Journal: bioRxiv

Article Title: Desmoplakin is a desmosomal mechanosensor

doi: 10.1101/2024.11.19.624364

Figure Lengend Snippet: (A) Schematic of desmosome structure. (B-D) Representative STED images of desmosomes showing a characteristic ‘railroad track’ pattern in (B) MCF7 WT cells, (C) MCF7 K19-KO cells, and (D) MCF7 K19-GfP cells. The desmosomes were immunolabeled for DPC (green) and Dsg2 (red). Scale bar: 500 nm. (E) The boxed area from the representative STED images of MCF7 WT cells shows a closer look at an individual DP railroad track with Dsg2 between the two parallel DP plaques. Scale bar: 200 nm. (F) Line-scan analysis of DPC and Dsg2 fluorescence intensity (indicated by the dashed line in E). (G) Quantification of DPC-DPC distance from WT and K19-KO cells showing that desmosomes in WT cells are wider than K19-KO cells. In all boxplots, the box represents the 25th and 75th percentiles with the median indicated and whiskers reach 1.5 times the interquartile range (IQR), defined as the difference between the 25th and 75th percentiles. Data points outside the whiskers are shown as outliers. Number of datapoints (n) = 630 (WT), 505 (K19-KO); Number of replicates (N) = 3. Mann-Whitney’s U test; ***, P<0.001. (H) Quantification of DPC-DPC distance in K19-KO, WT, and K19-GFP cells. Desmosomes in WT and K19-GFP cells are wider than K19-KO cells. n = 505 (K19-KO), 630 (WT), 716 (K19-GFP); N = 3. Kruskal-Wallis Test, followed by Dunn’s multiple comparison Test; ***, P<0.001.

Article Snippet: The following primary antibodies were used: human anti-DSG2 (MAB947, R&D systems), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2) (Santa Cruz Biotechnology), control mouse IgG (sc-2025) (Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Immunolabeling, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Desmoplakin is a desmosomal mechanosensor

doi: 10.1101/2024.11.19.624364

Figure Lengend Snippet: (A) Schematic of the desmosome under tension. The N-terminal of DP transitions from a closed conformation to an open conformation. (B) Quantification of desmosome half-unit widths (Dsg2-DPC distance) from WT and K19-KO cells. n = 1260 (WT), 1010 (K19-KO); N = 3. Mann-Whitney’s U test; ***, P<0.001. The distances between Dsg2 and DPC is significantly greater in the WT compared to the K19-KO cells. (C) Quantification of desmosome half-unit widths (Dsg2-DPN distance) from WT and K19-KO cells. n = 692 (WT), 826 (K19-KO); N = 3. Mann-Whitney’s U test; ns, P>0.05. The Dsg2-DPN distance in both cell lines are similar. (D) The mean values from the results in B and C are summarized in the bar chart to compare the DP length (DPN-DPC distance) between the WT and K19-KO cells. The DP length is 66 nm for the WT and 33 nm for the K19-KO, indicating that DP extends 33 nm in the WT cells. (E) Dispase assay after 24 h plating. The confluent cell sheets are treated with 4 mM EGTA for 1 h. The images show the intact cell sheets for WT, K19-KO, and K19-GFP rescued cells before stress and fragmented cell sheets after applying mechanical stress. (F) Quantification of the dispase assay from K19-KO, WT, and K19-GFP cells. n = 12 (KO), 12 (WT), 9 (K19-GFP); N = 3. Kruskal-Wallis Test, followed by Dunn’s multiple comparison Test; ***, P<0.001. The K19-KO cell sheets show a greater number of fragments compared to WT cell sheets, while K19-GFP cell sheets generate a similar number of fragments as WT. This indicates that elongation of DP strengthens intercellular adhesion in WT and K19-GFP cells.

Article Snippet: The following primary antibodies were used: human anti-DSG2 (MAB947, R&D systems), rabbit anti-DPC antibody (A303-356A, Bethyl Lab), rabbit anti-DPN antibody (25318-1-AP, Proteintech), chicken anti-GFP (600-901-215, Rockland), mouse anti-K19 antibody (A53-B/A2) (Santa Cruz Biotechnology), control mouse IgG (sc-2025) (Santa Cruz Biotechnology), and mouse anti-actin antibody (66009-1-Ig, Proteintech).

Techniques: Comparison

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: Membrane staining was performed by using antibodies to OBP2A (orb582625, Biorbyt Ltd., Cambridge, UK), FABP5 (39926, Cell Signaling Technology, MA, USA), PERK (5683, Cell Signaling Technology, MA, USA), Bip (3177, Cell Signaling Technology, MA, USA), PDI (3501, Cell Signaling Technology, MA, USA), involucrin (ab53112, abcam, Cambridge, UK), keratin 10 (ab76318, abcam, Cambridge, UK), corneodesmosin (13184, proteintech, IL, USA), desmoglein 2 (MAB947, R&D Systems, MO, USA), and β-actin (sc-47778, Santa Cruz Biotechnology, TX, USA) as primary antibodies and a Novex AP Rabbit Chemiluminescent Detection Kit or Novex AP Mouse Chemiluminescent Detection Kit (Invitrogen, CA, USA) as secondary antibodies.

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

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: Human Desmoglein-2 Antibody , R&D Systems , MAB947.

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