Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet: MiR-4769-3p inhibition promotes subcutaneous adipogenesis in SSc mice The mouse model of SSc was induced with bleomycin and then subcutaneously injected with Antago-miR-4769-3p or Antago-NC. (A) Histopathological changes in the skin tissues of mice in each group were assessed using H&E and Masson’s staining, and subcutaneous adipogenesis was assessed using oil red O staining (red indicates stained lipid droplets) (100×). (B and C) After treatment with Antago-miR-4769-3p or Antago-NC, the expression of miR-4769-3p in skin lesions of bleomycin-induced SSc mice was analyzed using real-time qPCR (B), and the levels of adipogenic proteins (PPAR-γ, FABP4, SREBP-1, and Lipin1) were measured using western blot analysis (C). Data are presented as mean ± SEM (n = 3); statistical analysis by ANOVA or Student’s t test: ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 versus the indicated group.

Article Snippet: Bleomycin , APExBIO , A8331.

Techniques: Inhibition, Injection, Staining, Expressing, Western Blot

Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet: USP18 inhibits the ubiquitination and degradation of VDAC2, synergistically promoting adipogenesis in 3T3-L1 cells (A) The levels of USP18 and HA in 3T3-L1 cells transfected with USP18-HA-OE fusion vector were measured using western blot analysis. (B) 3T3-L1 cells co-immunoprecipitated with HA antibody were analyzed by mass spectrometry for the binding proteins of USP18, and finally VDAC2 was focalized. (C) VDAC2 expression was evaluated by immunohistochemical staining in skin samples from SSc patients and healthy controls (dark brown indicates VDAC2) (100×). (D) 3T3-L1 cells co-immunoprecipitated with HA or VDAC2 antibodies were analyzed by coIP assay to verify the direct binding between USP18 and VDAC2. (E) VDAC2 expression in 3T3-L1 cells transfected with USP18-HA-OE fusion vector were measured using western blot analysis. (F and G) The mRNA and protein levels of VDAC2 in 3T3-L1 cells infected with si-USP18 or si-VDAC2 were analyzed using real-time qPCR (F) and western blot analysis (G). (H and I) After inducing adipogenic differentiation of 3T3-L1 cells infected with si-VDAC2 for 8 days, lipid droplet formation was assessed using oil red O staining (H) (red indicates stained lipid droplets) (200×), and the levels of adipogenic proteins (PPAR-γ, FABP4, SREBP-1, and Lipin1) were measured using western blot analysis (I). (J and K) The ubiquitination level of VDAC2 was analyzed by coIP assay in USP18-overexpressing cells (J) or skin lesions of bleomycin-induced SSc mice (K) after immunoprecipitation with VDAC2. Data are presented as mean ± SEM (n = 3); statistical analysis by ANOVA or Student’s t test: ∗∗ p < 0.01 and ∗∗∗∗ p < 0.0001 versus the indicated group.

Article Snippet: Bleomycin , APExBIO , A8331.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Mass Spectrometry, Binding Assay, Expressing, Immunohistochemical staining, Staining, Co-Immunoprecipitation Assay, Infection

Journal: iScience

Article Title: MiR-4769-3p suppresses adipogenesis in systemic sclerosis by negatively regulating the USP18/VDAC2 pathway

doi: 10.1016/j.isci.2024.110483

Figure Lengend Snippet:

Article Snippet: Bleomycin , APExBIO , A8331.

