Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: Cell reports

Article Title: Pyruvate metabolism controls chromatin remodeling during CD4 + T cell activation.

doi: 10.1016/j.celrep.2023.112583

Figure Lengend Snippet: Figure 2. ACSS2 inhibition or ACLY deficiency does not affect epigenome remodeling during T cell activation (A) Immunoblots of ACLY, ACSS2, PDCE1, and actin from human T cells activated for 8, 16, 24, or 48 h with anti-CD3/CD28 stimulation. (B and C) CD25 protein expression was measured by flow cytometry (FACS) and IL2ra gene expression was determined by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) in human CD4+ T cells activated with anti-CD3/CD28 in the presence and absence of ACSS2 inhibitor (15.6 mM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER FCCP, mitochondrial oxidative phosphorylation uncoupler Abcam # ab120081 Rotenone Merck Millipore # R8875 UK-5099 (Synonyms: PF-1005023) Med Chem Express # HY-15475 3PO R98% (HPLC) Sigma-Aldrich # SML1343 Sodium acetate Sigma-Aldrich # S1429 DCA Tocris Bioscience # 2755 Etomoxir sodium salt hydrate Sigma-Aldrich # E1905 ACSS2 inhibitor Selleck # S8588 6,8-Bis(benzylthio)-octanoic acid Sigma-Aldrich # SML0404 Sodium Pyruvate Thermo Fisher # 11360070 SB204990 Med Chem Express # HY-16450 BMS303141 Sigma-Aldrich # SML0784 HEPES Sigma-Aldrich #H4034-100G EDTA Sigma-Aldrich #EDS-100G DL-Dithiothreitol solution (DTT) Sigma-Aldrich #43816-10ML Bovine serum albumin (BSA) Sigma-Aldrich #A9647-500G D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML 13C6-glucose Cambridge Isotope Lab #CLM-1396 DMSO Sigma-Aldrich # D2650 Ficoll Paque Plus Sigma-Aldrich # GE17-1440-02 13C6-glucose Cambridge Isotope Lab #CLM-1396 Cell Trace Violet Invitrogen #C34557 Molecular ProbesTM 2-NBDG Invitrogen #N13195 SYBR Thermo fisher #S33102 iScript cDNA kit biorad #170-8891 Formaldehyde Sigma-Aldrich #252549-1L DAPI (1:2000) Invitrogen #D1306 Zombie NIRTM Fixable Viability Kit Biolegend #423106 Zombie GreenTM Fixable Viability Kit Biolegend #423112 7-AAD Invitrogen #A1310 D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML GolgiPlugTM (Protein Transport Inhibitor) BD Biosciences #555029 Tween 20 Sigma-Aldrich #P7949-500ML HALT protease inhibitor Thermo scientific #78439 Powdered milk Carl Roth #T145.2 PierceTM Protein A/G Magnetic Beads thermo fisher #88802 Proteinase K, recombinant, PCR Grade Sigma #3115828001 RNeasy Mini Kit Qiagen #74106 Seahorse XF DMEM medium Agilent Technologies #103575-100 Critical commercial assays MagniSortTM Human CD4+ T cell Enrichment Kit eBioscience #8804-6811-74 CD4 (L3T4) MicroBeads, mouse Miltenyi # 130-117-043 Duolink In Situ Detection Reagents Red Sigma DUO92008 Duolink Green PLA Sigma # DUO92014 Foxp3/Transcription Factor Staining Buffer Set eBioscience #00-5523-00 BD Cytofix/Cytoperm Fixation/Permeabilization kit BD Biosciences #554714 seahorse XFe24 Flux Packs Agilent #1023-40-100 Acetyl-CoA Assay Kit Biovision, Milpitas CA #K317-100 (Continued on next page) Cell Reports 42, 112583, June 27, 2023 19

