primary normal human epidermal keratinocytes nheks  (PromoCell)

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

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: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Western blot showing knockdown of endogenous RHBDL2 protein in HaCaT cells by multiple shRNAs (01, 00, 02) compared with wild-type (WT) and vector control (V) cells. B. Statistics of the duplicate proteomics experiments. Numbers of identified and quantified proteins ranked by their topology are shown for each of the SILAC experiments, their overlap and union. Type I membrane proteins (with a signal peptide and a single transmembrane helix), which are potential rhomboid substrates, represent about 25% of the secretome in each case and are shown in pale green. Blue, type II membrane proteins; turquoise, polytopic transmembrane proteins; grey, secreted proteins; black, intracellular proteins. C. Changes in membrane protein abundance in HaCaT keratinocyte secretome induced by RHBDL2 expression. In two independent reverse experiments, WT and R2kd HaCaT cells were isotopically labelled by heavy or light lysine and arginine, the media from both populations were pooled and the lectin-enriched glycoproteins were identified and quantified by MS analysis. The abundance ratios of all transmembrane proteins identified in both experiments (i.e. the overlap of the two datasets) were plotted against each other. Two-fold enrichment was set as a significance threshold (dotted line). Membrane proteins occurring in the grey quadrant showed consistent enrichment in both experiments and represent strong candidates for RHBDL2 substrates. D. HaCaT cells were stably transfected with constructs encoding fluorescent fusions of EGFR (GFP) and RHBDL2 (mCherry) and analysed by confocal microscopy. Scale bar = 10 µm. E. Media from WT, vector control (V) and RHBDL2 knockdown (R2kd) HaCaT and MDA-MB-468 cells was concentrated and probed with an antibody raised against the EGFR ectodomain to detect RHBDL2 dependent shedding at endogenous levels of expression. F. Conditioned media from HaCaT cells were divided equally and one half was subjected to high-speed ultracentrifugation (UC) to remove membranes including exosomes. The supernatant after ultracentrifugation and the untreated medium were immunoblotted using separate primary antibodies raised against the extracellular N-terminal (NT) or the intracellular C-terminal part (CT) of EGFR. Ultracentrifugation selectively depletes the full-length form of EGFR, which is reactive against the C-terminal antibody. G . Contribution of metalloproteases to EGFR shedding. N-terminally Strep-tagged EGFR was expressed in HEK293ET cells alongside HA-tagged RHBDL2 or a catalytically inactive mutant (S187A) and cultivated for 24 hrs in the presence or absence of 10 µM BB94. H. The candidate cleavage sites were identified by mass spectrometry (MS) of the purified ectodomain ( Fig. S1 ). Candidate P1 residues were mutated to proline to produce uncleavable mutants and tested by co-overexpression with the WT or inactive mutant enzyme (S187A) in HEK293ET cells. I. An RHBDL2 dependent C-terminal EGFR fragment (open arrow) can be produced by overexpression of the wild-type enzyme in HaCaT cells, but not the S187T inactive mutant (left). RHBDL2 overexpressing cells were incubated overnight with 10 nM PR-171 (PR), 10 µM lactacystin (LC), 10 µM chloroquine (CQ) or 100 nM bafilomycin A1 (BA1) to determine the fate of the fragment (middle). The fragment can also be observed by overnight treatment with bafilomycin A1 at endogenous levels of RHBDL2 expression (right).

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Western Blot, Knockdown, Plasmid Preparation, Control, Multiplex sample analysis, Membrane, Quantitative Proteomics, Expressing, Stable Transfection, Transfection, Construct, Confocal Microscopy, Mutagenesis, Mass Spectrometry, Purification, Over Expression, Produced, Incubation