Techniques: Virus, Over Expression, Plasmid Preparation, Recombinant, Staining, Modification, Transfection, Reverse Transcription, SYBR Green Assay, Luciferase, Reporter Gene Assay, Silver Staining, Mass Spectrometry, Software

Journal: Expert opinion on emerging drugs

Article Title: Emerging drugs for head and neck cancer

doi: 10.1517/14728214.2015.1031653

Figure Lengend Snippet: Current FDA-approved drugs for the treatment of HNSCC include five conventional chemotherapy drugs (cisplatin, methotrexate, 5-flurouracil, bleomycin, docetaxel) and one targeted agent (cetuximab). Emerging investigated drugs in clinical trials including other targeted agents, immunotherapy agents and gene therapy agents are represented here. Briefly, EGFR, VEGFR, c-MET and IGF-1R signals utilize a variety of downstream molecular pathways including the PI3K/Akt/mTOR, STAT and Ras/Raf/MEK/MAPK. Aberrant activation of these pathways are associated with tumor growth and angiogenesis. Agents targeting these pathways include monoclonal antibodies (cetuximab, zalutumumab, panitumumab, nimotuzumab, bevacizumab, ficlatuzumab and figitumumab), tyrosine kinase inhibitors (gefitinib, erlotinib, lapatinib, afatinib, dacomitinib), multikinase inhibitors (sorafenib, sunitinib), downstream inhibitors including Src family kinase inhibitors (dasatinib) and PI3K/Akt/mTOR inhibitors (BKM120, BYL719, everolimus, temsirolimus). Gene therapy includes Adp53/CRAdp53-based gene therapy (Gendicine, H-101, SCH-58500, ONYX-015) and EGFR antisense DNA. Immunotherapy includes non-specific immune stimulators/agents (nivolumab, IRX-2) and vaccine candidates (peptide-based vaccine, DNA vaccine and dendritic cell vaccine).

Article Snippet: Bleomycin , PCI Biotech , Marketed , DNA inhibitor.

Techniques: Clinical Proteomics, Activation Assay, Bioprocessing

Journal: Expert opinion on emerging drugs

Article Title: Emerging drugs for head and neck cancer

doi: 10.1517/14728214.2015.1031653

Figure Lengend Snippet: Competitive environment table of the major drugs and compounds currently applied or under development for HNSCC treatment.

Article Snippet: Bleomycin , PCI Biotech , Marketed , DNA inhibitor.

Techniques: DNA Synthesis, Virus

Journal: Cell Reports Medicine

Article Title: Signaling-induced systematic repression of miRNAs uncovers cancer vulnerabilities and targeted therapy sensitivity

doi: 10.1016/j.xcrm.2023.101200

Figure Lengend Snippet:

Article Snippet: Bleomycin , Hoelzel Diagnostika , Cat# ACS-1994B.

Techniques: Recombinant, Membrane, Protease Inhibitor, Isolation, Proliferation Assay, Luminescence Assay, Expressing, Sequencing, Comparison, Software

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Thromboxane–Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis

doi: 10.1164/rccm.202106-1503OC

Figure Lengend Snippet: TBXA2R (thromboxane–prostanoid receptor) is upregulated in fibroblasts during lung fibrosis. ( A ) Western blot nondiseased for TBXA2R in lung tissue from patients with idiopathic pulmonary fibrosis (IPF) compared with nondiseased control subjects. ( B ) Dot plot depicting TBXA2R gene expression in jointly analyzed single-cell RNA sequencing data from explanted lungs of 20 patients with pulmonary fibrosis and 10 nondiseased control subjects (primary data from Reference 6 ). ( C ) Representative immunostaining for TBXA2R in IPF and control lung sections. TBXA2R is red, and vimentin is green. ( D ) Western blot for TBXA2R expression in lungs of mice at baseline and 1–3 weeks after intratracheal bleomycin. ( E ) Immunostaining for TBXA2R in mouse lung sections at 3 weeks after treatment with vehicle (control) or bleomycin. TBXA2R is red. Scale bar, 50 μm. AT1 = alveolar type 1; AT2 = alveolar type 2; Bleo = bleomycin; cDC = classical dendritic cell; HAS1 = hyaluronan synthase 1; HSP70 = heat shock protein 70; KRT5 = keratin 5; KRT17 = keratin 17; MUC5AC = mucin 5AC, oligomeric mucus/gel-forming; MUC5B = mucin 5B, oligomeric mucus/gel-forming; NK = natural killer; pDC = plasmacytoid dendritic cell; PLIN2 = perilipin 2; SCGB1A1 = secretoglobin family 1A member 1; SCGB3A2 = secretoglobin family 3A member 2.

