1.617.2) in the absence or presence of antiviral drugs or blocking anti-ACE2 mAb (clone AC384) as indicated. SARS-CoV-2 RNA gene E (copy/μl) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition without drug with the noninfected DMSO control and comparing each infected condition with drug with the corresponding infected condition without drug. Only significant P -values are indicated above the infected conditions in the figure. ∗∗ P < .001 and ∗ P < .05. MOI, multiplicity of infection; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: SARS-CoV-2 Delta variant (B.1.617.2) binds to human epidermal keratinocytes but does not replicate. ( a ) Experimental design of SARS-CoV-2 Delta variant (B.1.617.2) infection. ( b, c ) A549-hACE2, ( b ) NHEK, and ( c ) N/TERT-2G cells unstimulated or stimulated with Poly (I:C) were noninfected (denoted as NI) or infected for 48 hours at the indicated MOI with SARS-CoV-2 Delta variant (B.1.617.2). SARS-CoV-2 E gene RNA levels normalized to GAPDH (2 -ΔCT ) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition with the noninfected control (denoted as NI). Only significant P -values ( P < .05) are indicated above the infected conditions in the figure. ∗∗∗∗ P < .0001, ∗∗∗ P < .001, and ∗∗ P < .01. ( d ) A549-hACE2, NHEK, and N/TERT-2G cells were noninfected (denoted as NI) or infected for 72 hours with SARS-CoV-2 Delta variant (B.1.617.2) in the absence or presence of antiviral drugs or blocking anti-ACE2 mAb (clone AC384) as indicated. SARS-CoV-2 RNA gene E (copy/μl) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition without drug with the noninfected DMSO control and comparing each infected condition with drug with the corresponding infected condition without drug. Only significant P -values are indicated above the infected conditions in the figure. ∗∗ P < .001 and ∗ P < .05. MOI, multiplicity of infection; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Variant Assay, Infection, Control, Blocking Assay

Quantification of ACE2 , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Quantification of ACE2 , dACE2 , TMPRSS2, NRP1, CTSL , BSG (CD147), DPP4 (CD26), AXL , KREMEN1 , and ASGR1 gene expression. mRNA levels normalized to GAPDH (2 -ΔCT ) in ( a ) NHDF, NHEKs, RHE, and human skin samples and in ( b ) Caco-2/TC-7, Calu-3, and HaCaT cell lines. Gene expression was quantified by RT-qPCR in triplicate. Data represent the mean ± SEM from ( a ) 3 to 14 independent experiments using biological samples from different individuals and ( b ) 3 or 4 independent experiments from cultured cells. NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; RHE, reconstructed human epidermis.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Gene Expression, Quantitative RT-PCR, Cell Culture

Differential ACE2, TMPRSS2, NRP1, CTSL, CD147, AXL, and DPP4 protein expression. Whole-cell lysates of the indicated cells were analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone 14H4), CTSL (mAb clone 33/1), CD147 (mAb clone HIM6), AXL (polyclonal goat IgG, AF154), and DPP4 (polyclonal goat IgG, AF1180). ( a ) Representative immunoblots from 2 to 4 independent experiments. ( b ) Densitometric quantification normalized to actin, expressed as mean ± SEM from 2 to 4 independent experiments. CTSL, cathepsin L.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Differential ACE2, TMPRSS2, NRP1, CTSL, CD147, AXL, and DPP4 protein expression. Whole-cell lysates of the indicated cells were analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone 14H4), CTSL (mAb clone 33/1), CD147 (mAb clone HIM6), AXL (polyclonal goat IgG, AF154), and DPP4 (polyclonal goat IgG, AF1180). ( a ) Representative immunoblots from 2 to 4 independent experiments. ( b ) Densitometric quantification normalized to actin, expressed as mean ± SEM from 2 to 4 independent experiments. CTSL, cathepsin L.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Expressing, Western Blot

