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

Journal: International Wound Journal

Article Title: Machine learning and single‐cell transcriptome profiling reveal regulation of fibroblast activation through THBS2/TGFβ1/P‐Smad 2/3 signalling pathway in hypertrophic scar

doi: 10.1111/iwj.14481

Figure Lengend Snippet: THBS2 promotes the proliferation, migration and expression of fibrotic marker protein. (A) qRT‐PCR showed that CTSK, CTHRC1 and THBS2 were expressed in HS. (B) qRT‐PCR showed that THBS2 was knocked down by siRNA in scar fibroblasts. (C) qRT‐PCR showed that the expression of Col1, Col3 and αSMA in fibroblasts with low THBS2 knockdown was decreased. (D) Western blotting showed that the expression of Col1, Col3 and αSMA decreased in fibroblasts with decreased THBS2 expression. (E) Cellular immunofluorescence showed decreased αSMA expression in fibroblasts with THBS2 knockdown. (F) Scratch test showed that the migration rate of fibroblasts with low THBS2 was decreased (scale bar = 100 μm). (G) CCK8 assay showed that the proliferation of THBS2 knockdown fibroblasts decreased. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. HS, hypertrophic scar; NS, normal skin.

Article Snippet: Subsequently, the PVDF membranes were washed with TBST before being incubated overnight at 4°C with the following rabbit anti‐human primary antibodies: GAPDH (10494‐1‐AP, Sanying, Wuhan, China, 1:1000), THBS2 (ab84469, Abcam, 1:1000), Col1 (14695‐1‐AP, Sanying, Wuhan, China, 1:1000), Col3 (22734‐1‐AP, Sanying, Wuhan, China, 1:1000), αSMA (14395‐1‐AP, Sanying, Wuhan, China, 1:1000), TGFβ1 (ab215715, Abcam), P‐Smad2/3 (8828 s, Cell Signalling Technology, 1:1000), Smad2/3 (8685s, Cell Signalling Technology, 1:1000).

Techniques: Migration, Expressing, Marker, Quantitative RT-PCR, Knockdown, Western Blot, Immunofluorescence, CCK-8 Assay

Journal: International Wound Journal

Article Title: Machine learning and single‐cell transcriptome profiling reveal regulation of fibroblast activation through THBS2/TGFβ1/P‐Smad 2/3 signalling pathway in hypertrophic scar

doi: 10.1111/iwj.14481

Figure Lengend Snippet: THBS2 activates scar fibroblasts through the TGFβ1/P‐Smad2/3 pathway. (A) GSEA plot demonstrates the association between THBS2 and the TGF‐β pathway. (B) qRT‐PCR showed the mRNA expression levels of Col1, Col3, αSMA and TGFβ1 in the control group, THBS2 (200 ng/mL), THBS2 (200 ng/mL) + SB‐431542 (10 ng/mL), THBS2 (200 ng/mL) + SB‐431542 (10 ng/mL) + TGFβ1 (10 ng/mL) treated fibroblasts. (C) Protein expression levels of Col1, Col3, αSMA and P‐Smad2/3 in the control group, THBS2, THBS2 + SB‐431542, THBS2 + SB‐431542 + TGFβ1‐treated fibroblasts by western blotting. (D) The proliferation ability of four groups of scar fibroblasts was tested by CCK8 proliferation test. (^: comparison between control and THBS2 groups; *: comparison between THBS2 and THBS2 + SB‐431542 groups). (E) The migration ability of four groups of scar fibroblasts was tested by cell scratch test (scale bar = 100 μm). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Subsequently, the PVDF membranes were washed with TBST before being incubated overnight at 4°C with the following rabbit anti‐human primary antibodies: GAPDH (10494‐1‐AP, Sanying, Wuhan, China, 1:1000), THBS2 (ab84469, Abcam, 1:1000), Col1 (14695‐1‐AP, Sanying, Wuhan, China, 1:1000), Col3 (22734‐1‐AP, Sanying, Wuhan, China, 1:1000), αSMA (14395‐1‐AP, Sanying, Wuhan, China, 1:1000), TGFβ1 (ab215715, Abcam), P‐Smad2/3 (8828 s, Cell Signalling Technology, 1:1000), Smad2/3 (8685s, Cell Signalling Technology, 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Comparison, Migration