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A-C . HaCaT cell lines were seeded equally and grown to confluent monolayers over 48 h. Cells were lysed on ice in the presence of protease and phosphatase inhibitors and samples were diluted to equal concentrations of total protein. Lysates were then probed for various components of EGFR signalling pathways by western blotting. D. HaCaT cell migration was analysed by time-lapse phase-contrast microscopy over the indicated timeframe. Left panel shows the time sequence of the cell colony outline during spreading of wild-type, R2kd and vector transfected cells. Colony outlines were superimposed from first image to last image. For clarity, outlines corresponding to initial 340 min are shown in 20 min increments. Right panel shows quantification of average distance migrated after 16 h. Asterisks correspond to P values (ns=P > 0.05, *=P ≤ 0.05,**= P ≤ 0.01, ***=P ≤ 0.001 and ****= P ≤ 0.0001). E. Gap-closure assay. WT, R2kd and vector HaCaT cells were grown to confluence around removable silicone inserts. After insert removal, gap closure was observed by time-lapse microscopy over 16 h. The average distance migrated is quantified in the box and whisker plot. F. Quantification of migration of WT-HaCaT, R2kd cells and rescue cells in which shRNA refractory wild type RHBDL2 (R2kd+WT) or its inactive S187T mutant (R2kd+mut) were re-introduced into R2kd cells. RHBDL2 overexpression was induced by 5 µg/mL cumate where indicated. G. Depletion of RHBDL2 potentiates invasion of HaCaT cells into a 3D collagen matrix. Spheroids of HaCaT keratinocytes, wild-type (WT), Vector control (V), RHBDL2 knockdown (R2kd) and rescue cells (R2kd+WT, R2kd+mut) were embedded in collagen and their invasion was measured after 72 h by comparing the total area of invaded cells relative to the area of the cell spheroid at 0 h. Invasion is quantified from 5-8 spheroids in each of 3 replicate experiments (total ≥20). Statistical analyses by Tukey’s multiple comparisons test were performed using Prism software (GraphPad Software Inc.). H. HaCaT cell proliferation rate after 48 h growth in 3D collagen was assayed using the Alamar Blue assay. The level of fluorescence is proportional to the metabolic activity and hence can be used to estimate the number of cells relative to each line. The box and whisker plot is generated from 4 replicate experiments. Statistical analyses by Tukey’s multiple comparisons test were performed using Prism software (GraphPad Software Inc.).

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Western Blot, Migration, Microscopy, Sequencing, Plasmid Preparation, Transfection, Time-lapse Microscopy, Whisker Assay, shRNA, Mutagenesis, Over Expression, Control, Knockdown, Software, Alamar Blue Assay, Fluorescence, Activity Assay, Generated

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. RHBDL2 mRNA comparison by qPCR in the Ker-CT and HaCaT cells. Gene expression was normalized to GAPDH and the level of RHBDL2 expression in HaCaT cells was used to normalize RHBDL2 expression in all other cell lines, also in panel F. The mRNA analysis was done from three biological replicates, each in three technical replicates. Error bars show standard deviation. B. and C. Immunoblotting of conditioned medium and lysate after 4 h incubation with 5 mM calcium and 1 µM ionomycin detecting shedding of EGFR in primary NHEK-Ad and immortalized keratinocytes Ker-CT that is inhibited by a RHBDL2 ketoamide inhibitor compound 11 (50 µM) confirming RHBDL2 dependency. Tubulin is was used as a loading control. D. Immunoblotting of conditioned medium after 4 h incubation of Ker-CT keratinocytes with 100 µM PLCγ activator m-3M3FBS in the absence and presence of RHBDL2 inhibitor compound 11 (50 µM). E. Immunoblotting of conditioned medium after 4 h incubation of Ker-CT with thapsigargin (2 µM), bradykinin (10 µM) or 5 mM calcium as a positive control that induces RHBDL2 dependent EGFR shedding. F. RHBDL2 mRNA detection by qPCR in the N/TERT keratinocyte derived RHBDL2 knockout (R2ko) cell lines with comparison to the HaCaT and HaCaT RHBDL2 knockdown (R2kd) cells. The mRNA analysis was done from three biological replicates, each in three technical replicates. Error bars show standard deviation. G. Immunoblotting of conditioned medium and lysate of N/TERT keratinocytes to confirm RHBDL2 dependency of EGFR shedding in these cells and to validate the RHBDL2 KO (CB and CC) generated in N/TERT keratinocytes. Constitutive (48 h) or calcium (5 mM) stimulated (4 h) shedding of EGFR is inhibited by compound 11 (50 µM) in the WT, confirming its RHBDL2 dependence.

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Comparison, Gene Expression, Expressing, Standard Deviation, Western Blot, Incubation, Control, Positive Control, Derivative Assay, Knock-Out, Knockdown, Generated

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Hematoxylin and eosin staining of human skin equivalent sections generated from wild type (WT) and RHBDL2 deficient (clones CB and CC) N/TERT keratinocytes. B. Quantification of qualitative assessment of morphology of human skin organotypic cultures is based on histological analysis. Detailed scoring of individual parameters in each layer of WT or RHBDL2 deficient clones was rated on a scale 1 to 18 where 18 denotes highest differentiation ( Fig. S6 ). Differentiation scores between groups were compared by one-way ANOVA; adjusted P-value: ****<0.0001, ***≤0.001, N=4 for each group.

