primary normal human epidermal keratinocytes  (PromoCell)

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture restores gene expression of skin barrier molecules filaggrin and loricrin in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were left unstimulated (control) or stimulated with a psoriasis-like cytokine mixture (IL-1β, IL-17A, IL-22 and TNF-alpha, each 10 ng/mL) either in the absence (Psoriasis) or in the presence of the plant extract (Psoriasis + Extract) for 21 h. Gene expression levels of ( a ) FLG , ( b ) LOR and ( c ) IVL were determined by real-time PCR. Statistical significance was tested by a, c) one-way ANOVA with subsequent Sidak’s multiple comparison test or b) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12 * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Control, Real-time Polymerase Chain Reaction, Comparison

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture downregulates inflammatory markers in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of ( a ) IL1A , ( b ) IL1B, ( c ) IL1RN , ( d ) CXCL8 , ( e ) TNFA , ( f ) IL17C , ( g ) IL36G , ( h ) CSF2 , ( i ) VEGFA were measured. Statistical significance was tested by a, b, c, e, h) one-way ANOVA with subsequent Sidak’s multiple comparison test or d, f, g) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12, * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant). Protein expression levels of ( j ) IL-8 and k) TNFα were measured in the supernatants of the cells by ELISA. Values for unstimulated control and plant extract-treated cells were below the detection limit of 31,3 pg/mL (IL-8) or 62,5 pg/mL (TNFα) and not detectable; therefore, no statistical analysis was performed. n.d.= non-detectable.

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Comparison, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture lowers upregulated antimicrobial peptide expression in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of ( a ) S100A7 , ( c ) DEFB4A and ( e ) DEFB103A were measured. Statistical significance was tested by ( a , c ) one-way ANOVA with subsequent Sidak’s multiple comparison test or ( e ) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12). Protein expression levels of ( b ) psoriasin and ( d ) hBD2 were measured in the supernatants of the cells by ELISA. Statistical significance was tested by b) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12) or d) one-way ANOVA with subsequent Sidak’s multiple comparison test ( n = 6, * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Expressing, Gene Expression, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture reduces NFKBIZ and NFKBIA gene expression and IκBζ protein levels. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of ( a ) NFKBIZ and ( c ) NFKBIA were measured and statistical significance was tested by one-way ANOVA with subsequent Sidak’s multiple comparisons test ( n = 12; * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant). ( b ) Protein expression levels of IκBζ were assessed by western blot using an IκBζ antibody. Detection of pan-actin serves as a loading control. Uncropped blots are shown in Supplementary Figure .

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture activates the AhR in the 2D psoriasis model. ( a ) CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of CYP1A1 were measured and statistical significance was tested by Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12). ( b ) To determine AhR activation, NHEKs were transfected with the pGudLuc6.1 plasmid containing firefly luciferase which expression depends on AhR activation and the pGL4.74 [ hRLuc /TK] reference plasmid containing renilla luciferase. One day after transfection, cells were stimulated as described in ( a ). After cell lysis, activation of AhR was determined by measurement of relative luciferase activities. Statistical significance was tested by one-way ANOVA with subsequent Sidak’s multiple comparison test ( n = 12; ** p < 0.01; *** p < 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Lysis, Comparison

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Downregulation of AhR inhibits the filaggrin-inducing effects but not the anti-inflammatory effects of the plant extract in the 2D psoriasis model. NHEKs were transfected with control or AhR siRNA and stimulated as described in Fig. . Gene expression levels of ( a ) CYP1A1 , ( b ) FLG , ( c ) NFKBIZ , ( d ) TNFA , ( e ) IL36G ( f ) CXCL8 and ( g ) DEFB4A were measured. Statistical significance was determined by ( a – f ) one-way ANOVA with subsequent Sidak’s multiple comparison test or ( c ) Kruskal-Wallis with subsequent Dunn’s multiple comparison test (n = 9; * p < 0.05; ** p < 0.01; *** p 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Transfection, Control, Gene Expression, Comparison

