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: Microbiology Spectrum

Article Title: Higher affinities of fibers with cell receptors increase the infection capacity and virulence of human adenovirus type 7 and type 55 compared to type 3

doi: 10.1128/spectrum.01090-23

Figure Lengend Snippet: Affinity analysis of receptor DSG2 binding fiber knobs of HAdV-3, HAdV-7, and HAdV-55. (a) SDS-PAGE of the purified recombinant fiber knobs of HAdV-3, HAdV-7, HAdV-55, and HAdV-5. Purified protein in loading buffer was incubated at room temperature for 5 min and then incubated on ice (native, N) or denatured at 98°C for 5 min (boiled, B). The image labeled “Knob: Ad3” is identical to the image labeled “HAdV-B3-Knob” in our previously published article . The Ad3-knob protein used in both articles is the same one prepared from the same batch. (b) Comparison of the affinities of knobs binding DSG2 by ELISA. The plates were coated with 1.5 µg/mL DSG2 in HBS-N (Ca 2+ ) and then incubated with serially diluted Ad3K, Ad7K, Ad55K, and Ad5K. Afterward, the HRP-labeled anti-His antibody was added. Finally, the substrate was added to read A450. (c) Affinity measurement and comparison of receptor DSG2 binding with fiber knobs of HAdV-3, HAdV-7, and HAdV-55 by SPR. (d and e) Knob competition experiments. Serially diluted recombinant knobs in phosphate-buffered saline (PBS) were added to A549 cells and incubated on ice. Subsequently, EGFP-expression HAdV rAd3EGFP (d) or rAd55EGFP (e) was added. After washing twice with cold PBS, the cells were cultured in a fresh medium for 2 days. Cells were photographed, and the number of fluorescent cells was determined. HAdV-5 fiber knob was used as the negative control.

Article Snippet: Humanized DSG2 KI homozygous C57BL/6 mice, Ho-hDSG2-C57, with human DSG2 cDNA inserted into the DSG2 gene, were generated by Cyagen Biosciences (Suzhou, China) with TALEN (Transcription Activator-Like Effector Nuclease) knock-in technology.

Techniques: Binding Assay, SDS Page, Purification, Recombinant, Incubation, Labeling, Comparison, Enzyme-linked Immunosorbent Assay, Saline, Expressing, Cell Culture, Negative Control

Journal: Microbiology Spectrum

Article Title: Higher affinities of fibers with cell receptors increase the infection capacity and virulence of human adenovirus type 7 and type 55 compared to type 3

doi: 10.1128/spectrum.01090-23

Figure Lengend Snippet: Humanized DSG2-KI mice intranasally infected with rAdV3E, rAdV3E-K7, and rAdV3E-K55. (a) Hematoxylin and eosin staining of lung and liver tissues. Arrows indicate pathological changes. (b) Lung injury scores and (c) liver injury scores. (d) Viral DNA load in lung tissues. Each symbol represents an individual mouse, and the horizontal lines indicate mean values or mean ± standard deviation. No significant differences were found ( P > 0.05).

Article Snippet: Humanized DSG2 KI homozygous C57BL/6 mice, Ho-hDSG2-C57, with human DSG2 cDNA inserted into the DSG2 gene, were generated by Cyagen Biosciences (Suzhou, China) with TALEN (Transcription Activator-Like Effector Nuclease) knock-in technology.

Techniques: Infection, Staining, Standard Deviation