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Staining, Generated, Clone Assay

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet:

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Quantitative Proteomics, Membrane, Mass Spectrometry

Journal: Frontiers in Immunology

Article Title: A skin isolate of Micrococcus luteus negates the Staphylococcus aureus- induced release of type 2 cytokines from keratinocytes

doi: 10.3389/fimmu.2026.1711723

Figure Lengend Snippet: The ability to negate S. aureus -induced cytokine secretion from keratinocytes is a strain specific effect of M. luteus CFCS. (A, B) NHEK were treated with FSA or co-treated with FSA and skin bacterial CFCS for 24 h before quantifying IL-33 and TSLP in cell culture medium using ELISA. Stimulation of NHEK with FSA caused an increase in IL-33 and TSLP release. (B) Co-treatment with skin isolated M. luteus FAML CFCS negated FSA-induced release of IL-33 and TSLP. (C) Co-treatment with the M. luteus type strain NCTC 2665 had no effect on FSA-induced IL-33 and TSLP release. Data are expressed as mean ± SEM (n≥3). P values determined by one-way ANOVA *P ≤ 0.05 **P ≤ 0.01 ***P ≤ 0.001 ****P ≤ 0.0001 compared with FSA treated NHEK; ns, non-significant.

Article Snippet: Primary Normal Human Epidermal Keratinocytes (NHEK) (PromoCell, Heidelberg, Germany) were isolated from the epidermis of juvenile foreskin from pooled donors and grown in KGM supplemented with KGM SupplementMix (Promocell) at 37 ̊C in a humidified atmosphere of 5% CO 2 .

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Frontiers in Immunology

Article Title: A skin isolate of Micrococcus luteus negates the Staphylococcus aureus- induced release of type 2 cytokines from keratinocytes

doi: 10.3389/fimmu.2026.1711723

Figure Lengend Snippet: The efficacious molecule secreted by M. luteus FAML is a putative protein. M. luteus FAML was cultured for 1, 2, 4, 6 and 24 h before harvesting CFCS. NHEK were co-cultured with FSA and M. luteus FAML CFCS for 24 h before measuring (A) TSLP and (B) IL-33 in cell culture medium using ELISA. (C) M . luteus FAML CFCS collected at 24 h lost activity against FSA-induced IL-33 and TSLP release in NHEK after heat treatment (HT) to 85°C. (D) Proteins within M. luteus FAML CFCS were precipitated using acetone, then reconstituted in cell culture medium before testing for activity using the same model. Activity was retained within the protein precipitate (PP). Data are expressed as mean ± SEM (n≥4). P values determined by one-way ANOVA *P ≤ 0.05 **P ≤ 0.01 ***P ≤ 0.001 ****P ≤ 0.0001 compared with FSA treated NHEK; ns, non-significant.

Article Snippet: Primary Normal Human Epidermal Keratinocytes (NHEK) (PromoCell, Heidelberg, Germany) were isolated from the epidermis of juvenile foreskin from pooled donors and grown in KGM supplemented with KGM SupplementMix (Promocell) at 37 ̊C in a humidified atmosphere of 5% CO 2 .

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: A skin isolate of Micrococcus luteus negates the Staphylococcus aureus- induced release of type 2 cytokines from keratinocytes

doi: 10.3389/fimmu.2026.1711723

Figure Lengend Snippet: Recombinant PADP negates FSA-induced IL-33 and TSLP release from NHEK. NHEK were co-cultured with FSA and rPADP for 24 h before measuring IL-33 and TSLP in the cell culture medium using ELISA. The rPADP negated FSA-induced IL-33 and TSLP release in NHEK. Data are expressed as mean ± SEM (n≥3). P values determined by one-way ANOVA *P ≤ 0.05 **P ≤ 0.01 ****P ≤ 0.0001 compared with FSA treated NHEK.

Article Snippet: Primary Normal Human Epidermal Keratinocytes (NHEK) (PromoCell, Heidelberg, Germany) were isolated from the epidermis of juvenile foreskin from pooled donors and grown in KGM supplemented with KGM SupplementMix (Promocell) at 37 ̊C in a humidified atmosphere of 5% CO 2 .

Techniques: Recombinant, Cell Culture, Enzyme-linked Immunosorbent Assay