Validation of flow cytometry protocols and anti-ACE2 antibodies. ( a ) Comparison of cell detachment protocols. ( b ) Comparison of anti-ACE2 antibody staining by flow cytometry. ( c, d ) Binding of 2019-nCoV Spike Protein S1 (RBD) protein and blocking with anti-ACE2 antibody (mAb clone AC384) assessed by ( c ) flow cytometry and ( d ) immunofluorescence (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of 3 independent experiments. ( e ) Cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in A549 cells. Data are representative of 2 independent experiments.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Validation of flow cytometry protocols and anti-ACE2 antibodies. ( a ) Comparison of cell detachment protocols. ( b ) Comparison of anti-ACE2 antibody staining by flow cytometry. ( c, d ) Binding of 2019-nCoV Spike Protein S1 (RBD) protein and blocking with anti-ACE2 antibody (mAb clone AC384) assessed by ( c ) flow cytometry and ( d ) immunofluorescence (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of 3 independent experiments. ( e ) Cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in A549 cells. Data are representative of 2 independent experiments.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Biomarker Discovery, Flow Cytometry, Comparison, Staining, Binding Assay, Blocking Assay, Immunofluorescence, Expressing

Cell surface expression of ACE2, TMPRSS2, NRP1, CD147, AXL, and DPP4 receptors is associated with exclusive binding of SARS-CoV-2 Spike Protein S1 (RBD) to ACE2-expressing cells. ( a ) Flow cytometry analysis of cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in the indicated cell lines and primary skin cells. Data are representative of 2–4 independent experiments. ( b, c ) Representative immunofluorescence microscopy images showing ( b ) ACE2 expression (mAb clone Poly5036) and ( c ) binding of 2019-nCoV Spike Protein S1 (RBD) in red. Cell nuclei are stained with DAPI (white). Bar = 10 μm. The proportions of cells expressing ACE2 and binding to spike are shown. Data are representative of at least 2 independent experiments. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Cell surface expression of ACE2, TMPRSS2, NRP1, CD147, AXL, and DPP4 receptors is associated with exclusive binding of SARS-CoV-2 Spike Protein S1 (RBD) to ACE2-expressing cells. ( a ) Flow cytometry analysis of cell surface expression of ACE2 (mAb clone Poly5036), TMPRSS2 (mAb clone S20014A), NRP1 (mAb clone U21-1283), CD147 (mAb clone HIM6), AXL (mAb clone 108724), and DPP4 (mAb clone BA5b) in the indicated cell lines and primary skin cells. Data are representative of 2–4 independent experiments. ( b, c ) Representative immunofluorescence microscopy images showing ( b ) ACE2 expression (mAb clone Poly5036) and ( c ) binding of 2019-nCoV Spike Protein S1 (RBD) in red. Cell nuclei are stained with DAPI (white). Bar = 10 μm. The proportions of cells expressing ACE2 and binding to spike are shown. Data are representative of at least 2 independent experiments. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Expressing, Binding Assay, Flow Cytometry, Immunofluorescence, Microscopy, Staining

Differentiation of human keratinocytes moderately upregulates ACE2. NHEKs and N/TERT-2G cells were cultured as undifferentiated cells (denoted as undiff.) or differentiated cells (denoted as Diff.). ( a ) ACE2 and K RT 10 mRNA levels relative to control (2 -ΔΔCT ), quantified by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 2 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) ACE2, CTSL, and K10 protein expression analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), CTSL (mAb clone 33/1), and K10 (rabbit polyclonal Poly19054) in ( b ) NHEK and ( c ) N/TERT-2G cells. Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. CTSL, cathepsin L; K, keratin; NHEK, normal human epidermal keratinocyte.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: Differentiation of human keratinocytes moderately upregulates ACE2. NHEKs and N/TERT-2G cells were cultured as undifferentiated cells (denoted as undiff.) or differentiated cells (denoted as Diff.). ( a ) ACE2 and K RT 10 mRNA levels relative to control (2 -ΔΔCT ), quantified by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 2 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) ACE2, CTSL, and K10 protein expression analyzed by western blot using the following antibodies: ACE2 (mAb clone AC384), CTSL (mAb clone 33/1), and K10 (rabbit polyclonal Poly19054) in ( b ) NHEK and ( c ) N/TERT-2G cells. Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using the Wilcoxon signed-rank test to compare the 2 conditions. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. CTSL, cathepsin L; K, keratin; NHEK, normal human epidermal keratinocyte.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Cell Culture, Control, Quantitative RT-PCR, Expressing, Western Blot

