Journal: Experimental & molecular medicine

Article Title: Loss of KDM5B ameliorates pathological cardiac fibrosis and dysfunction by epigenetically enhancing ATF3 expression.

doi: 10.1038/s12276-022-00904-y

Figure Lengend Snippet: Fig. 2 KDM5B deficiency protects the heart from dysfunction and adverse cardiac remodeling after MI. a Echocardiographic measurement of the LVEF, LVFS, left ventricular end-diastolic internal dimension (LVIDd), and left ventricular end-systolic internal dimension (LVIDs) of KDM5B-KO or WT mice at baseline (Day 0) and on the indicated day after MI or sham operation (n = 6 mice per group). b–e Representative Masson’s trichrome staining images and quantitation of the scar size (b, c) or Sirius red staining images and quantitation of the fibrosis area (d, e) in myocardial tissues from KDM5B-KO or WT mice on Day 28 after MI. n = 6 mice per group. Scale bar, 1.6 mm (upper), 200 μm (bottom). f Q-PCR analysis of Col1a1 and Col3a1 mRNA levels in myocardial tissues from KDM5B-KO or WT mice on Day 14 after MI or sham operation (n = 6 mice per group). g Representative immunofluorescence staining of α-SMA (red) and Col III (green) in myocardial tissues from KDM5B-KO or WT mice on Day 14 after MI (n = 6 mice per group). Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s t test (c, e) or ANOVA (a, f) was performed.

Article Snippet: Antibodies against collagen type III (NB600-594) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Staining, Quantitation Assay

Journal: Experimental & molecular medicine

Article Title: Loss of KDM5B ameliorates pathological cardiac fibrosis and dysfunction by epigenetically enhancing ATF3 expression.

doi: 10.1038/s12276-022-00904-y

Figure Lengend Snippet: Fig. 3 KDM5B deficiency prevents pressure overload-induced cardiac dysfunction and cardiac fibrosis. a Representative echocardio- graphic M-mode images of left ventricles from KDM5B-KO or littermate control WT mice on Day 28 after AngII or normal saline (NS) infusion. b, c Echocardiographic measurement of the LVEF (b) and LVFS (c) of KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). d, e The ratio of heart weight to body weight (HW/BW) (d) and the ratio of heart weight to tibia length (HW/TL) (e) of KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). f–i Representative Masson’s trichrome images and quantitation of perivascular (f, g) or interstitial (h, i) fibrosis in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). Scale bar, 200 μm (left), 100 μm (right). j Q-PCR analysis of Acta2, Col1a1, Col3a1 and Ctgf mRNA expression levels in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion (n = 6 mice per group). k Immunoblot analysis of Col I and Col III protein expression in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII or NS infusion. l Representative immunofluorescence staining of α-SMA (red) and Col III (green) in myocardial tissues from KDM5B-KO or WT mice on Day 28 after AngII infusion. Scale bar, 50 μm. *p < 0.05, **p < 0.01. Unpaired Student’s t test (g, i) or one-way ANOVA (b–e, j) was performed.

Article Snippet: Antibodies against collagen type III (NB600-594) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Control, Saline, Quantitation Assay, Expressing, Western Blot, Staining

Journal: Experimental & molecular medicine

Article Title: Loss of KDM5B ameliorates pathological cardiac fibrosis and dysfunction by epigenetically enhancing ATF3 expression.

doi: 10.1038/s12276-022-00904-y

Figure Lengend Snippet: Fig. 4 KDM5B promotes fibrotic responses and the transition of cardiac fibroblasts. a, b Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression (a) (n = 6 per group) or immunoblot analysis of Col I and Col III protein expression (b) in KDM5B-deficient (KO) or littermate control WT cardiac fibroblasts stimulated with TGF-β (10 ng/ml) for 24 h. c, d Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression (c) (n = 6 per group) or immunoblot analysis of Col I and Col III protein expression (d) in Kdm5b-silenced or control siRNA-transfected cardiac fibroblasts stimulated with TGF-β (10 ng/ml) for 24 h. e, f Q-PCR analysis of Col1a1, Col3a1, Fn1 and Ccn2 mRNA expression (e) (n = 6 per group) or immunoblot analysis of Col I and Col III protein expression (f) in cardiac fibroblasts treated with the KDM5B inhibitor GSK467 or DMSO followed by stimulation with TGF-β (10 ng/ml) for 24 h. g–i Representative immunofluorescence staining of α-SMA (red) in cardiac fibroblasts with KDM5B-deficiency (g), KDM5B knockdown (h) or GSK467 treatment (i) and the corresponding control cardiac fibroblasts (g–i) stimulated with TGF-β (10 ng/ml) for 24 h. Similar results were obtained from three independent experiments (g–i). Scale bar, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired Student’s t test (a, c, e) was performed.

Article Snippet: Antibodies against collagen type III (NB600-594) were obtained from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Western Blot, Control, Transfection, Staining, Knockdown

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Transcriptional Profiling of Muscle in Females With Distal Radius Fracture and Functional Sarcopenia

doi: 10.1093/gerona/glae002

Figure Lengend Snippet: Transcripts With Altered Gene Expression in the EXP Compared to NORM

Article Snippet: Recombinant human pro-collagen COL1A1 (R&D System, Minneapolis, MN, USA, 6220-CL) 75 μg/mL and recombinant human pro-collagen COL3A1 (R&D System, 7294-CL) 75 μg/mL were incubated with 5 μg/mL recombinant human BMP-1 (R&D, 1927-ZN) for 2 hours.

Techniques: Gene Expression

Journal: Scientific Reports

Article Title: Enhanced antiviral and antifibrotic effects of short hairpin RNAs targeting HBV and TGF-β in HBV-persistent mice

doi: 10.1038/s41598-017-04170-1

Figure Lengend Snippet: AAV-shRNA treatment decreases the expression of liver fibrosis biomarkers in HBV-persistent mice. Mice received AAV2-shRNA treatment, and serum levels of TGF-β ( a ), collagen I ( b ), and collagen III ( c ) were measured by ELISA during a 6 month period. ( d – g ) Reverse transcription quantitative PCR measurements of Tgf-β1 ( d ), Col I ( e ), Col III ( f ), and α-SMA ( g ) mRNA levels in the liver. ( h ) Western blot analysis of α-SMA in liver samples. Statistical analyses were performed using a two-way analysis of variance. P < 0.05: significant difference. Ns: the difference was not significant. Data represent the mean ± SD (n = 4).

Article Snippet: The levels of TGF-β1, collagen I, and collagen III in the liver and serum samples were determined using commercially available ELISA kits including Mouse TGF-β1 Quantikine ELISA kit, Mouse Collagen type I (Col I) ELISA kit, and Mouse Collagen type III (Col III) ELISA kit (R&D Systems, Minneapolis, MN).

Techniques: shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

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