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Cytometry, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 1 The expression of DEF6 is increased in hypertrophic hearts and cardiomyocytes. A mRNA levels of DEF6 in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 5). B Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the LV myocardium of mice subjected to sham or 4 weeks of TAC surgery (n = 4). C mRNA levels of DEF6 in NRCMs administrated with PBS or 24 h of PE (50 μM) (n = 5). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in NRCMs administrated with PBS or 24 h of PE (n = 4). *P < 0.05, ***P < 0.001 vs. sham or PBS. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Student’s t test (A, C) or Mann–Whitney U test (B, D).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Expressing, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 2 Ablation of DEF6 mitigates TAC-induced cardiac hypertrophy. A Strategy to construct KO mice and the sequencing results of WT and KO mice. B Protein levels of cardiac DEF6 in WT and KO mice (n = 5). C Comparisons of HW, HW/BW, LW/BW, and HW/TL in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). D Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area from groups (n = 6). E RT-PCR analyses of the hypertrophic markers in the indicated groups (n = 4). F–H Comparisons of the LVEDd, LVESd, LVPWd, FS, and EF values in WT and KO mice subjected to sham or 4 weeks of TAC surgery (n = 10). I Left, PSR-stained LV sections in WT and KO mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). J RT-PCR analysis of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by One-way ANOVA (C, D, F–I) or Kruskal–Wallis test (E, J).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Construct, Sequencing, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 3 Overexpression of DEF6 aggravates TAC-induced cardiac hypertrophy. A mmunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in the hearts of mice injected with AAV9-vector or AAV9-DEF6 (n = 4). B Comparisons of HW, HW/BW, LW/ BW, and HW/TL in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). C Left, gross hearts and H&E-stained LV sections of each groups. Scale bars, 0.3 cm and 50 μm, respectively. Right, Comparisons of cardiomyocyte cross-sectional area between groups (n = 6). D RT-PCR analyses of the hypertrophic markers in each groups (n = 4). E–G Comparisons of LVEDd, LVESd, LVPWd, FS, and EF in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery (n = 10). H Left, PSR-stained LV sections in AAV9-vector- and AAV9-DEF6-infected mice subjected to sham or 4 weeks of TAC surgery. Scale bars, 50 μm. Right, comparisons of LV collagen volume between groups (n = 6). I RT-PCR analyses of the fibrotic markers in each groups (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AAV9- vector or AAV9-vector sham, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AAV9-vector TAC. Data are displayed as mean ± SD. Statistical analysis were conducted by Mann–Whitney U test (A) or One-way ANOVA (B, C, E–H) or Kruskal–Wallis test (D, I).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Over Expression, Expressing, Injection, Plasmid Preparation, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 4 DEF6 exacerbates PE-induced cardiomyocyte hypertrophy. A Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCMs infected with AdshRNA or AdshDEF6 (n = 3). B Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with AdshRNA or AdshDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). C RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). D Immunoblot analyses (left) and results of quantification (right) of DEF6 protein expression in cultured NRCVs infected with Advector or AdDEF6 (n = 3). E Left, immunofluorescence staining (α-actinin, red) in cultured NRCMs infected with Advector or AdDEF6 and administrated with PBS or 24 h of PE. Scale bar, 20 μm. Right, comparisons of the cardiomyocyte surface areas in cultured NRCVs of each groups (n ≥48 cells per group). F RT-PCR analyses of the hypertrophic markers in cultured NRCVs of each groups (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. AdshRNA or AdshRNA PBS or Advector or Advector PBS, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. AdshRNA PE or Advector PE. Data are displayed as mean ± SD. Statistical analysis were conducted by two-tailed Mann–Whitney U test (A, D) or One-way ANOVA (B, E) or Kruskal–Wallis test (C, F).

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Western Blot, Expressing, Cell Culture, Infection, Staining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY

Journal: Cell death & disease

Article Title: DEF6(differentially exprehomolog) exacerbates pathological cardiac hypertrophy via RAC1.

doi: 10.1038/s41419-023-05948-0

Figure Lengend Snippet: Fig. 6 Prohypertrophic effect of DEF6 depends on Rac1-MEK-ERK signaling. A Co-IP of DEF6 was performed with anti-Flag and probed by Western blots with anti-HA (left); Co-IP of Rac1 was performed with anti-HA and probed by Western blots with anti-Flag (right). B In vitro GST pulldown assays for the interaction of purified Flag-DEF6 and GST-HA-Rac1 (left), as well as Flag-Rac1 and GST-HA-DEF6 (right). C The activity of Rac1 changes in the same direction as the DEF6 expression. D Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with Advector or AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM) (n = 3). E Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with Advector and AdDEF6 and treated with PBS or NSC23766 (50 μM, 24 h) under 24 h of PE treatment (50 μM). (n ≥48 cells per group). F RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). G Immunoblot analyses of total and activated MEK1/2, ERK1/2 in cultured NRCMs infected with AdshRNA or AdshDEF6 and with Adcontrol or AdRac1(G12V) under PE 24 h of PE treatment (50 μM) (n = 3). H Immunofluorescence staining (α-actinin, red) (left) and comparison of cardiomyocyte surface areas (right) of NRCMs infected with the indicated adenovirus and administrated with 24 h of PE (50 μM) (n ≥48 cells per group). I RT-PCR analysis of the hypertrophic markers in cultured NRCVs of each groups (n = 3). •P < 0.05, P < 0.01,•P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, *P < 0.05, **P < 0.01, ***P < 0.001 vs. Advector PBS PE or AdshRNA Adcontrol PE, ###P < 0.001 vs. AdDEF6 PBS PE or AdshDEF6 Adcontrol PE, and n.s. indicates no significance. Data are displayed as mean ± SD. Statistical analysis were conducted by Kruskal–Wallis test.