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: The plant extract mixture exhibits antioxidant effects in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . ( a ) Gene expression levels of NQO1 were measured and statistical significance was tested by one-way ANOVA with subsequent Sidak’s multiple comparisons test ( n = 12). ( b ) Intracellular reactive oxygen species were measured by DCFDA-based assay. The mean fluorescence value of the control cells was set to 100%, and the relative intracellular ROS levels of the other samples were calculated as percentages of the control. Statistical significance was tested by Kruskal-Wallis test with subsequent Dunn’s multiple comparison test ( n = 12, * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant). ( c ) NHEKs were transfected and stimulated as described in Fig. . Gene expression levels of NQO1 were measured and statistical significance was determined by one-way ANOVA with subsequent Sidak’s multiple comparison test ( n = 9; * p < 0.05; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Fluorescence, Control, Comparison, Transfection

Journal: bioRxiv

Article Title: Comparative analysis of varicella-zoster virus and herpes simplex virus 1 interaction with epidermal terminal differentiation in primary human keratinocytes models of differentiation

doi: 10.64898/2026.04.08.717198

Figure Lengend Snippet: A. Schematic of epidermal differentiation and the differentiation stages at which viral infections were implemented; undifferentiated (Undiff.), early differentiated (Early diff.) and late differentiated (Late diff.). B. Schematic of plan of infection with VZV at MOI 0.1 of Undiff., Early diff. and Late diff. NHEKs. “Undiff.”, “Early diff.” and “Late diff.” labelling refers to the state of NHEKs differentiation at the time of infection (day 0). The “Undiff.” condition includes the NHEKs undergoing differentiation by calcium switch after infection (Differentiating_sync.) C. Analysis by qPCR of cell-associated VZV genome copy number normalised to number of cells, upon infection with VZV of Undiff., Early diff. and Late diff. NHEKs, and evaluated at 24h and 120h p.i. The data are generated from quantification of the VZV ORF31 gene (gB) normalised to RPP30 gene (host internal control) and are reported as the mean of three technical replicates. D. Analysis by qPCR of cell-associated VZV genome copy number normalised to number of cells upon infection with VZV of Late diff. NHEKs, and evaluated at 5 days (120h) and 7 days p.i. The data are generated from quantification of the VZV ORF31 gene (gB) normalised to RPP30 gene (host internal control) and are reported as the mean of three technical replicates. E. Analysis by qRT-PCR of ORFC2 (encoding the IE62 protein) and ORFC8 (encoding the gE protein) in VZV infection of Undiff., Early diff. and Late diff. NHEKs, and reported at 24h and 120h p.i. The data are reported as mean of 2 -dCT of three technical replicates, where the normalisation was performed against GAPDH . F. Analysis by qRT-PCR of ORFC2 and ORFC8 in VZV infection of Late diff. NHEKs, and evaluated at 120h and 7 days p.i. The data are reported as mean of 2 -dCT of three technical replicates, where the normalisation was performed against GAPDH . G. Western Blotting analysis of gE and IE62 expression at 120h p.i. following infection of Undiff., Early diff. and Late diff. NHEKs with VZV. GAPDH was used as loading control . H. Fast red staining of gE at 120h p.i. following infection of Undiff., Early diff. and Late diff. NHEKs with VZV. Graph shows the quantification of number of VZV plaques in at least two fields of view per condition and is reported as mean ± SD. I. IE62 and gE (green) immunofluorescence staining of Undiff. and Early diff. NHEKs infected with VZV and analysed at 120h p.i. DAPI (blue) was used for staining of nuclei. Graphs show quantification of integrated density of IE62 and gE signals in at least two fields of view per condition ± SD. Data in C., E. and G. are representative of n=3 independent experiments (for the Undiff. and Early Diff. conditions), data in I. are representative of n=2 independent experiments. Statistical significance was evaluated in H. by one-way ANOVA with Dunnett’s multiple comparisons test, in I. by two-tailed t test. Statistical significance is indicated as *P< 0.05, **P< 0.01, ***P< 0.001. Scale bar, 50 μm. Undiff., undifferentiated; Early diff., early differentiated; Late diff., late differentiated; p.i., post infection; NHEKs, normal human epidermal keratinocytes.

Article Snippet: Primary normal human epidermal keratinocytes (NHEKs) isolated from juvenile foreskin and pooled from different donors were purchased from Promocell or Thermo Fisher Scientific and grown are previously described [ , ].

