Journal: Redox Biology

Article Title: Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues

doi: 10.1016/j.redox.2024.103102

Figure Lengend Snippet: PXDN and laminin expression are modulated in human lung fibroblasts (HLF) and bone-marrow-derived macrophages (BMDM) upon TGF-β1 stimulation. (A – C) HLFs were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for LAMA1 (A) or PXDN (B) mRNA or PXDN protein expression (C). (D – G) BMDM from C57BL6/NJ mice were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for Pxdn (D) , Lama1 (E) , or Lamb1 (F) mRNA, or Laminin α/β1 protein expression by immunofluorescence imaging (G). Scale bar: 50 μm. Values are the mean of at least three independent biological replicates ± SEM. Differences among groups were evaluated by Student's T-test.

Article Snippet: HLF (ATCC PCS-201-013) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast growth kit-low serum (ATCC PCS-201-041) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Imaging

Journal: Reproductive Sciences

Article Title: The Essential Role of GATA6 in the Activation of Estrogen Synthesis in Endometriosis

doi: 10.1177/1933719118756751

Figure Lengend Snippet: A, Differences in estradiol (E2) production in OSIS experimental groups. N = 4. Asterisk (*) indicates significantly lower concentration compared to other groups tested (P < .05). B, Differences in estradiol (E2) production in normal endometrial stromal cells (NoEMs) experimental groups. N = 4. Asterisk (*) indicates a significantly higher concentration compared to other groups tested (P < .05). C, Differences in estradiol (E2) production in skin fibroblast (BJ) experimental groups. N = 4.

Article Snippet: Human foreskin fibroblast cells (BJ cells), a nonimmortalized stromal cell line with a normal diploid karyotype, were obtained from ATCC and used as controls (cat. CRL-2522; Manassas, Virginia; n = 7).

Techniques: Concentration Assay

Journal:

Article Title: The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences

doi: 10.1073/pnas.0601069103

Figure Lengend Snippet: Genes border XIST RNA at interphase. Genomic probes for genes, α-satellite, and subtelomeric sequences were cohybridized with XIST RNA in normal diploid fibroblasts, WI38 (CCL 75) and TIG-1 (AG06173). The signals were scored relative to XIST RNA (see Fig. 1 A–C for examples of each category). Genes that escape X inactivation are denoted by an asterisk; TIMP1 escapes X inactivation weakly and is denoted by an ∗. (A) Clearly, all Xi-linked genes examined, regardless of activity and position on the metaphase chromosome, maintain a bordering relationship with XIST RNA the majority of the time, whereas the α-satellite sequences were found mostly inside of the XIST RNA domain. (B) By using a different protocol to preserve the 3D architecture of the nucleus (see Materials and Methods), the IN category was subdivided to include IN (Hole) (see Fig. 1G for an example). The genes were scored as touching the border of XIST RNA the majority of the time, whereas the α-satellite sequences were found mostly inside of the XIST RNA domain.

Article Snippet: Normal female diploid fibroblast (lung) cell lines used were CCL 75 (WI-38; American Type Culture Collection) and AG06173A (TIG1; Coriell Cell Repositories, Camden, NJ).

Techniques: Activity Assay

Journal:

