Journal: Investigative Ophthalmology & Visual Science

Article Title: Defining the Role of Mitochondrial Fission in Corneal Myofibroblast Differentiation

doi: 10.1167/iovs.63.4.2

Figure Lengend Snippet: Intracellular mediators underlying anti-fibrotic effects of Mdivi-1. ( A ) Representative Western blot depicting phospho- and total-SMAD3 expression levels in protein extracts from cultured cat corneal fibroblasts. Cells were incubated ± 10 µM Mdivi-1, ± 1 ng/mL TGF-β1 and finally 2.3 µM SB431542 with 1 ng/mL TGF-β1, as indicated. ( B ) Plot of densitometric ratios for phospho-SMAD3 expression relative to total-SMAD2/3, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 116.3, P < 0.0001. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 alone are denoted: * P < 0.05, # P < 0.01. ( C ) Representative Western blots of corneal fibroblast extracts for phosphor-p54 and -p46 and total-JNK expression under the conditions indicated. ( D ) Plot of densitometric ratios for p-p54 JNK expression relative to total-p54 JNK, normalized to the TGF-β1 treatment. One-way repeated measures ANOVA: F(3,11) = 7.48, P = 0.01884. Significances relative to the TGF-β1 condition were computed and are denoted as in B . ( E ) Representative Western blot of corneal fibroblast extracts showing phospho-p38, phospho-ERK, and phospho-AKT expression levels under the conditions indicated. ( F ) Plot of densitometry ratios for phosphorylated proteins relative to β-actin, normalized to the TGF-β1 treatment. One-way repeated measures ANOVAs demonstrated significant effects of treatments for all 3 molecules: p38 F(3,11) = 37.52, P = 0.0003; pERK F(3,11) = 11.12, P = 0.0073; pAKT F(3,11) = 8.85, P = 0.0127. Significances from post-hoc Tukey's HSD tests relative to TGF-β1 are indicated as in B and D . For all graphs, data shown are means ± SD from 3 experimental replicates.

Article Snippet: Blots were incubated overnight at 4°C containing primary antibodies to the following targets at the dilutions indicated: Type 1 collagen (COL1; 1:2000; no. LF-68, kindly provided by Dr. Larry W. Fisher, NIH, Bethesda, MD, USA), total Fibronectin (t-FN; 1:2000; #H-300; Santa Cruz Inc.), α-SMA (1:10,000; no. MA5-11547; Thermo Fisher Scientific, Waltham, MA, USA), total SMAD 2/3 (1:2000; no. 8685; Cell Signaling Technology, Danvers, MA, USA), phosphorylated small mothers against decapentaplegic 3 (SMAD3) (S423+S425; 1:1000; no. P00059-1; Boster Technology, Pleasanton, CA, USA), phospho-p46/54 JNK Thr183/Tyr185 (1:1000; no. 4671; Cell Signaling Technology), phospho-p38 MAPK Thr180/Tyr182 (1:1000; no. 9215; Cell Signaling Technology), phospho-p44/42 MAPK Thr202/Tyr204 (1:1000; no. 9106; Cell Signaling Technology), phospho-AKT Ser473 (1:1000; no. 4060; Cell Signaling Technology), total-p46/54 JNK (1:1000; no. 9252; Cell Signaling Technology), p-Drp1 Ser616 (1:500; no. 3455; Cell Signaling Technology), total-Drp1 (1:1000; no. 611112; BD Biosciences, San Jose, CA, USA), Opa1 (1:1000; no. 612606; BD Biosciences), Mfn1 (1:1000; ab57602; Abcam, Boston, MA, USA), Mfn2 (1:1000; no. M6319; Sigma Aldrich), and β-actin-HRP (1:5000; no. sc-47778; Santa Cruz Inc.).

Techniques: Western Blot, Expressing, Cell Culture, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Losing Regulation of the Extracellular Matrix is Strongly Predictive of Unfavorable Prognostic Outcome after Acute Myocardial Infarction