Article Snippet: Bleomycin (Hospira Inc.) was purchased from the Vanderbilt University Medical Center pharmacy.

Techniques: Western Blot, Control, Gene Expression, Single Cell, RNA Sequencing, Immunostaining, Expressing

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Thromboxane–Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis

doi: 10.1164/rccm.202106-1503OC

Figure Lengend Snippet: Deleting TBXA2R (thromboxane–prostanoid receptor) protects against experimental lung fibrosis. ( A ) Representative Masson’s trichrome–stained lung sections from wild-type (WT) and TBXA2R iKO ( Rosa26-CreER + TBXA2R f / f ) mice at 21 days after intratracheal bleomycin (Bleo). ( B ) Morphometric evaluation of lung fibrosis on lung sections. n = 3 for control WT and TBXA2R iKO mice, and n = 5 for the Bleo treatment groups. ( C ) Hydroxyproline content in the right upper lobe. n = 3 for control WT mice, n = 2 for control TBXA2R iKO mice, n = 11 for the Bleo-treated WT mice, and n = 8 for the Bleo-treated TBXA2R iKO mice. Statistical comparison was performed using the Wilcoxon rank sum test (also P < 0.05 by two-way ANOVA with Tukey’s honestly significant difference test). ( D ) Representative Masson’s trichrome–stained lung sections from WT mice at 21 days after treatment with intratracheal Bleo or vehicle (Veh), with or without thromboxane synthesis inhibitor (Oza) beginning on Day 7. ( E ) Morphometric evaluation of lung fibrosis on lung sections. n = 6 for each group. ( F ) Hydroxyproline content in right upper lobe. n = 6 for each group. Error bars in all panels denote SEM. N.S. = not significant by Wilcoxon rank sum test; Oza = ozagrel; Rt = right.

Article Snippet: Bleomycin (Hospira Inc.) was purchased from the Vanderbilt University Medical Center pharmacy.

Techniques: Staining, Control, Comparison

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Thromboxane–Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis

doi: 10.1164/rccm.202106-1503OC

Figure Lengend Snippet: F2-isoprostanes (F2-IsoPs) activate TBXA2R signaling in fibrosis. ( A ) TxB2 was measured in the lungs of mice at baseline and 1–3 weeks after bleomycin (Bleo). n = 3 per group. * P < 0.05 compared with baseline. ( B ) F2-IsoPs measured in the lungs of mice at baseline and 1–3 weeks after intratracheal Bleo. n = 3 or 4 per group. * P < 0.05 compared with baseline. ( C ) Primary lung fibroblasts from Rosa-creER / Tbxa2r f / f mice were treated ex vivo with 4OH-tamoxifen to obtain TBXA2R-deleted fibroblasts (TBXA2R iKO ) and BrdU incorporation was measured at 48 hours under full-serum (10%) conditions. n = 6 mice per group. * P < 0.05 compared with wild-type (WT) fibroblasts. ( D ) F2-IsoP (100 nM) stimulation of WT and Tbxa2r iKO fibroblasts under reduced serum conditions (2.5%) measured at 48 hours. n = 4 per group. * P < 0.05. ( E ) Immunocytochemistry evaluating for α-SMA (α-smooth muscle actin) expression 48 hours after F2-IsoP treatment. α-SMA, red; DAPI, blue. Scale bars, 40× image with 100 μm. ( F ) Western blots for α-SMA and collagen 1 expression at 48 hours after F2-IsoP treatment. ( G ) Densitometry for collagen 1. n = 3 lungs per condition. All comparisons were significant both by nonparametric (Wilcoxon rank sum) and parametric (ANOVA with post hoc test) measures. Error bars denote SEM. BrdU = bromodeoxyuridine; Hsp70 = heat shock protein 70; N.S. = not significant; TxB2 = thromboxane B2; TBXA2R = thromboxane–prostanoid receptor.

