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:

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: 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