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
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![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).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5211/pmc13125211/pmc13125211__41598_2026_50000_Fig5_HTML.jpg)
