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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