Article Title: The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences

doi: 10.1073/pnas.0601069103

Figure Lengend Snippet: The Barr body is composed of silent repetitive elements. (A) Cot-1 DNA (red) and Cot-1 RNA (green) were simultaneously detected in normal diploid fibroblasts (TIG-1). Cot-1 DNA (C) is found throughout the nucleoplasm but is more intense over the DAPI-stained Barr body (B). (D) Cot-1 RNA is also found throughout the nucleoplasm but is not transcribed from the Barr body. (E) A line scan through the Xi shows that the pixel intensity and location of the DAPI and Cot-1 DNA signal overlap, whereas the Cot-1 RNA signal is depleted throughout the Barr body. (F) Cot-1 DNA (red) and Cot-1 RNA (green) is detected inside the Barr body. (H) Cot-1 DNA is more intense over the DAPI stained Barr body [It is not clear exactly why the interphase Xi is stained differentially with DAPI. Some evidence suggests the intense staining of the Xi DNA represents differences in folding rather than just condensation (24) (G), whereas the Cot-1 RNA is depleted in this region (I). (J) A line scan through the Barr body shows that the Cot-1 DNA signal is more intense over the Barr body. (K–M) X paint (green) (L) vs. Cot-1 RNA (M). Although the Barr body identified by DAPI (K) (arrowhead) and chromosome painting (L) (arrowhead) is devoid of Cot-1 transcription (M) (arrowhead), Xa (L) (arrow) is actively transcribing Cot-1 RNA (M) (arrow). (N) Ongoing RNA Pol II-mediated transcription is shown using the H5 antibody against the elongating form of the RNA Pol II enzyme (red). The DAPI DNA fluorescence (P) reveals that the heterochromatic Barr body is deficient in RNA Pol II activity (O).

Article Snippet: Normal female diploid fibroblast (lung) cell lines used were CCL 75 (WI-38; American Type Culture Collection) and AG06173A (TIG1; Coriell Cell Repositories, Camden, NJ).

Techniques: Staining, Fluorescence, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Oncostatin M decreases interleukin-1 β secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo

doi: 10.1111/j.1582-4934.2011.01412.x

Figure Lengend Snippet: Impact of OSM on gene expression (A, B) and protein secretion (C, D) in stimulated HSFs. HSFs were stimulated for 24 hrs with a combination of LPS (100 ng/ml) and GM-CSF (1.4 nM). Human recombinant OSM (10 ng/ml) was added with or without 10 μg/ml of neutralizing anti-OSM, or isotype-matched non-specific antibody. Levels of IL-1β (A), IL-6, CXCL8 and CCL2 mRNA (B) were measured by real-time PCR. Results (mean ± S.E.M., n ∇ 6) are expressed as multiples relative to stimulated cells (dotted line). Protein levels of IL-1β (C), IL-6, CCL2 and CXCL8 (D) in supernatants were determined by ELISA (mean ± S.E.M., n ∇ 6). *Significantly different ( P < 0.05) from stimulated cells. # Significantly different from the stimulated cell in the presence of OSM. Control: unstimulated cells.

Article Snippet: Normal human primary synovial fibroblasts (HSFs) were purchased from Asterand (Detroit, MI, USA).

Techniques: Gene Expression, Recombinant, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Oncostatin M decreases interleukin-1 β secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo

doi: 10.1111/j.1582-4934.2011.01412.x

Figure Lengend Snippet: Impact of PMN-derived OSM on gene expression and protein secretion in stimulated HSFs. PMNs were stimulated with a combination of LPS (100 ng/ml) and GM-CSF (1.4 nM) for 4 hrs. (A) OSM concentrations in supernatants of resting or LPS/GM-CSF-stimulated PMNs were measured by ELISA (mean ± S.E.M., n ∇ 3). (B) HSFs were incubated for 24 hrs with supernatants of resting or stimulated PMNs with or without anti-OSM neutralizing antibody (10 μg/ml). Expression levels are presented relative to the levels for cells stimulated in the absence of anti-OSM (as reciprocals in the case of decreases), as measured using real-time PCR (mean ± S.E.M., n ∇ 4). (C) IL-1β, IL-6, CCL2 and CXCL8 concentrations were quantified in cell-free supernatants by ELISA. Results are expressed as mean ± S.E.M. ( n ∇ 4). *Significantly different from SRP. # Significantly different ( P < 0.05) from SSP.

Article Snippet: Normal human primary synovial fibroblasts (HSFs) were purchased from Asterand (Detroit, MI, USA).

Techniques: Derivative Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Oncostatin M decreases interleukin-1 β secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo

doi: 10.1111/j.1582-4934.2011.01412.x

Figure Lengend Snippet: Response of HSF mRNA levels to LIF and IL-6. HSFs were stimulated with a combination of LPS and GM-CSF for 24 hrs in the absence or presence of OSM, LIF or IL-6 (10 ng/ml each). Levels of IL-1β, IL-6, CXCL8 and CCL2 mRNA were quantified by real-time PCR. Results (mean ± S.E.M., n ∇ 3) are expressed as multiples relative to stimulated cells (dotted line). * P < 0.05, ** P < 0.01 from the stimulated condition.