TLR3-mediated activation upregulates ACE2 expression in human epidermal keratinocytes. NHEK and N/TERT-2G cells were stimulated with Poly (I:C) and IFN-α + β for 24 hours. ( a ) ACE2 and CTSL mRNA levels relative to control (2 -ΔΔCT ), measured by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 3 independent experiments. Data were analyzed using the Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) Western blot analysis of ACE2 (mAb clone AC384) and CTSL (mAb clone 33/1). Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. ( d ) Western blot analysis of TMPRSS2 (mAb clone S20014A) expression. Shown is immunoblot representative of 2 independent experiments. ( e ) Representative immunofluorescence microscopy images showing ACE2 expression (mAb clone Poly5036) (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of at least 2 independent experiments. CTSL, cathepsin L; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; TLR3, toll-like receptor 3.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: TLR3-mediated activation upregulates ACE2 expression in human epidermal keratinocytes. NHEK and N/TERT-2G cells were stimulated with Poly (I:C) and IFN-α + β for 24 hours. ( a ) ACE2 and CTSL mRNA levels relative to control (2 -ΔΔCT ), measured by RT-qPCR in triplicate and normalized to GAPDH. Data represent the mean ± SEM from 3 independent experiments. Data were analyzed using the Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ( b, c ) Western blot analysis of ACE2 (mAb clone AC384) and CTSL (mAb clone 33/1). Shown are representative immunoblots and densitometric quantification, normalized to actin and expressed as mean ± SEM from 3 to 6 independent experiments. Data were analyzed using Wilcoxon signed-rank test, comparing each stimulation condition with the unstimulated control. Only significant P -values ( P < .05) are indicated in the figure. ∗ P < .05. ( d ) Western blot analysis of TMPRSS2 (mAb clone S20014A) expression. Shown is immunoblot representative of 2 independent experiments. ( e ) Representative immunofluorescence microscopy images showing ACE2 expression (mAb clone Poly5036) (red). Cell nuclei are stained with DAPI (white). Bar = 10 μm. Data are representative of at least 2 independent experiments. CTSL, cathepsin L; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; TLR3, toll-like receptor 3.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Activation Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Microscopy, Staining

SARS-CoV-2 Delta variant (B.1.617.2) binds to human epidermal keratinocytes but does not replicate. ( a ) Experimental design of SARS-CoV-2 Delta variant (B.1.617.2) infection. ( b, c ) A549-hACE2, ( b ) NHEK, and ( c ) N/TERT-2G cells unstimulated or stimulated with Poly (I:C) were noninfected (denoted as NI) or infected for 48 hours at the indicated MOI with SARS-CoV-2 Delta variant (B.1.617.2). SARS-CoV-2 E gene RNA levels normalized to GAPDH (2 -ΔCT ) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition with the noninfected control (denoted as NI). Only significant P -values ( P < .05) are indicated above the infected conditions in the figure. ∗∗∗∗ P < .0001, ∗∗∗ P < .001, and ∗∗ P < .01. ( d ) A549-hACE2, NHEK, and N/TERT-2G cells were noninfected (denoted as NI) or infected for 72 hours with SARS-CoV-2 Delta variant (B.1.617.2) in the absence or presence of antiviral drugs or blocking anti-ACE2 mAb (clone AC384) as indicated. SARS-CoV-2 RNA gene E (copy/μl) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition without drug with the noninfected DMSO control and comparing each infected condition with drug with the corresponding infected condition without drug. Only significant P -values are indicated above the infected conditions in the figure. ∗∗ P < .001 and ∗ P < .05. MOI, multiplicity of infection; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Journal: JID Innovations