Techniques: Infection, Generated, Control, Quantitative RT-PCR, Western Blot, Expressing, Staining, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Comparative analysis of varicella-zoster virus and herpes simplex virus 1 interaction with epidermal terminal differentiation in primary human keratinocytes models of differentiation

doi: 10.64898/2026.04.08.717198

Figure Lengend Snippet: A. Schematic of plan of infection with HSV-1 at MOI 0.005 of undifferentiated (Undiff.), early differentiated (Early diff.) and late differentiated (Late diff.) NHEKs. “Undiff.”, “Early diff.” and “Late diff.” labelling refers to the state of NHEKs differentiation at the time of infection (day 0). The “Undiff.” condition includes the NHEKs undergoing spontaneous asynchronous differentiation after infection (Differentiating_async.) B. Analysis by qPCR of cell-associated HSV-1 genome copy number normalised to number of cells, upon infection with HSV-1 of Undiff., Early diff. and Late diff. NHEKs, and evaluated at 24h, 48h and 72h p.i. The data are generated from quantification of the HSV-1 UL27 gene (gB) normalised to RPP30 gene (host internal control) and are reported as mean of n=3 independent experiments ± SEM. C. Analysis by qPCR of cell-associated HSV-1 genome copy number normalised to number of cells, upon infection with HSV-1 of Late diff. NHEKs, and reported at 72h and 120h p.i. The data are generated from quantification of the HSV-1 UL27 gene (gB) normalised to RPP30 gene (host internal control) and are reported as the mean of three technical replicates. D. ICP0 (pink) immunofluorescence staining of Undiff., Early diff. and Late diff. NHEKs infected with HSV-1 and analysed at 4h, 24h and 72h p.i. The reported images include GFP (green) signal deriving from the GFP protein tagged to the late HSV-1 tegument protein UL46 and DAPI (grey) staining of nuclei. E. Analysis by qRT-PCR analysis of RL2 gene (encoding the ICP0 protein) and UL48 gene (encoding the VP16 protein) expression in HSV-1 infection of Undiff., Early diff. and Late diff. NHEKs, and reported at 24h, 48h and 72h p.i. The data are reported as mean of 2 -dCT of n=3 independent experiments ± SEM, where the normalisation was performed against GAPDH . F. Western Blotting analysis of ICP0 and VP16 expression at 24h, 48h and 72h p.i. following infection of Undiff., Early diff. and Late diff. NHEKs with HSV-1. GAPDH was used as loading control. Data are representative of n=4 independent experiments (Undiff. condition), n=3 independent experiments (Early diff. condition) and n=2 independent experiments (Late diff. condition). G. Schematic of the model of HSV-1 infection implemented in undifferentiated NHEKs, which either remained undifferentiated throughout the infection and until the last time point (72h) (Undifferentiated) or started undergoing spontaneous differentiation during the last time points of infection (Differentiating_async). H. Analysis by qRT-PCR of KRT5 and KRT10 mRNAs in uninfected NHEKs at 72h confirming the undifferentiated and differentiating status of the keratinocytes. The data are reported as fold change (FC) (2 -ddCT ) to the undifferentiated condition and are the mean of three technical replicates, where the normalisation was performed against GAPDH . I. Keratinocytes where infected when undifferentiated and then either maintained their undifferentiated status throughout the infection (Undifferentiated) or were allowed to spontaneously differentiate by 72h p.i. (Differentiating). Cell-associated HSV-1 genome copy number was analysed at 72h p.i. in both conditions. The data are generated from quantification of the HSV-1 UL27 gene (gB) normalised to RPP30 gene (host internal control), are reported as the mean of three technical replicates and are representative of n=3 independent experiments. J. Anlysis by qRT-PCR of RL2 and UL48 mRNAs in HSV-1 infected Undifferentiated and Differentiating NHEKs at 72h p.i. The data are reported as mean of 2 -dCT of three technical replicates, where the normalisation was performed against GAPDH . Data are representative of n=3 independent experiments. Statistical significance was evaluated in B. and E. by 2way ANOVA with Tukey’s multiple comparisons test and indicated as *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001. In D. , dotted lines mark nuclei at 4h p.i. Insets contain zoomed-in areas of the ICP0 signal inside the nuclei at 4h p.i. Scale bar, 10 μm. Undiff., undifferentiated; Early diff., early differentiated; Late diff., late differentiated; p.i., post infection; NHEKs, normal human epidermal keratinocytes.

Article Snippet: Primary normal human epidermal keratinocytes (NHEKs) isolated from juvenile foreskin and pooled from different donors were purchased from Promocell or Thermo Fisher Scientific and grown are previously described [ , ].