Article Snippet: Normal human primary synovial fibroblasts (HSFs) were purchased from Asterand (Detroit, MI, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: Journal of Cellular and Molecular Medicine

Article Title: Oncostatin M decreases interleukin-1 β secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo

doi: 10.1111/j.1582-4934.2011.01412.x

Figure Lengend Snippet: Signalling pathways mediating the OSM-effects on the IL-1β, CXCL8, IL-6 and CCL2 expression in stimulated HSFs. (A) HSFs were stimulated or not with a combination of LPS and GM-CSF for 10 min. in the absence or presence of OSM (10 ng/ml) and its neutralizing antibody (10 μg/ml), as indicated, prior to lysis and subsequent immunoblot analysis using primary antibodies raised against phospho-ERK1/2, phospho-STAT-1 (Tyr 701) and total STAT-1 (as a loading control). Immunoblots shown are representative of three independent experiments; bands were quantified by densitometric analysis and results presented as mean ± S.E.M. When indicated, cells were pre-incubated for 15 min. with the MEK inhibitor U0126 (10 μM) or the Jak inhibitor (0.5 μM) before stimulation in the presence of OSM. * P < 0.05 from the control condition (fixed at 1). (B) Otherwise identical conditions were used to study the consequences of a 24-hr stimulation in the presence of OSM and the metabolic inhibitors, on the mRNA expression of IL-1β, CXCL8, IL-6 and CCL2 by real-time PCR. Results (mean ± S.E.M., n ∇ 4) are expressed as multiples relative to stimulated cells (dotted line). * P < 0.05 from the stimulated condition, # P < 0.05 from the s. + OSM condition. (C) Schematic representation of the OSMRβ signalling in LPS and GM-CSF stimulated HSFs. OSM receptor engagement induces activation/phosphorylation of STAT-1 and also increases ERK1/2 phosphorylation. This leads to a STAT-1-dependent negative regulation of the IL-1β and CXCL8 genes transcription and to the up-regulation of the IL-6 and CCL2 genes.

Article Snippet: Normal human primary synovial fibroblasts (HSFs) were purchased from Asterand (Detroit, MI, USA).

Techniques: Expressing, Lysis, Western Blot, Control, Incubation, Real-time Polymerase Chain Reaction, Activation Assay, Phospho-proteomics

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

doi: 10.1165/rcmb.2019-0051OC

Figure Lengend Snippet: Glutaminase (Gls) 1 is upregulated in lung fibroblasts of mice with bleomycin (BLM)-induced pulmonary fibrosis. (A) Fibroblast tdTomato+ mice were instilled intratracheally with saline or BLM. At 3 weeks after instillation, lungs were harvested and single–lung cell suspension prepared. tdTomato+ fibroblasts were obtained by FACS sorting and total RNA from these purified cells. The expression of the indicated genes was determined by real-time PCR. n = 3; mean ± SE; *P < 0.05, **P < 0.01, and ***P < 0.001. (B) Fibroblast tdTomato+ mice were treated as in A. Lungs were harvested and frozen tissue sections prepared. In situ immunofluorescence assay was performed with anti-Gls1 and anti-RFP primary antibodies. Nuclei were stained by DAPI. Scale bars: 100 μm. Col1A1 = collagen type I α 1 chain; Fn = fibronectin; SMA = smooth muscle actin.

Article Snippet: Primary mouse normal lung fibroblast line MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC).