Article Title: Human keratinocytes exhibit limited potential for SARS-CoV-2 infection despite ACE2 and mature cathepsin L expression

doi: 10.1016/j.xjidi.2025.100447

Figure Lengend Snippet: SARS-CoV-2 Delta variant (B.1.617.2) binds to human epidermal keratinocytes but does not replicate. ( a ) Experimental design of SARS-CoV-2 Delta variant (B.1.617.2) infection. ( b, c ) A549-hACE2, ( b ) NHEK, and ( c ) N/TERT-2G cells unstimulated or stimulated with Poly (I:C) were noninfected (denoted as NI) or infected for 48 hours at the indicated MOI with SARS-CoV-2 Delta variant (B.1.617.2). SARS-CoV-2 E gene RNA levels normalized to GAPDH (2 -ΔCT ) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition with the noninfected control (denoted as NI). Only significant P -values ( P < .05) are indicated above the infected conditions in the figure. ∗∗∗∗ P < .0001, ∗∗∗ P < .001, and ∗∗ P < .01. ( d ) A549-hACE2, NHEK, and N/TERT-2G cells were noninfected (denoted as NI) or infected for 72 hours with SARS-CoV-2 Delta variant (B.1.617.2) in the absence or presence of antiviral drugs or blocking anti-ACE2 mAb (clone AC384) as indicated. SARS-CoV-2 RNA gene E (copy/μl) were calculated from triplicates of 1 or 2 replicates and expressed as mean values ± SD. Data were analyzed using the Kruskal–Wallis 1-way ANOVA test, comparing each infected condition without drug with the noninfected DMSO control and comparing each infected condition with drug with the corresponding infected condition without drug. Only significant P -values are indicated above the infected conditions in the figure. ∗∗ P < .001 and ∗ P < .05. MOI, multiplicity of infection; NHEK, normal human epidermal keratinocyte; Poly (I:C), polyinosinic:polycytidylic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: The following TaqMan MGB-FAM-labelled probes (Applied Biosystems, Thermo Fisher Scientific) were used for qPCR analysis: ACE2 (Hs01085340_m1), ACE2 + dACE2 (Hs01085333_m1), TMPRSS2 (Hs01120965_m1), NRP1 (Hs00826128_m1), CTSL (Hs00964650_m1), BSG (CD147) (Hs00936295_m1), DPP4 (Hs00897386_m1), AXL (Hs01064444_m1), KREMEN1 (Hs00230750_m1), ASGR1 (Hs01005019_m1), and reference gene GAPDH (Hs02758991_g1).

