Journal: STAR Protocols

Article Title: Protocol for odorant-binding protein 2A gene knockdown by dual siRNA transfection in a three-dimensional human epidermal equivalent model

doi: 10.1016/j.xpro.2025.103626

Figure Lengend Snippet:

Article Snippet: Normal human epithelial keratinocytes , Kurabo , KK-4009.

Techniques: Recombinant, Transfection, Saline, Sterility, Cell Culture, Protease Inhibitor, DC Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Dermis equivalents were 3D printed onto the apical side of transwell inserts using the RegenHU 3D Discovery bioprinter (image courtesy of RegenHU). Keratinocytes were pipetted onto the apical surface of the dermis. In the submerged model, the tissues were infected at the apical surface. In the ALI model, tissues were brought to ALI and then infected at the basolateral surface (created with BioRender.com ). (B) H&E and IHC images of differentiated ALI tissues. K10 (cyan) and K14 (red) identify keratinocytes in the suprabasal and basal layer of the epidermis respectively (scale bar 50µm) (C) Submerged tissues were infected at various MOI and then imaged at specified times. Fibroblasts express tdTomato (orange) while infected cells express GFP (green) (scale bar 1mm). (D) GFP and tdTomato signal at each MOI and timepoint (*** P < 0.001, **** P < 0.0001 by ordinary one-way ANOVA). (E) Maximum projection of infected tissues from the top (column 1) or side (column 2) view. H&E (column 3) and IHC (column 4) staining of infected submerged or ALI models (scale bar 1mm (column 1) or 50µm (column 2, 3, 4).

Article Snippet: Neonatal Normal Human Epithelial Keratinocytes (NHEK N , ScienCell 2100) were cultured in KBM Gold basal medium (Lonza, Cat. # 00192151) supplemented with the KGM Gold Keratinocyte Growth Medium BulletKit (Lonza, Cat. #00192152).

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Punch biopsies from six donors were collected and dissociated by enzymatic and mechanical processes. Vero cells, keratinocytes (B), and fibroblasts (C) were infected with GFP-expressing HSV-1, and live cell images were taken every two hours. (D) Keratinocytes, fibroblasts, and Vero cells were infected with GFP-expressing HSV-1 and then treated with acyclovir at the specified doses. Representative live cell images were taken at the peak of GFP expression. (Scale bar 500µm). (E) Dose-response curve of acyclovir in keratinocyte cultures compared to Vero cells (grey line). (F) Dose-response curve of acyclovir in fibroblast cultures compared to Vero cells (grey line). (G) IC 50 values for each donor in each cell type (*** P < 0.001, * P < 0.05, linear mixed model).

Article Snippet: Neonatal Normal Human Epithelial Keratinocytes (NHEK N , ScienCell 2100) were cultured in KBM Gold basal medium (Lonza, Cat. # 00192151) supplemented with the KGM Gold Keratinocyte Growth Medium BulletKit (Lonza, Cat. #00192152).

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Dose-response curves for the 11 top candidate antivirals compared to acyclovir (ACV) (keratinocytes blue and grey, respectively; fibroblasts green and black, respectively). (B) IC 50 values for each top candidate antiviral compared between keratinocytes (blue) and fibroblasts (green). Striped bars (FMP, VRD) indicate candidate antivirals that failed to reduce GFP expression by at least 50% consistently. (C) Maximum inhibition for each top candidate antiviral is compared between keratinocytes (blue) and fibroblasts (green). Statistical significance was determined by linear mixed model (*** P < 0.001, ** P < 0.01, * P < 0.05) for (B) and (C) . (D) CC 50 dose-response curves for all twelve candidate antivirals compared to their respective IC 50 to IC 80 dose ranges. Keratinocyte data is from 20HPI (grey), while fibroblast data is from 48HPI (red).

Article Snippet: Neonatal Normal Human Epithelial Keratinocytes (NHEK N , ScienCell 2100) were cultured in KBM Gold basal medium (Lonza, Cat. # 00192151) supplemented with the KGM Gold Keratinocyte Growth Medium BulletKit (Lonza, Cat. #00192152).

Techniques: Expressing, Inhibition

Journal: bioRxiv

Article Title: Identification of potent HSV antivirals using 3D bioprinted human skin equivalents

doi: 10.1101/2024.12.04.626896

Figure Lengend Snippet: (A) Pairwise comparisons of IC 50 values for candidate antivirals in the four models tested. (B) Pairwise comparisons of CC 50 values for candidate antivirals in the four models tested. (C) Fold change was determined by dividing the IC 50 value of each candidate antiviral in keratinocytes by the IC 50 of the same candidate antiviral in submerged models. (D) Fold change was determined by dividing the IC 50 value of each candidate antiviral in fibroblasts by the IC 50 of the same candidate antiviral in ALI models. (E) IC 50 values for each candidate antiviral were pooled (keratinocytes and fibroblasts, submerged and ALI), then IC 50 values for candidate antivirals in 2D were divided by IC 50 values in 3D. (C) (D) (E) Green bars indicate candidate antivirals that were more potent in 3D, while blue bars indicate candidate antivirals that are potent in 2D.