Techniques: Saline, Suspension, Purification, Expressing, Real-time Polymerase Chain Reaction, In Situ, Immunofluorescence, Staining

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

doi: 10.1165/rcmb.2019-0051OC

Figure Lengend Snippet: Transforming growth factor (TGF)-β1 induces Gls1 in mouse lung fibroblasts. (A) Primary normal mouse lung fibroblast line MLg cells were cultured in Eagle’s minimum essential medium (EMEM) supplemented with 1% FBS for 24 hours. The cells were then treated with or without 10 ng/ml mouse TGF-β1 for 24 hours. Total RNA from these cells was purified. The expression of the indicated genes was determined by real-time PCR. n = 4; mean ± SD; **P < 0.01 and ***P < 0.001. (B) MLg cells were treated as in A. The expression of the indicated genes was determined by Western blotting. (C) MLg cells were treated as in A. Gls1 expression was determined by immunofluorescence microscopy. Mitochondria were visualized by MitoTracker Deep Red FM. Nuclei were visualized by DAPI. Scale bars: 50 μm. A representative of two to three independent experiments is shown. Con = control.

Article Snippet: Primary mouse normal lung fibroblast line MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC).

Techniques: Cell Culture, Purification, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Microscopy, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

doi: 10.1165/rcmb.2019-0051OC

Figure Lengend Snippet: Knockdown of Gls1 inhibits collagen expression at the protein, but not the mRNA, level in TGF-β1–treated mouse lung fibroblasts. (A) Primary normal mouse lung fibroblast MLg cells were transfected with 20 nM control siRNA or Gls1 siRNA, followed by treatment with or without 10 ng/ml TGF-β1 for 48 hours. Levels of the indicated proteins were determined by Western blotting. (B) MLg cells were transfected as in A, followed by treatment with TGF-β1. The expression of the indicated genes was determined by real-time PCR. n = 3; mean ± SD; ***P < 0.001. A representative of two independent experiments is shown. si = siRNA.

Article Snippet: Primary mouse normal lung fibroblast line MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

doi: 10.1165/rcmb.2019-0051OC

Figure Lengend Snippet: Pharmaceutical inhibition of Gls1 diminishes TGF-β1–induced collagen production in mouse lung fibroblasts. (A) Primary normal mouse lung fibroblast MLg cells were cultured in EMEM supplemented with 1% FBS for 24 hours. The cells were then pretreated with or without 5 μM CB-839 for 1 hour, followed by treatment with or without 10 ng/ml TGF-β1 for 48 hours. Levels of the indicated proteins were determined by Western blotting. (B) MLg cells were treated with TGF-β1 for 24 hours, washed, and treated again with or without 5 μM CB-839 for 24 hours. The cells were then treated with 5 μg/ml cycloheximide (CHX) for 0, 1.5, and 3 hours. Levels of the indicated proteins were determined by Western blotting and densitometric analyses performed using ImageJ. (C) MLg cells were treated as in A. The expression of the indicated genes was determined by real-time PCR. n = 3; mean ± SD. A representative of two to three independent experiments is shown.

Article Snippet: Primary mouse normal lung fibroblast line MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

doi: 10.1165/rcmb.2019-0051OC

Figure Lengend Snippet: Conditional ablation of Gls1 in fibroblasts attenuates BLM-induced pulmonary fibrosis. (A) Primary mouse lung fibroblasts were isolated from Gls1 fl/fl and fibroblast Gls1−/− mice. Total RNA was purified and the expression of Gls1 determined by real-time PCR. n = 3 and 4; mean ± SE; **P < 0.01. (B) Gls1 fl/fl and fibroblast Gls1−/− mice were instilled intratracheally with saline or BLM. At 3 weeks after instillation, lungs were harvested and hydroxyproline levels in the lungs determined. n = 3, 7, 4, 6 for groups Gls1 fl/fl saline, Gls1 fl/fl BLM, fibroblast Gls1−/− saline and fibroblast Gls1−/− BLM, respectively; mean ± SE; *P < 0.05 and ***P < 0.001. (C and D) Gls1 fl/fl and fibroblast Gls1−/− mice were treated as in B. Lungs were harvested and tissue sections prepared. (C) Hematoxylin and eosin and (D) Masson’s trichrome stainings were performed. Scale bars: 500 μm in C and 100 μm in D. (E) Gls1 fl/fl and fibroblast Gls1−/− mice were treated as in B. Primary mouse lung fibroblasts were isolated and the expression of the indicated genes determined by real-time PCR. n = 3, 4, 4, 4 for groups Gls1 fl/fl saline, Gls1 fl/fl BLM, fibroblast Gls1−/− saline and fibroblast Gls1−/− BLM, respectively; mean ± SE; *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Primary mouse normal lung fibroblast line MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC).