Techniques: Variant Assay, Infection, Control, Blocking Assay

Humoral epitope dominance from immunization of C57BL/6 or Ig‐humanized mice with CoV‐1 or CoV‐2 Spike MVA. C57BL/6 and Ig‐humanized mice were immunized with 2 × 10 8 PFU IV of CoV‐1, CoV‐2 or control MVA on days 0 and 14 and the immune response examined on day 28. (a) Composite model of the CoV‐2 RBD (gray, PDB 7msq) showing the co‐binding of ACE2 to the Class 1/2 epitope (blue, PDB 6m0j), S309 antibody Fab to the Class 3 epitope (orange, PDB 7r6w), EY6A antibody Fab to the Class 4 epitope (purple, PDB 6zcz), and S2H97 antibody Fab to the Class 5 epitope (green, PDB 7m7w). (b) Representative flow cytometric plots of spleen B cells from a CR3022 immunoglobulin transgenic mouse expressing membrane IgM encoding the Class 4 antibody on most B cells, showing staining with CoV‐2 RBD followed by a cocktail of each of the fluorescent antibodies and ACE2‐Fc indicated on the Y and Y axis. The gating strategy applied to identify B cells that bind CoV‐2 RBD (red box) through epitopes competed by the indicated fluorescent conjugates (dashed boxes) is shown. (c) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (d) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (e) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (f) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (g) Mean Fluorescence Intensity (MFI) of serum antibody response blocking the binding of fluorescent ACE2 (Class1/2) or canonical antibodies to the Class 3, 4, or 5 epitopes to CoV‐2 conjugated to the surface of sheep red blood cells. Data points represent individual mice. Columns show mean ± SEM. A two‐way ANOVA was applied to figures A‐K and a one‐way ANOVA to figure H. If ANOVA revealed significant differences, unpaired t ‐tests were applied to compare between different groups. Welch's correction was applied if variances between groups were statistically different. ns = not significant * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data pooled from two independent experiments.

Journal: Immunology and Cell Biology

Article Title: Humoral epitope dominance and immune imprinting by SARS ‐ CoV ‐1 and SARS ‐ CoV ‐2 vaccines

doi: 10.1111/imcb.70072

Figure Lengend Snippet: Humoral epitope dominance from immunization of C57BL/6 or Ig‐humanized mice with CoV‐1 or CoV‐2 Spike MVA. C57BL/6 and Ig‐humanized mice were immunized with 2 × 10 8 PFU IV of CoV‐1, CoV‐2 or control MVA on days 0 and 14 and the immune response examined on day 28. (a) Composite model of the CoV‐2 RBD (gray, PDB 7msq) showing the co‐binding of ACE2 to the Class 1/2 epitope (blue, PDB 6m0j), S309 antibody Fab to the Class 3 epitope (orange, PDB 7r6w), EY6A antibody Fab to the Class 4 epitope (purple, PDB 6zcz), and S2H97 antibody Fab to the Class 5 epitope (green, PDB 7m7w). (b) Representative flow cytometric plots of spleen B cells from a CR3022 immunoglobulin transgenic mouse expressing membrane IgM encoding the Class 4 antibody on most B cells, showing staining with CoV‐2 RBD followed by a cocktail of each of the fluorescent antibodies and ACE2‐Fc indicated on the Y and Y axis. The gating strategy applied to identify B cells that bind CoV‐2 RBD (red box) through epitopes competed by the indicated fluorescent conjugates (dashed boxes) is shown. (c) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (d) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual C57BL/6 mice immunized with CoV‐2 MVA or CoV‐1 MVA. (e) Percentage of CoV‐2 RBD‐binding B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (f) Percentage of total B cells from the GC (left) or PB (right) compartment which specifically block staining with fluorescent conjugates against the Class 1/2, Class 3, Class 4, or Class 5 epitope, in individual Ig‐humanized mice immunized with CoV‐2 MVA or CoV‐1 MVA. (g) Mean Fluorescence Intensity (MFI) of serum antibody response blocking the binding of fluorescent ACE2 (Class1/2) or canonical antibodies to the Class 3, 4, or 5 epitopes to CoV‐2 conjugated to the surface of sheep red blood cells. Data points represent individual mice. Columns show mean ± SEM. A two‐way ANOVA was applied to figures A‐K and a one‐way ANOVA to figure H. If ANOVA revealed significant differences, unpaired t ‐tests were applied to compare between different groups. Welch's correction was applied if variances between groups were statistically different. ns = not significant * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data pooled from two independent experiments.

Article Snippet: CoV‐2 variant and CoV‐1 RBD proteins and ACE2‐Fc were ordered from Sinobiological.

Techniques: Control, Binding Assay, Transgenic Assay, Expressing, Membrane, Staining, Blocking Assay, Fluorescence