doi: 10.3390/ijms21176219

Figure Lengend Snippet: Protein expressions of apoptosis and fibrosis in LV myocardium by 60 days after the AMI procedure. ( A ) Protein expressions of mitochondrial (mito)-Bax, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( B ) Protein expression of cleaved caspase 3 (c-Csp3), * vs. other groups with different symbols (†, ‡), p < 0.0001. ( C ) Protein expression of cleaved poly (ADP-ribose) polymerase (c-PARP), * vs. other groups with different symbols (†, ‡), p < 0.0001. ( D ) Protein expression of phosphorylated (p)-Smad3, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( E ) Protein expression of transforming growth factor (TGF)-ß, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( F–I ) Illustrating the H&E stain for identification of lung injury score (200×). ( J ) Analytical result of the number of alveolar sacs, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( K ) Analytical result of the crowded score, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( L ) Analytical result of wall thickness, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test ( n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). SC = sham-operated control; MT DO = double knock out of matrix metalloproteinase (MMP)-9 and tissue plasminogen (tPA) in mice; B6 (i.e., wild type) = C57BL/6 mice. LV = left ventricular; AMI = acute myocardial infarction.

Article Snippet: Then, the membranes were incubated with the indicated primary antibodies (matrix metalloproteinase (MMP)-2 (1:1000, Cell Signaling, Beverly, MA, USA), MMP-9 (1:1000, Abcam), phosphorylated (p)-nuclear factor (NF)-κB (1:1000, Cell Signaling), tumor necrosis factor (TNF)-α (1:1000, Cell Signaling), cleaved caspase 3 (1:1000, Cell Signaling), mitochondrial Bax (1:1000, Abcam), mitochondrial Bax (1:1000, Abcam), cleaved caspase 3 (1:1000, Cell Signaling), cleaved poly (ADP-ribose) polymerase (C-PARP) (1:1000, Cell Signaling), phosphorylated (p)-Smad3 (1:1000, Cell Signaling), transforming growth factor (TGF)-ß (1:1000, Abcam), NOX-1 (1:1500, Sigma, Darmstadt, Germany), NOX-2 (1:1000, Sigma), brain natriuretic peptide (BNP) (1:1500, Abcam), ß-myosin heavy chain (ß-MHC) (1:1000, Santa Cruz, Dallas, TX, USA), α-MHC (1:1000, Santa Cruz), cytosolic cytochrome C (1:2000, BD, Franklin Lakes, NJ, USA), mitochondrial cytochrome C (1:2000, BD), phosphorylated (p)-γ-H2AX (1:1000, Cell Signaling) and GAPDH (I: 10000, Cell Signaling)) for 1 h at room temperature.

Techniques: Expressing, Staining, Comparison, Control, Knock-Out

Journal: Kidney international

Article Title: Inhibition of transforming growth factor β1 signaling in resident interstitial cells attenuates profibrotic gene expression and preserves erythropoietin production during experimental kidney fibrosis in mice.

doi: 10.1016/j.kint.2021.02.035

Figure Lengend Snippet: Figure 4 | Lineage tracing for platelet-derived growth factor receptor b–positive (PDGFR-bD) cells was performed using PDGFR- bCreERT2/D mT/mG mice as controls without transforming growth factor b receptor 2 (TGFb-R2) deletion and in mice with tamoxifen- inducible deletion of TGFb-R2 in PDGFR-bD cells (TGFb-R2ko) with the mT/mG reporter. After tamoxifen treatment, all cells with Cre activation start to express green fluorescent protein (GFP). (Top row) Immunohistochemistry for a-smooth muscle actin (a-SMA) (red; left), GFP (green; middle), and merge (right) for lineage tracing of matrix-producing PDGFR-bþ cells on kidney tissue of a TGFb-R2ko mT/mG mouse after 5-day unilateral ureteral occlusion (UUO). Costaining of a-SMA showed a good overlap with GFP in a majority of myofibroblasts identified by a-SMA. These cells were targeted by the deletion of TGFb-R2. Nuclei were counterstained with 40,6-diamidino-2-phenylindole (blue). Bars ¼ 50 mm. (Bottom row) Immunohistochemistry for the active nuclear transcription-factor phosphorylated SMAD3 (pSMAD3) (red) and GFP (green) on kidney sections of PDGFR-bCreERT2/þ mT/mG mice (left) and TGFb-R2ko mice (right) after 5-day UUO. In control kidneys, a robust nuclear pSMAD3 signal as an indicator for an activated TGFb pathway could be detected in tubules and GFPþ interstitial cells (arrows) after 5-day UUO. In TGFb-R2ko mice, GFPþ interstitial cells showed no activated phosphorylated SMAD3 (pSMAD3) signal (arrows), whereas the tubular pSMAD3 signal was preserved. Bars ¼ 20 mm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Article Snippet: Chicken anti– green fluorescent protein (ab13970, Abcam) and rabbit anti– phosphorylated SMAD3 (#9520S, Cell Signaling Technology) immunohistochemistry was performed analogously.