Article Snippet: Neonatal Normal Human Epithelial Keratinocytes (NHEK N , ScienCell 2100) were cultured in KBM Gold basal medium (Lonza, Cat. # 00192151) supplemented with the KGM Gold Keratinocyte Growth Medium BulletKit (Lonza, Cat. #00192152).

Techniques:

Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet: Expression of OBPs in human epidermal keratinocytes (A) qPCR analysis of OBPs in keratinocytes. GAPDH was used for RNA quality control. M: Marker. (B) Comparison of relative expression of OBPs ( n = 4). Anova F value = 18.73, p < 0.0006 for OBP2A , Anova F value = 22.01, p < 0.0003 for OBP2B , Anova F value = 2.861, p < 0.109 for LCN1 . (C) Immunofluorescence staining of OBP2A in human skin. Cell nuclei appear in blue. Bars = 20 μm. SC: stratum corneum. Bars and lines represent mean ± SD. ∗: p < 0.05, ∗∗∗∗: p < 0.0001, ns: not significant in ANOVA with Scheffé’s method. See also Figure S1 .

Article Snippet: Normal human epithelial keratinocytes were purchased from Kurabo (Osaka, Japan) and cultured in EPILIFE-KG2 (Kurabo, Osaka, Japan).

Techniques: Expressing, Control, Marker, Comparison, Immunofluorescence, Staining

Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet: Effects of OBP2A on the reduction of cell viability induced by NE, OA, and POA (A) Western-blot analysis of OBP2A in cells. (B) Relative value of OBP2A western-blot signal ( n = 3). (C) ELISA analysis of OBP2A in cells ( n = 4). (D, E, and F) Viability of keratinocytes treated with NE, OA, or POA ( n = 12). The cells were treated with scrambled siRNA or OBP2A siRNA. Anova F value = 47.41, p < 0.0001 for NE, Anova F value = 18.54, p < 0.0001 for OA, Anova F value = 19.04, p < 0.0001 for POA. (G, H, and I) Docking simulation of NE, OA, or POA to OBP2A. Gray: carbon atom, Red: oxygen atom, green: OBP2A (PDB ID: 4RUN ). NE: trans -2-nonenal, OA: oleic acid, POA: palmitoleic acid. Bars and lines represent mean ± SD. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.0005, ∗∗∗∗: p < 0.0001, ns: not significant in Student’s t test (B and C) and in ANOVA with Scheffé’s method (D, E, and F). See also Figure S2 .

Article Snippet: Normal human epithelial keratinocytes were purchased from Kurabo (Osaka, Japan) and cultured in EPILIFE-KG2 (Kurabo, Osaka, Japan).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet: Effect of IL-4, IL-13, and urushiol on OBP2A expression and IL-1α secretion in keratinocytes (A and C) qPCR analysis of OBP2A in cells treated with IL-4 or IL-13 ( n = 4). (B and D) ELISA analysis of OBP2A in cells treated with IL-4 or IL-13 ( n = 8). (E and F) ELISA analysis of OBP2A in the 3DE-model treated with IL-4 or IL-13 ( n = 8). (G) ELISA analysis of IL-1α in cells treated with urushiol (Uru) ( n = 8). Anova F value = 15.63, p < 0.0001. (H) Docking simulation of urushiol to OBP2A. Gray: carbon atom, red: oxygen atom, green: OBP2A (PDB ID: 4RUN ). Bars and lines represent mean ± SD. ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.0005, ns: not significant in Student’s t test (A, B, C, D, E, and F) and in ANOVA with Scheffé’s method (G).

Article Snippet: Normal human epithelial keratinocytes were purchased from Kurabo (Osaka, Japan) and cultured in EPILIFE-KG2 (Kurabo, Osaka, Japan).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: OBP2A regulates epidermal barrier function and protects against cytotoxic small hydrophobic molecules

doi: 10.1016/j.isci.2024.111093

Figure Lengend Snippet:

Article Snippet: Normal human epithelial keratinocytes were purchased from Kurabo (Osaka, Japan) and cultured in EPILIFE-KG2 (Kurabo, Osaka, Japan).

Techniques: Imaging, Recombinant, Transfection, Saline, Sterility, Cell Culture, Protease Inhibitor, DC Protein Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Quantitation Assay, Fluorescence, Sequencing, Software