Techniques: Isolation, Purification, Expressing, Real-time Polymerase Chain Reaction, Saline

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis

doi: 10.1165/rcmb.2019-0051OC

Figure Lengend Snippet: Gls1 inhibitor is therapeutically effective in mice with BLM or active TGF-β1–induced pulmonary fibrosis. (A) Schematic illustration of the experimental design. C57BL/6 mice were instilled intratracheally with saline or BLM. At 7 days after the instillation, the mice were injected intraperitoneally with CB-839 (28 mg/kg body weight dissolved in 30 μl DMSO) or the vehicle DMSO once a day for 2 weeks. At the end of the treatment, mice were killed and lungs harvested. (B) Lung hydroxyproline levels were determined. n = 3, 7, 3, 7 for groups vehicles saline, vehicles BLM, CB-839 saline and CB-839 BLM, respectively; mean ± SE; *P < 0.05 and ***P < 0.001. (C and D) Experiments were performed as in A. Lungs were harvested and tissue sections prepared. (C) Hematoxylin and eosin and (D) Masson’s trichrome stainings were performed. Scale bars: 500 μm in C and 100 μm in D. (E) C57BL/6 mice were instilled intratracheally with control adenovirus (ad-con) or adenovirus expressing active TGF-β1 (Ad-TGF-β1) (6 × 109 pfu/kg body weight). At 7 days after the instillation, the mice were injected intraperitoneally with CB-839 or the vehicle DMSO once a day for 2 weeks. Mice were then killed and lung hydroxyproline levels determined. n = 3 each; mean ± SE; *P < 0.05 and ***P < 0.001. (F) Experiments were performed as in E. Lung tissue sections were prepared and Masson’s trichrome staining performed. Scale bar: 100 μm. (G) Fibroblast Gls1−/− mice were instilled intratracheally with BLM. At 7 days after the instillation, the mice were injected intraperitoneally with CB-839 or the vehicle DMSO once a day for 2 weeks. Mice were then killed and lung hydroxyproline levels determined. n = 5 and 7; mean ± SE. i.p. = intraperitoneal; i.t. = intratracheal.

Article Snippet: Primary mouse normal lung fibroblast line MLg (CCL-206) was purchased from the American Type Culture Collection (ATCC).

Techniques: Saline, Injection, Control, Expressing, Staining

Journal: Nutrients

Article Title: Essential Oils, Pituranthos chloranthus and Teucrium ramosissimum , Chemosensitize Resistant Human Uterine Sarcoma MES-SA/Dx5 Cells to Doxorubicin by Inducing Apoptosis and Targeting P-Glycoprotein

doi: 10.3390/nu13051719

Figure Lengend Snippet: Effects of Pituranthos chloranthus (PC) and Teucrium ramosissimum Desf. (TR) extracts on normal primary human uterine fibroblast cells (HUF) and primary murine Bone Marrow-Derived Macrophages (BMDM) viability. After treatment of primary HUF and murine BMDM with increasing concentrations (0–100 µg/mL) of PC and TR for 72 h, the percentage of viable cells was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ( A ) Dose–response curves of PC-treated HUF (left panel) and TR-treated HUF (right panel). ( B ) Dose–response curves of PC-treated BMDM (left panel) and TR-treated BMDM (right panel). Data are expressed as a mean percentage of control growth ± Standard Deviation (SD) of two representative experiments ( n = 6 replicates per concentration).

Article Snippet: Primary human uterine fibroblast normal cells (HUF) were obtained from the ATCC.

Techniques: Derivative Assay, MTT Assay, Control, Standard Deviation, Concentration Assay