Article Snippet: Bleomycin (Hospira Inc.) was purchased from the Vanderbilt University Medical Center pharmacy.

Techniques: Ex Vivo, BrdU Incorporation Assay, Immunocytochemistry, Expressing, Western Blot

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Thromboxane–Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis

doi: 10.1164/rccm.202106-1503OC

Figure Lengend Snippet: TBXA2R signaling regulates bleomycin (Bleo)–induced fibrosis, even when started 7 and 14 days after injury. ( A ) Experimental schema showing the timing of ifetroban (Ifet) start and date of sacrifice for panels. The top two lines correspond to B – D in this figure and the bottom line to E – K . ( B–D ) Wild-type mice were treated with intratracheal Bleo (0.04 U) or vehicle (Veh), with or without Ifet (25 mg/kg/day). Ifet treatment (Ifet[tr]) was delivered from Day 7 to Day 21 after Bleo. Ifet prevention (Ifet[pr]) was started 3 days before Bleo treatment and continued to Day 21. ( B ) Representative Masson’s trichrome–stained lung sections. ( C ) Morphometric evaluation of lung fibrosis using these lung sections. ( D ) Hydroxyproline content in right upper lobe. * P < 0.05 by Wilcoxon signed rank test. ( E ) Change in weight after Bleo treatment. Data are presented as mean ± SEM. Significance was determined using ANOVA with repeated measures. ( F ) Representative Masson’s trichrome–stained lung sections from wild-type mice at 28 days after treatment with intratracheal Bleo or Veh with or without Ifet treatment begun at Day 14. ( G ) Morphometric evaluation of lung fibrosis on lung sections. Data are presented as mean ± SEM. ( H ) Hydroxyproline content quantified from the right upper lobe. Data are presented as mean ± SEM. * P = 0.05 by Wilcoxon signed rank test. ( I ) Western blot for Timp-1 (tissue inhibitor of metalloproteinase-1), phospho-Smad2/3, and total Smad2/3 in lung tissue. ( J ) Densitometry for Timp-1. * P < 0.05 by Wilcoxon signed rank test. ( K ) Densitometry for total and phospho-Smad2/3 ( n = 5 per group). * P < 0.05 by Wilcoxon signed rank test. Error bars denote SEM. BW = body weight; P-Smad2/3 = phospho-Smad2/3; rt = right; TBXA2R = thromboxane–prostanoid receptor; T-Smad2/3 = total Smad2/3.

Article Snippet: Bleomycin (Hospira Inc.) was purchased from the Vanderbilt University Medical Center pharmacy.

Techniques: Staining, Western Blot

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Thromboxane–Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis

doi: 10.1164/rccm.202106-1503OC

Figure Lengend Snippet: TBXA2R antagonism attenuates Hermansky-Pudlak syndrome (HPS)-associated fibrosis and radiation-induced fibrosis. ( A and B ) HPS1, HPS2, or wild-type control mice were treated with ifetroban (Ifet) or vehicle, followed by intratracheal bleomycin (0.025 U), and lungs were harvested on Day 7. Sircol assay was conducted to quantify collagen content in lungs of HPS1 mice or HPS2 mice. n = 3 or 4 per group unchallenged mice and 9–17 per group HPS mice. ( C ) Representative histology for HPS2 mice with or without Ifet pretreatment. ( D and E ) Sircol assay quantifying collagen content ( D ) and representative histology from lungs of mice exposed to thoracic irradiation (17 Gy) together with Ifet ( n = 5) or vehicle ( n = 12) and harvested at 4 months after irradiation ( E ). Top row, scale bars, 4× image with 1000 μm; bottom row, scale bars, 40× with 100 μm. * P < 0.05. For all quantitative figure elements, data are presented as mean ± SEM, with data from each individual mouse overlaid as circles. Statistical analysis was performed using ANOVA, followed by Tukey’s honestly significant difference test for comparisons between individual groups (* P < 0.05). Bleo = bleomycin; Rad = radiation; TBXA2R = thromboxane–prostanoid receptor; Veh = vehicle; WT = wild-type.

