Journal: Molecular Therapy. Nucleic Acids

Article Title: USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea

doi: 10.1016/j.omtn.2020.07.032

Figure Lengend Snippet: Immunohistochemical Analysis of Collagen III after Wounding (A–C) Frozen sections of corneas 6 weeks after wounding were immunostained for collagen III (green), DAPI (blue). (A) UnWnd, (B) Wnd-NTC with magnified inset, and (C) Wnd-US09 with magnified inset. Scale bar, 0.5 mm. (D) Compared to UnWnd, Wnd-NTC demonstrated a 276.2-fold increase in collagen III immunostaining (p < 0.01), which was reduced by 71.7% (p < 0.05) with US09 treatment. The comparison between UnWnd and Wnd-US09 was not significant. (E) By qPCR, days 1 and 2 combined, compared to UnWnd, Wnd-NTC demonstrated a 4.26-fold increase in USP10 gene expression (p < 0.001). Compared to Wnd-NTC, USP10 expression with Wnd-US09 treatment was reduced by 56.8% (p < 0.01). N = 4 rabbits per condition. At 6 weeks, compared to UnWnd, Wnd-NTC demonstrated a 35.7-fold increase in USP10 gene expression (p < 0.05). Compared to Wnd-NTC, USP10 expression with Wnd-US09 treatment was reduced by 91.2% (p < 0.05). N = 6 rabbits per condition.

Article Snippet: On the next day sections were rehydrated in PBS for 15 min, treated with blocking buffer (10% normal goat serum in PBS, Jackson ImmunoResearch Laboratories) for 20 min, and then incubated with primary antibodies (FN-EDA [Sigma, F6140], collagen III [Novus Biologicals, NBP105119B], α-SMA [Sigma, C6198], CD45 Thermo Fisher Scientific, MA5-28392]) at 1:250 for 1 h in a moist chamber at room temperature (RT).

Techniques: Immunohistochemical staining, Immunostaining, Comparison, Gene Expression, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea

doi: 10.1016/j.omtn.2020.07.032

Figure Lengend Snippet: Immunohistochemical Analysis of α-SMA, Cell Proliferation, and Thickness after Wounding (A–C) Frozen sections of corneas 6 weeks after wounding were immunostained for α-SMA (green), DAPI (blue). (A) UnWnd, (B) Wnd-NTC with magnified inset, and (C) Wnd-US09 with magnified inset. Scale bar, 0.5 mm. (D) Compared to UnWnd, Wnd-NTC demonstrated a 5.77-fold increase in α-SMA immunostaining (p < 0.05). Compared to Wnd-NTC, α-SMA immunostaining after Wnd-US09 treatment was reduced by 83.6% (p < 0.05). The comparison between UnWnd and Wnd-US09 was not significant. (E and F) Cell proliferation was analyzed by the object counter plugin in ImageJ software. “Inside the wound” is denoted by the anterior cornea demarcated by the collagen III scar. “Outside the wound” is the posterior cornea beneath the scar. These counts were normalized by the total area of each portion to generate a nuclei density measurement. (E) Compared to UnWnd, Wnd-NTC demonstrated a 1.61-fold increase in cell proliferation (p < 0.01). Compared to Wnd-NTC, cell proliferation after Wnd-US09 treatment was reduced by 29.9% (p < 0.05). The comparison between UnWnd and Wnd-US09 was not significant. (F) Cell proliferation below the scar, in the stroma down to the endothelium. All relationships were not significant. (G) Corneal thickness was measured at pixel resolution in these thresholded images as the distance across the nonzero region, and thickness is averaged across the entire cornea. Wnd-NTC trended toward a slight decrease in thickness (p = 0.05). Wnd-US09 treatment restored corneal thickness to non-wounded parameters. N = 6 rabbits per condition.

Article Snippet: On the next day sections were rehydrated in PBS for 15 min, treated with blocking buffer (10% normal goat serum in PBS, Jackson ImmunoResearch Laboratories) for 20 min, and then incubated with primary antibodies (FN-EDA [Sigma, F6140], collagen III [Novus Biologicals, NBP105119B], α-SMA [Sigma, C6198], CD45 Thermo Fisher Scientific, MA5-28392]) at 1:250 for 1 h in a moist chamber at room temperature (RT).

Techniques: Immunohistochemical staining, Immunostaining, Comparison, Software

Journal: PLoS ONE

Article Title: Topically Delivered Adipose Derived Stem Cells Show an Activated-Fibroblast Phenotype and Enhance Granulation Tissue Formation in Skin Wounds

doi: 10.1371/journal.pone.0055640

Figure Lengend Snippet: GFP-expressing ASCs were analyzed 7 days after transplantation in wounds. Chicken anti-GFP and mouse anti-α-SMA antibodies were used to detect GFP and α-SMA. Nuclei were stained with DAPI. (A): Low magnification of wounds. The areas analyzed in were indicated by ‘a’ and ‘b’. (B–D): Higher magnifications of the indicated regions in A (white squares; labeled as i, ii, iii). (E–G): Higher magnifications of the indicated regions in B–D (white squares; labeled as iv, v, vi). Merged images of α-SMA (red) and GFP (green) indicate that α-SMA is expressed in ASCs. Scale bars: 500 µm (A), 50 µm (B–G).

Article Snippet: For immunofluorescence microscopy, chicken anti-GFP (1∶200 dilution, Life Technologies), α-SMA (1∶200 dilution, Santa Cruz Biotechnology), mouse anti-collagen III (col3, 1∶200 dilution, Novus Biologicals, Littleton, CO), mouse anti-CD31 (1∶25 dilution, Abcam), mouse anti-Ki67 (1∶20 dilution, Novocastra, Buffalo Grove, IL), and mouse anti-PCNA (1∶100 dilution, BD Biosciences, San Jose, CA) antibodies were used as primary antibodies.

Techniques: Expressing, Transplantation Assay, Staining, Labeling