Techniques: Infection, Generated, Control, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Comparative analysis of varicella-zoster virus and herpes simplex virus 1 interaction with epidermal terminal differentiation in primary human keratinocytes models of differentiation

doi: 10.64898/2026.04.08.717198

Figure Lengend Snippet: A. Analysis of qRT-PCR expression of KRT10 (encoding the K10 protein) in VZV infection (at the MOI of 0.1) of Undiff., Early diff. and Late diff. NHEKs, and reported at 24h, 120h p.i. and at 7 days p.i. (in the Late diff.). The data are reported as fold change (FC) (2 -ddCT ) to each uninfected control from the mean of three technical replicates, where the normalisation was performed against GAPDH . Similar trends were observed in other 2 independent experiments for the Undiff. and Early Diff. conditions using different MOIs. B. Western Blotting analysis of K10 expression at 120h p.i. following infection of Undiff., Early diff. and Late diff. NHEKs with VZV at the MOI of 0.1. GAPDH was used as loading control. Numbers at the bottom of the blots indicate K10 densitometry expressed as fold change to each uninfected control, following normalisation against GAPDH . Data are representative of n=3 independent experiments for the Undiff. and Early Diff. conditions. C. K10 (red) and gE (green) immunofluorescence staining of Undiff., Early diff. and Late diff. NHEKs infected with VZV at the MOI of 0.1 and analysed at 120h p.i. DAPI (blue) was used for staining of nuclei. Data are representative of n=2 independent experiments for the Undiff. and Early Diff. conditions. D. Analysis by qRT-PCR of KRT10 mRNA expression in HSV-1 infection (at the MOI of 0.005) of Undiff., Early diff. and Late diff. NHEKs, and reported at 24h, 48h and 72h p.i. The data are reported as fold change (FC) (2 -ddCT ) to each uninfected control from the mean of n=3 independent experiments ± SEM, where the normalisation was performed against GAPDH . E. Western Blotting analysis of K10 expression at 24h, 48h and 72h p.i. following infection of Undiff., Early diff. and Late diff. NHEKs with HSV-1 at the MOI of 0.005. GAPDH was used as loading control. Data are representative of n=4 independent experiments for the Undiff. condition, n=3 independent experiments for the Early diff. condition and n=2 independent experiments for the Late diff. condition. In the Undiff. and Early diff. conditions 16μg of proteins were run. In the Late diff. condition 5μg of proteins were run to compensate for the high concentration of K10 in this condition. F. K10 (yellow) and VP16 (pink) immunofluorescence staining, of Undiff., Early diff. and Late diff. NHEKs infected with HSV-1 at the MOI of 0.005, and analysed at 72h p.i. DAPI (blue) was used for staining of nuclei. G. Analysis by qRT-PCR at 72h p.i. of expression of KRT10 mRNA in HSV-1 infection (at the MOI of 0.005) of Undiff. NHEKs treated either with foscarnet or with vehicle alone at the time of infection. The data are reported as fold change (FC) (2 -ddCT ) to each uninfected control, from the mean of three technical replicates, where the normalisation was performed against GAPDH . The data are representative of n=2 independent experiments. H. Keratin 10 (green) and gE (red) immunofluorescence staining of ex-vivo human skin tissues infected with VZV (10 4 PFU) and analysed 11 days p.i. DAPI (blue) was used for staining of nuclei. I. Keratin 10 (red) immunofluorescence staining of ex-vivo human skin tissues infected with HSV-1 (10 4 PFU) and analysed 11 days p.i. GFP signal (green) marks the expression of the GFP-tagged late HSV-1 protein UL46. DAPI (blue) was used for staining of nuclei. In H. and I. dotted lines indicate the epidermal-dermal junction. Scale bars, 50 μm. Undiff., undifferentiated; Early diff., early differentiated; Late diff., late differentiated; p.i., post infection; UNINF, uninfected.

Article Snippet: Primary normal human epidermal keratinocytes (NHEKs) isolated from juvenile foreskin and pooled from different donors were purchased from Promocell or Thermo Fisher Scientific and grown are previously described [ , ].

Techniques: Quantitative RT-PCR, Expressing, Infection, Control, Western Blot, Immunofluorescence, Staining, Concentration Assay, Ex Vivo