Article Snippet: The cardiomyocytes were cultured in DMEM/F12 medium (Gibco, C11330) added with 10% fetal bovine serum (FBS), 5-bromodeoxyuridine (0.1 mM), and 1% penicillin/streptomycin for 24 h. The NRCMs were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 6 h. Subsequently, the medium was replaced with serum-free DMEM/F12, and 12 h later, the cardiomyocytes were stimulated with phosphatebuffered saline (PBS) or PE (50 μM) for 24 h. For rescue experiments, the cardiomyocytes were treated with Rac1 inhibitor NSC23766 (50 μM, 24 h) (S8031, Selleck) before adenovirus infection [31].

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Activity Assay, Expressing, Cell Culture, Infection, Staining, Comparison, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Myosin VI Reduces Proliferation, but Not Differentiation, in Pluripotent P19 Cells

doi: 10.1371/journal.pone.0063947

Figure Lengend Snippet: (A) Mouse embryonal P19 cells were stably transfected with Myo6 expression vector, followed by selection by G418 and subsequent determination of Myo6 mRNA expression with RT-PCR analysis. Several clones of stable transfectants were cultured with ATRA for 4 days for subsequent (B) micrographic observation and (C) MTT reduction determination. Each value represents the mean ± S.E. in 4 different experiments. *P<0.05, **P<0.01, significantly different from the control value in stable EV transfectants.

Article Snippet: After 24 h, and every 48 h thereafter for 2 weeks, the culture medium was replaced with fresh medium containing 500 μg/ml of G418 (Invitrogen).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Selection, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Cell Culture, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A : cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. *** P < 0.001. B : this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C : PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. ** P < 0.005. D : PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. * P < 0.05; ** P < 0.005. E : PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. *** P < 0.005. F : PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. *** P < 0.001.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Isolation, Derivative Assay, Inhibition, Thymidine Incorporation Assay, Western Blot, Activation Assay, Proliferation Assay, Positive Control, Incubation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Increased expression of p38 MAPK α-isoform in animal models of PH. A : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the BCA method. Equal concentrations were then loaded on a gel and blotted for p38 MAPKα and β-actin for loading control. There are 3 wells for each condition. Immunoblot shown is best representative of 3 experiments using 3 different animals with each condition. B : densitometry of immunoblot in A . Values are mean arbitrary values from 3 immunoblots expressed relative to the value for β-actin. * P < 0.05 by ANOVA. C : lung sections (5 mm) were prepared from normal, CH, and MCT animals. Sections were stained for p38 MAPKα using 1:400 dilution. Magnification: ×20. Bar represents = 150 mm. Arrows identify blood vessels. D : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then used in a p38 MAPK activity assay using immunoprecipitation and the phosphorylation of activating transcription factor-2 (ATF-2) as a read out. Immunoblot shown is representative of 3 experiments using 3 different animals. E and F : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phosphorylated p38 MAPK and β-actin for loading control. Immunoblot shown is representative of 3 experiments using 3 different animals with each condition. Densitometry is shown for remaining blots. * P < 0.05.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Expressing, Protein Concentration, Control, Western Blot, Staining, Activity Assay, Immunoprecipitation, Phospho-proteomics

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH is prevented by administration of SB203580, a p38 MAPKα inhibitor. A and B : animals were exposed to a hypobaric hypoxic environment for 2 wk. Some animals received daily injections of SB203580. Hemodynamics ( A ) and hematocrit ( B ) were measured after 2 wk. RVSP, right ventricular (RV) systolic pressure. Data represent mean values ± SE. Total animals n = 5–6 per group. C : hearts were isolated from the animals and the RV was dissected out from the left ventricle (LV) and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. The total RV weight was also plotted. Values are means ± SD; n = 5 per group. ** P < 0.05. D : noninvasive systemic blood pressure taken by tail-cuff measurement. E and F : rats were exposed to hypoxia ± SB203580. The lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five random fields per slide were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized and are presented as numbers per total number of vessels present in each field. Groups analyzed by ANOVA for overall change with posttest analysis; n = 5 animals. *** P < 0.001, for A – C and E .

