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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

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

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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

Techniques: Expressing, Western Blot

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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

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

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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

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

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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

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

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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

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

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: Among the antihuman ACE2 mAbs reported to work in flow cytometry ( ; ), we identified 3 clones—AC18F (AdipoGen), 535919 (R&D Systems), and Poly5036 (BioLegend—that effectively stained A549-hACE2 cells but not the parental A549 cells ( b).

Techniques: Variant Assay, Infection, Control, Blocking Assay