Article Snippet: Bleomycin (Hospira Inc.) was purchased from the Vanderbilt University Medical Center pharmacy.

Techniques: Control, Irradiation

Journal:

Article Title:

doi:

Figure Lengend Snippet: Standard-dose bleomycin increases pulmonary vascular permeability in EC- S1pr1 −/− mice. ( A ) Evans blue dye assay was performed on Day 0 and Day 7 after standard-dose intratracheal (IT) bleomycin (1.0 U/kg). ( B ) Quantification of vascular permeability was calculated as an Evans blue (EB) index defined as the ratio of lung Evans blue to plasma Evans blue. n = 3–7 mice per group. ( C ) Hematoxylin and eosin and Masson’s trichrome staining of lungs was performed on Day 7 after standard dose bleomycin. Representative images are shown from n = 3 mice per group. Scale bars, 50 μm. ( D ) Survival of mice receiving standard-dose (1.0 U/kg) bleomycin. n = 5 mice per group. * P < 0.05 comparing survival of EC- S1pr1 −/− mice to littermate controls S1pr1 f/f . *** P < 0.001 comparing EB index in EC- S1pr1 −/− mice to littermate controls S1pr1 f/f . H&E = hematoxin and eosin; Trich = trichrome.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Permeability, Clinical Proteomics, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Low-dose bleomycin induces fibrosis in EC- S1pr1 −/− mice. ( A ) Survival of mice receiving low-dose (0.5 U/kg) bleomycin. n = 5 mice per group. ( B ) Total protein content in the BAL was quantified at Days 0, 7, 14, and 21 after low-dose bleomycin. BAL from EC- S1pr1 −/− mice was compared with intact littermate controls S1pr1 f/f . n = 3–5 mice per group. ( C ) Hematoxylin and eosin and Masson’s trichrome staining of lungs were performed on Day 21 after low-dose bleomycin. Representative images are shown from n = 3 mice per group. Scale bars, 50 μm. ( D ) Fibrosis was quantified at Days 0, 7, 14, 21, and 42 after low-dose IT bleomycin by measuring hydroxyproline levels in the mouse lungs. n = 3–15 mice per group. ( E ) Ashcroft scoring performed on trichrome stained lung sections from EC- S1pr1 −/− mice at Days 0 and 14 or 21 was compared with intact littermate controls S1pr1 f/f . n = 3–4 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of total protein or hydroxyproline in EC- S1pr1 −/− mice compared with intact littermate control mice S1pr1 f/f . OHP = hydroxyproline.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Staining, Control

Journal:

Article Title:

doi:

Figure Lengend Snippet: Endothelial-specific S1pr1 deletion leads to reduced intercellular junction proteins and increased extravascular coagulation. ( A ) Mice were injected with FITC-labeled albumin to identify albumin extravasated into the lung tissue after bleomycin. Sequential sectioning and staining of the lung tissue were performed with Masson’s trichrome stain. Representative images are shown from n = 3 mice per group. Scale bars, 100 μm. ( B ) D-dimer ELISA was performed on BAL fluid collected from mice on days 0, 7, 14, and 21 after low-dose IT bleomycin (0.5 U/kg). n = 4 mice per group. ( C ) D-dimer ELISA was performed on homogenized lung tissue collected from mice on days 0, 7, 14, and 21 after low-dose IT bleomycin (0.5 U/kg). n = 4 mice per group. ( D ) Junctional proteins were assessed through immunoblotting for VE-Cadherin in lung tissue homogenates harvested at days 0 and 14 after low-dose bleomycin challenge. Densitometry was performed to quantify differences in protein expression at each time point. n = 2–3 mice per group. * P < 0.05 and *** P < 0.001 for comparisons of D-dimer in the BAL and lung tissue of EC- S1pr1 −/− mice as compared with intact littermate controls S1pr1 f/f .