Techniques: Derivative Assay, Activation Assay, Immunohistochemistry, Control

Journal: Neural Regeneration Research

Article Title: Fibrotic scar formation after cerebral ischemic stroke: Targeting the Sonic hedgehog signaling pathway for scar reduction

doi: 10.4103/NRR.NRR-D-24-00999

Figure Lengend Snippet: Inhibiting Smad3 phosphorylation diminishes upregulated-effect of Shh signaling activation on MFN2 expression. (A) Timeline of in vivo experiments. Created with BioRender.com. (B–D) Western blot analysis of the expression of MFN2 and P-Smad3 protein and quantification data in the Sham, MCAO/R, MCAO/R + rAd-Shh, MCAO/R + rAd-Shh + SIS3 and MCAO/R + rAd-Shh + DMSO groups ( n = 3). (E) Neurological scores after stroke onset in the Sham, MCAO/R, MCAO/R + rAd-Shh, MCAO/R + rAd-Shh + SIS3, and MCAO/R + rAd-Shh + DMSO groups (Kruskal-Wallis H test). (F) Representative immunofluorescence images of MFN2 (green)/α-SMA (red)-positive cells, Fn (red) and TUNEL (red) staining in the Sham, MCAO/R, MCAO/R + rAd-Shh, MCAO/R + rAd-Shh + SIS3, and MCAO/R + rAd-Shh + DMSO groups. Scale bars: 20 µm or 50 µm. (G, H) Representative images at 7 days following MCAO/R injury and quantitative analysis of infarct volumes in the Sham, MCAO/R, MCAO/R + rAd-Shh, MCAO/R + rAd-Shh + SIS3 and MCAO/R + rAd-Shh + DMSO groups ( n = 3). Scale bars: 5 mm. (I) Timeline of in vitro experiments. Created with BioRender.com. (J–L) Western blot analysis of the expression of MFN2 and P-Smad3 proteins and quantification data in the Con, rAd-Shh, rAd-Shh + SIS3 and rAd-Shh + DMSO groups ( n = 3). (M–O) Western blot analysis of the expression of MFN2 and P-Smad3 proteins and quantification data in the Con, SAG, SAG + SIS3, and SAG + DMSO groups ( n = 3). (P) Representative immunofluorescence images of α-SMA (red)-positive cells in the Con, rAd-Shh, rAd-Shh + SIS3, and rAd-Shh + DMSO groups. Scale bars: 20 µm. (Q, R) The migration ability of fibroblasts in the rAd-Shh, rAd-Shh + SIS3, and rAd-Shh + DMSO groups was evaluated by Transwell assay. Scale bar: 50 µm. (S) Representative immunofluorescence images of α-SMA (red)-positive cells in the Con, SAG, SAG + SIS3, and SAG + DMSO groups. Scale bars: 20 µm. (T, U) The migration ability of fibroblasts in the Con, SAG, SAG + SIS3, and SAG + DMSO groups was evaluated by Transwell assay. Scale bar: 50 µm. Data are expressed as mean ± SD. * P < 0.05, vs . Sham or Con group; # P < 0.05, vs. MCAO/R or rAd-Shh or SAG group; & P < 0.05, vs . the MCAO/R + rAd-Shh or rAd-Shh + SIS3 or SAG + SIS3 group; Δ P < 0.05, vs . the MCAO/R + rAd-Shh + SIS3 group (one-way analysis of variance followed by Tukey’s post hoc test). Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; Fn: fibronectin; IF: immunofluorescence; MCAO/R: middle cerebral artery occlusion/reperfusion; MFN2: mitofusin 2; P-Smad3: phosphorylated-mothers against decapentaplegic homolog 3; rAd-HK: adenoviral control vector; rAd-Shh recombinant adenovirus-sonic hedgehog; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; WB: western blot; α-SMA: alpha-smooth muscle actin.

Article Snippet: SIS3 (Smad3 phosphorylation inhibitor; 10 μL, 0.8 mg/mL, MCE, HY-13013) was injected intracerebroventricularly at 1 day after MCAO/R surgery.

Techniques: Phospho-proteomics, Activation Assay, Expressing, In Vivo, Western Blot, Immunofluorescence, TUNEL Assay, Staining, In Vitro, Migration, Transwell Assay, Control, Plasmid Preparation, Recombinant