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Isolation, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH in 2 in vivo animal models is reversed by the administration of SB203580, a p38 MAPKα inhibitor. A : animals were exposed to a hypobaric hypoxic environment for 2 wk and then p38 MAPK inhibition was commenced. Hemodynamics and RVSP were measured after 4 wk. Data represent mean values ± SE. Total animals n = 5–6 per group. ** P < 0.01; *** P < 0.001, for normal relative to all other conditions. B : hearts were isolated form the animals and the RV dissected out from the LV and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. Values are means ± SE; n = 6–7. ** P < 0.01; *** P < 0.005. C : hematocrit ratio. D : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. ** P < 0.01; *** P < 0.001. E : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 7 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. ** P < 0.01; *** P < 0.001. F and G : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 6–7 per group. * P < 0.05; *** P < 0.001, for F . * P < 0.05; ** P < 0.01, for G . H : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. * P < 0.05; *** P < 0.001. I : lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 MCT control.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: In Vivo, Inhibition, Isolation, Staining, Control, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: PH in a reversal strategy in 2 in vivo animal models by administration of PH-797804, a more selective p38 MAPKα inhibitor. A and B : animals were exposed to CH and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RV hypertrophy (RVH) were measured after 4 wk. Data represent mean values ± SE. * P < 0.05; ** P < 0.01 for A . ** P < 0.01; *** P < 0.001, for B . C : the lungs were removed after experiment and sections (5 mm) were cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05 for complete muscularized group in drug-treated vs. day 14 hypoxic control. D : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. E and F : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 14–15 per group. *** P < 0.001 for E . * P < 0.05; ** P < 0.01 for F . G : the lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05, for complete muscularized group in drug-treated vs. day 14 hypoxic control. H : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for drug-treated vs. day 14 MCT control.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: In Vivo, Inhibition, Staining, Control, Injection

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B : supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. * P < 0.05 ** P < 0.005. C : PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D : PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. ** P < 0.01; *** P < 0.01, for C and D . E : PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. * P < 0.05; ** P < 0.005; *** P < 0.0001. F : PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. **** P < 0.0001. G : PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. ** P < 0.01. H and I : PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. *** P < 0.005; **** P < 0.001, for I .

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, DNA Synthesis, Marker

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A : lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B : lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05 ** P < 0.01. C and D : lungs from CH 28-day controls and SB203580 -treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. ** P < 0.001. E : lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05; ns is not significant by ANOVA. F : PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. *** P < 0.005. G : PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. * P < 0.01; *** P < 0.005. H : fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. ** P < 0.01, *** P < 0.001.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Inhibition, In Vivo, Isolation, Protein Concentration, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Cell Culture, Derivative Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

doi: 10.1152/ajplung.00038.2015

Figure Lengend Snippet: Phospho-p38 MAPK and p38 MAPKα expression in explanted lungs from patients with idiopathic pulmonary arterial hypertension (IPAH). A : sections of 5 mm were taken. Then, normal control lung and IPAH lung are stained for phospho-p38 MAPK at dilution of 1:300. The isotype on IPAH lung is also shown. This dilution was optimally assessed for. Objective: ×20. Bar = 150 mm. B : high-power microscopy shows that there is strong staining for phospho-p38 MAPK in the intima, media, and the adventitia (arrows). Objective lens: ×20 and ×40. Bar = 50 mm. C : sections of control lung ( A ) and IPAH lung ( B ) were stained for p38 MAPKα at dilution of 1:300. Objective lens × 20. Bar = 150 mm. D : staining for p38 MAPKa showed increased cytosolic staining in the IPAH lung ( right ) compared with control lung ( left ). E : high-power view (×40) of staining with isotype and p38 MAPKα in a vessel in IPAH lung. This shows staining throughout the vessel layers but especially in adventitia and fibroblast cells (arrow). Bar = 50 mm. F : low-power view of a plexiform lesion. Staining for p38 MAPKα using 1:300 dilution. Objective lens × 20. Bar = 150 mm. G : histological scoring shows increased p38 MAPKα staining throughout the vascular wall. With the use of a well-validated histological scoring system (Allred), the vascular wall cells were scored for intensity of staining. The intensity multiplied by the number of vessels with that intensity determines the values. Values shown are from 5 random high-power fields from 2 slides. **** P < 0.0001 by ANOVA. ** P < 0.001 for individual IPAH vs. control columns.

Article Snippet: The p38 MAPK antagonist SB203580 was obtained from Selleck Chemicals and the dose used was 20 mg/kg given intraperitoneally once daily.

Techniques: Expressing, Control, Staining, Microscopy