Journal: bioRxiv

Article Title: Epidermal loss of PRMT5 leads to the emergence of an atypical basal keratinocyte-like cell population and defective skin stratification

doi: 10.1101/2024.11.08.620904

Figure Lengend Snippet: ( A ) A histogram summarizing the observed frequency (Y-axis) of the possible genotypes, generated from the experimental cross, at different embryonic (e.g., E15.5, E18.5) and postnatal (e.g., P21) stages (X-axis). The expected frequency for each genotype (i.e., 25%) is indicated by the dashed line. Note, ‘n =’ refers to the total number of embryos/pups scored at the indicated stage, ns refers to ‘not significant’, and *** = p < 0.0001, Likelihood Ratio (RStudio package XNomial). ( B-I ) Brightfield lateral view images of a CTRL (B-D, I) or cKO (E-G, J) embryo at E13.5 (B, E), E15.5 (C, F), and E18.5 (D, I-J) stages. Ventral is to the right. Embryos in I and J have also been stained with toluidine blue as an assessment of skin permeability, with the number of embryos displaying permeable skin quantified in the bottom right corner (I-J). ( H ) A bar graph qualitatively summarizing no (purple), mild (cyan), or severe (orange) skin defects in CTRL and cKO samples. Note, gross anatomy images underwent post-acquisition white balancing using GIMP. Scale bar = 2mm.

Article Snippet: Primary antibodies include: α-CDKN1A (1:50, ProteinTech, 10355-1-AP), α-CLDN1 (1:60, Abcam, ab15098), α-COL4 (1:500, Invitrogen, PA1-85320), α-eGFP (1:500, Novus Biologicals, NB600-308), α-FLG (1:500, Biolegend, 905804), α-IGFBP2 (1:200, Bioss, bs-1108R), α-KI67 (1:200, Abcam, ab16667), α-KRT6 (1:500, Biolegend, PRB-169F), α-KRT10 (1:500, Biolegend, 905404), α-KRT14 (1:20, SantaCruz, sc-53253), α-KRT17 (1:1000, Proteintech, 17516-1-AP), α-NF (0.5ug/ml, Hybridoma, AB531793), α-PRMT5 (1:200, Millipore, 07-405), α-TRP63 (1:500, Cell Signaling, 13109S), and α-VCL (1:300, Sigma, V9131).

Techniques: Generated, Staining, Permeability

Journal: bioRxiv

Article Title: Epidermal loss of PRMT5 leads to the emergence of an atypical basal keratinocyte-like cell population and defective skin stratification

doi: 10.1101/2024.11.08.620904

Figure Lengend Snippet: ( A-M ) scRNA-seq violin plots for Cdkn1a (A), Igfbp2 (E), Trp63 (H), and Krt17 (K) in the novel (19) and basal (3/5) keratinocyte clusters. Corresponding α-CDKN1A (B-C), α-IGFBP2 (F-G), α-TRP63 (I-J), and α-KRT17 (L-M) immunofluorescence from E15.5 (B, C, F, G, I, J) or E17.5 (L, M) CTRL (B, F, I, L) or Prmt5 cKO (C, G, J, M) embryos. Integrative Genomics Viewer (IGV) reveals a suggestive (FC=1.74, p_ adj=0.51) locus specific increase in chromatin accessibility at the Cdkn1a promoter (D). ( N ) A schematic summarizing the normal progression of keratinocytes in CTRL (top) and atypical progression in Prmt5 cKOs (bottom), including the molecular features involved. Sections shown are the craniofacial epidermis, lateral to the oral cavity, in a coronal plane. White dashed lines demarcate the boundary of the dermis and epidermis. Nuclei are counterstained with DAPI. All section immunofluorescence images are Z-stacks compiled with ‘Max Intensity’ in FIJI. Scale bar = 10 µm.

Article Snippet: Primary antibodies include: α-CDKN1A (1:50, ProteinTech, 10355-1-AP), α-CLDN1 (1:60, Abcam, ab15098), α-COL4 (1:500, Invitrogen, PA1-85320), α-eGFP (1:500, Novus Biologicals, NB600-308), α-FLG (1:500, Biolegend, 905804), α-IGFBP2 (1:200, Bioss, bs-1108R), α-KI67 (1:200, Abcam, ab16667), α-KRT6 (1:500, Biolegend, PRB-169F), α-KRT10 (1:500, Biolegend, 905404), α-KRT14 (1:20, SantaCruz, sc-53253), α-KRT17 (1:1000, Proteintech, 17516-1-AP), α-NF (0.5ug/ml, Hybridoma, AB531793), α-PRMT5 (1:200, Millipore, 07-405), α-TRP63 (1:500, Cell Signaling, 13109S), and α-VCL (1:300, Sigma, V9131).

Techniques: Immunofluorescence