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Coagulation, Injection, Labeling, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal:

Article Title:

doi:

Figure Lengend Snippet: Immune cell populations in the BAL of mice lacking endothelial S1pr1 after bleomycin. ( A ) Total cell counts were quantified from BAL obtained on Day 0 and Day 7 after low-dose IT bleomycin. n = 3–4 mice per group. Flow cytometry was performed on BAL collected 7 days after bleomycin challenge in EC- S1pr1 −/− and control mice to assess the number of ( B ) alveolar macrophages, ( C ) dendritic cells, ( D ) NK cells, ( E ) monocyte-derived dendritic cells (DCs), ( F ) inflammatory monocytes, ( G ) CD4 + T cells, ( H ) CD8 + T cells, and ( I ) T regulatory cells. In contrast, no differences were detected in the quantities of BAL ( J ) B cells, ( K ) eosinophils, ( L ) γδ T cells, or ( M ) neutrophils. n = 3–4 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of BAL cells in EC- S1pr1 −/− mice as compared with intact control mice S1pr1 f/f . γδ T cells = GD T cells.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Flow Cytometry, Control, Derivative Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Bleomycin-induced lung injury reduces endothelial cell populations in the lung. ( A ) Immunoblotting was performed using protein from lung homogenates of C57BL/6 wild-type mice (WT) at Days 0, 1, 3, 7, and 14 after IT bleomycin (0.5 U/kg). Blots were probed for S1PR1, and protein expression levels were compared using GAPDH as a loading control. n = 2–3 mice per group. Densitometry was performed using Image J software (NIH). ( B ) Flow cytometry to identify CD31 + cells was performed on lung tissue from WT mice on Days 0, 7, and 14 after standard-dose IT bleomycin. n = 4 mice per group. ( C ) Flow cytometry of CD31 + cells expressing S1PR1. n = 4 mice per group. ( D ) The percentage of endothelial cells expressing S1PR1 at each time point after bleomycin was quantified. ( E ) Mean fluorescence intensity of S1PR1 in CD31 + cells after bleomycin was quantified. ( F ) Plasma S1P levels in WT mice were quantified at Days 0, 3, 7, 10, and 14 after IT bleomycin. n = 3–4 mice per group. ( G ) Immunoflourescence co-staining on lungs from naive and bleomycin treated mice at Day 14 was performed for S1PR1 (red), endothelial CDH5/VE-cadherin (green), and nuclear DAPI (blue). White box indicates a fibrotic region. Scale bars, 50 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of cells in WT mice at various time points after bleomycin injury.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Western Blot, Expressing, Control, Software, Flow Cytometry, Fluorescence, Clinical Proteomics, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Increased circulating S1P is insufficient to protect mice from bleomycin-induced pulmonary fibrosis. ( A ) Transgenic mice overexpressing human apolipoprotein M (ApoM Tg + ) were bred, and Evans blue dye assay was performed at Day 7 after standard-dose IT bleomycin. ( B ) Evans blue indices were calculated using Evans blue dye indices in the lungs and plasma of ApoM Tg + mice and littermate controls (WT). n = 4 mice per group. ( C ) BAL total protein was quantified at Day 14 after standard dose bleomycin. ( D ) Histology at Days 0 and 14 after standard-dose IT bleomycin were stained with hematoxylin and eosin and Masson’s trichrome staining. Representative images are shown from n = 3 mice per group. Scale bars, 100 μm. ( E ) Quantification of fibrosis was performed with hydroxyproline assay. ( F ) S1P levels were quantified by mass spectrometry from circulating plasma at baseline (Day 0), Day 7, and Day 14 after standard-dose bleomycin. n = 3–6 mice per group. n = 3–8 mice per group. *** P < 0.001, and **** P < 0.0001 for comparisons of S1P between ApoM-Tg + and control mice.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Transgenic Assay, Clinical Proteomics, Staining, Hydroxyproline Assay, Mass Spectrometry, Control