Journal: bioRxiv

Article Title: NOX4 inhibition promotes the remodeling of dystrophic muscle

doi: 10.1101/2022.01.08.475493

Figure Lengend Snippet: ( A ) Representative immunofluorescent images from Nox4 WT : mdx and Nox4 KO : mdx gastrocnemius (gastroc) muscles stained with anti-NOX4 antibody. Muscle fibrosis was histologically assessed in the diaphragm and gastroc muscles from 3 month-old Nox4 WT : mdx and Nox4 KO : mdx mice using picrosirius red staining (n = 4). ( B ) Representative images and ( C-D ) histological quantifications reveal no significant differences in muscle fibrosis between the groups at this age. Data are presented as box-and-whisker plots with error bars representing minimum and maximum values. Data were analyzed using unpaired, two-tailed Welch’s T-tests (α = 0.05; effect size is presented as Cohen’s d ). Scale bar represents 100 µm.

Article Snippet: Picrosirius red staining was performed as previously described ( ) following decalcification of muscle sections using Formical-2000 (StatLab).

Techniques: Muscles, Staining, Whisker Assay, Two Tailed Test

Journal: bioRxiv

Article Title: NOX4 inhibition promotes the remodeling of dystrophic muscle

doi: 10.1101/2022.01.08.475493

Figure Lengend Snippet: ( A ) Representative picrosirius red staining and image quantification of ( B ) diaphragm and ( C ) gastrocnemius muscles from 6 month-old D2.WT (n = 6), NOX4 wild-type (NOX4-WT) and knockout (NOX4-KO) littermates from a NOX4 KO line generated on the D2. mdx background (Nox4 KO : mdx ; n = 12), and D2. mdx mice that received vehicle or GK831 (GKT) treatments beginning at 3 months of age (n = 12-14). Fibrosis quantifications for 3 month-old D2. mdx mice are included to show baseline values for GKT treatment groups. Muscle function was assessed for the ( D ) diaphragm and ( E ) extensor digitorum longus (EDL) muscles of NOX4-WT (+/+) and NOX4-KO (-/-) littermates of the Nox4 KO : mdx mouse line. ( F ) Biochemical quantification of collagen content was performed on gastrocnemius muscles of vehicle and GKT-treated D2. mdx mice. Data are presented as box-and-whisker plots with error bars representing minimum and maximum values. ( D-F ) Percent values indicate the difference between mean values of the two groups. Data were analyzed using ( B-C ) ANOVA followed by Tukey post-hoc tests (α = 0.05; effect size is reported as η 2 ) or ( D-F ) unpaired, two-tailed Welch’s T-tests (α = 0.05; effect size is reported as Cohen’s d ); *p < 0.05 vs. D2.WT values; #p < 0.05 vs. 3 mo D2. mdx values; $p < 0.05 vs. respective control group values.

Article Snippet: Picrosirius red staining was performed as previously described ( ) following decalcification of muscle sections using Formical-2000 (StatLab).

Techniques: Staining, Muscles, Knock-Out, Generated, Whisker Assay, Two Tailed Test, Control

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: Apabetalone blocks TGF-β1-induced HRMC activation. ( A ): HRMCs were treated with TGF-β1 ± BETi or TGFBRi for 24 h followed by gene expression analysis by real-time PCR ( n = 5). ( B ): Representative images of HRMCs treated with TGF-β1 ± apabetalone for 48 h, followed by immunofluorescence microscopy for α-SMA (green); nuclei were stained with DAPI (blue); apa = apabetalone. ( C ): Fluorescence intensity of α-SMA was quantified as percent of the image area. ( D ): Collagen gel contraction was evaluated after 4 days of treatment ( n = 6). ( E ) Collagen deposition was evaluated by picrosirius red staining after 5 days of treatment ( n = 4). Data in bar graphs are the mean ± SD. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. *** p < 0.001, ** p < 0.01, NS not significant. ACTA2: α-SMA gene. α-SMA: alpha smooth muscle actin. TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation. TGFBRi: small molecule inhibitor of the TGF-β receptor.

Article Snippet: Collagen was stained with picrosirius red solution (Abcam # ab246832) for 60 min, followed by destaining with 0.1 N HCl.

Techniques: Activation Assay, Gene Expression, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy, Staining, Fluorescence, Comparison

Journal: bioRxiv

Article Title: IL-11 neutralising therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH

doi: 10.1101/470062

Figure Lengend Snippet: a , IL-11 is upregulated in hepatic stellate cells (HSCs) stimulated with TGFβ1 (n=3). b , IL-11 protein is secreted from HSCs stimulated with TGFβ1 (ELISA, n=3). c , Human precision cut liver slices were stimulated with TGFβ1 and IL-11 protein was measured in supernatant (ELISA, n=3). d , IL6R, gp130, and IL11RA expression in HSCs (TPM, transcripts per million). e, f , Representative fluorescence images (scale bars, 200 µm) of HSCs and automated fluorescence quantification for ( e ) ACTA2 +ve cells and ( f ) Collagen I immunostaining following incubation without stimulus (-), with TGFβ1, PDGF, or IL-11. g , Collagen secretion supernatants of HSC stimulated with TGFβ1, PDGF, or IL-11 (Sirius red assay. n≥7). h , Dose-dependent matrigel invasion of HSCs induced by IL-11(n=3). i , Hyper IL-11 induces IL-11 protein secretion from HSCs (ELISA, n=3). a-c, e-g, i , TGFβ1 (5 ng ml -1 ), Hyper IL-11 (0.2 ng ml -1 ), PDGF (20 ng ml -1 ), IL-11 (5 ng ml -1 ); 24 h stimulation; h , 48 h stimulation. j , Schematic of mice receiving daily subcutaneous injection of either saline (control) or rmIl-11 (100 µg kg -1 ). k , Relative liver hydroxyproline content, l , mRNA expression of pro-fibrotic markers, and m , serum ALT levels (k, l , control, n=7; rmIl-11, n=10; m , control n=8; rmIl-11, n=11). a, b, g, h, i, k, m Data are represented as mean ±s.d; c-f, l , Box-and-whisker plots show median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers). a-d, i, k-m , Two-tailed Student’s t -test; e-h , two-tailed Dunnett’s test. FC: fold change; I/A: intensity/area.

Article Snippet: Total secreted collagen in the cell culture supernatant was quantified using Sirius red collagen detection kit (9062, Chondrex).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Immunostaining, Incubation, Injection, Whisker Assay, Two Tailed Test