Journal: Europace

Article Title: Angiotensin II-mediated up-regulation of connective tissue growth factor promotes atrial tissue fibrosis in the canine atrial fibrillation model

doi: 10.1093/europace/eus052

Figure Lengend Snippet: Polymerase chain reaction primers used for amplification of inflammation and fibrosis-related genes

Article Snippet: After blocking with 5% non-fat milk, the membrane was incubated with anti-rabbit COL1 antibody (sc-8784; Santa Cruz Biotechnology, Inc., CA, USA), anti-rabbit COL3 antibody (LSL-LB-1387; Cosmo Bio Co., Ltd., Japan) or anti-mouse GAPDH antibody (GTX28245; GeneTex ® Inc., USA) and subsequently with the secondary antibody (Amersham ECL Plus Western blotting reagent pack; GE Healthcare UK Ltd.).

Techniques: Polymerase Chain Reaction, Amplification

Journal: Europace

Article Title: Angiotensin II-mediated up-regulation of connective tissue growth factor promotes atrial tissue fibrosis in the canine atrial fibrillation model

doi: 10.1093/europace/eus052

Figure Lengend Snippet: Messenger ribonucleic acid expressions of extracellular matrix and inflammation-related molecules. In the pacing control group, mRNA expressions of extracellular matrix-related genes (connective tissue growth factor, collagen type 1 and 3, and fibronectin 1) were up-regulated, especially in the right atrium in comparison with the non-pacing group, and this up-regulation was suppressed in the pacing + olmesartan group. In contrast, messenger ribonucleic acid expression of transforming growth factor-β did not differ among the three groups. See text for details. RA, right atrium; LA, left atrium; CTGF, connective tissue growth factor; COL1, collagen type 1; COL3, collagen type 3; FN1, fibronectin 1; TGF-β, transforming growth factor-β; MCP-1, monocyte chemotactic protein-1. * P < 0.05 vs. non-pacing group; † P < 0.05 vs. pacing control group.

Article Snippet: After blocking with 5% non-fat milk, the membrane was incubated with anti-rabbit COL1 antibody (sc-8784; Santa Cruz Biotechnology, Inc., CA, USA), anti-rabbit COL3 antibody (LSL-LB-1387; Cosmo Bio Co., Ltd., Japan) or anti-mouse GAPDH antibody (GTX28245; GeneTex ® Inc., USA) and subsequently with the secondary antibody (Amersham ECL Plus Western blotting reagent pack; GE Healthcare UK Ltd.).

Techniques: Control, Comparison, Expressing

Journal: Europace

Article Title: Angiotensin II-mediated up-regulation of connective tissue growth factor promotes atrial tissue fibrosis in the canine atrial fibrillation model

doi: 10.1093/europace/eus052

Figure Lengend Snippet: Western blot analysis of protein levels of collagens. ( A ) Representative examples of the bands and ( B ) a summary of all samples. Glyceraldehyde-3-phosphate dehydrogenase protein level was measured as an internal control. The protein level of collagen type 1 did not show any difference, but collagen type 3 was up-regulated in the pacing control group in comparison with the non-pacing group, and this up-regulation was suppressed in the pacing + olmesartan group. See text for details. RA, right atrium; LA, left atrium; COL1, collagen type 1; COL3, collagen type 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P < 0.05 vs. non-pacing group; † P < 0.05 vs. pacing control group.

Article Snippet: After blocking with 5% non-fat milk, the membrane was incubated with anti-rabbit COL1 antibody (sc-8784; Santa Cruz Biotechnology, Inc., CA, USA), anti-rabbit COL3 antibody (LSL-LB-1387; Cosmo Bio Co., Ltd., Japan) or anti-mouse GAPDH antibody (GTX28245; GeneTex ® Inc., USA) and subsequently with the secondary antibody (Amersham ECL Plus Western blotting reagent pack; GE Healthcare UK Ltd.).

Techniques: Western Blot, Control, Comparison