Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: 27-Hydroxycholesterol accelerates cellular senescence in human lung resident cells.

doi: 10.1152/ajplung.00351.2015

Figure Lengend Snippet: Fig. 8. Effects of 27-OHC on prostaglandin (PG) synthesis and effects of cyclooxygenase (COX) inhibitors on 27-OHC-enhanced cellular senes- cence. HFL-1 cells were exposed to various concentrations of 27-OHC for 2 wk. COX-1 and COX-2 expression was evaluated by Western blotting (A and B). PGE2 and PGD2 release was quantified by enzyme-linked immunoassay (ELISA) (C and D). Cells were treated with 27-OHC in the presence or absence of indo- methacin, a nonselective COX inhibitor; cele- coxib, a selective COX-2 inhibitor; and PF- 04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist. Cells were harvested and assayed for 3-NT formation (E) and SA--gal activity (F and G). Relative intensity was calcu- lated by dividing each protein band intensity by the -actin band intensity. Values are means SE (n 4). *P 0.05 and **P 0.01 vs. control group. †P 0.05 and ††P 0.01 vs. 27-OHC-treated group.

Article Snippet: Commercially available reagents were obtained as follows: mouse monoclonal anti-p53 antibody, mouse monoclonal anti-p16 antibody, mouse monoclonal anti-cyclooygenase-1 (COX-1) antibody, mouse monoclonal anti-COX-2 antibody, mouse monoclonal anti- -actin antibody, and mouse monoclonal anti-lamin A/C antibody were from Santa Cruz Biotechnology (Dallas, TX); rabbit polyclonal anti-p21 antibody, rabbit polyclonal anti-phosphorylated (p)-retinoblastoma (pRB) antibody and rabbit polyclonal anti-p-p53 antibody were from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antisterol 27-hydroxylase antibody and 27-OHC were from Avanti Polar Lipids (Alabaster, AL); mouse anti-CD68 antibody was from BioLegend (San Diego, CA); fibronectin enzyme-linked immunosorbent assay (ELISA) kit, goat fluorescein isothiocyanate polyclonal secondary antibody, and rabbit Dylight 650 polyclonal secondary antibody were from Abcam (Cambridge, UK); indomethacin, a nonselective COX inhibitor, and celecoxib, a selective 27-OHC-2 inhibitor, were from Sigma (St. Louis, MO); manganese (III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a peroxynitrite scavenger, was from Calbiochem (La Jolla, CA); diaminofluorescein-2, a fluorescent indicator, was from Sekisui Medical (Tokyo, Japan); PF-04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist and EIA prostaglandin kit were from Cayman Chemical (San Diego, CA); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), and antibiotic-antimycotic were purchased from Invitrogen Life Technologies (Grand Island, NY).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: 27-Hydroxycholesterol accelerates cellular senescence in human lung resident cells.

doi: 10.1152/ajplung.00351.2015

Figure Lengend Snippet: Fig. 1. Detection of p53 in lung fibroblasts. Lung tissues were obtained from healthy controls who had never smoked, healthy exsmokers, and exsmokers with chronic obstructive pulmonary disease (COPD). Local- ization of p53, a senescence-associated protein, was investigated in the lung tissues by immunostaining. Representative photographs of p53 immunoreactivity are shown (A). The boxed area indicates a region of higher magnification. Arrows indicate p53-immunopo- sitive lung fibroblasts. Red, p53; green, vimentin (marker of fibroblasts). The p53 and vimentin double- immunopositive cells were semiquantified using Image J (B). Four strains of primary lung fibroblasts were obtained from the patients. Cells were treated with 27-hydroxycholesterol (27-OHC), and the nuclear frac- tion was obtained. Translocation of phosphorylated p53 (p-p53) into the nucleus was evaluated by Western blotting (C). Relative intensity was calculated by divid- ing the p-p53 band intensity by the lamin A/C band intensity. Values are means SE. **P 0.01 vs. healthy control who had never smoked (HNS). ††P 0.01 vs. healthy exsmoker (HES).

Article Snippet: Commercially available reagents were obtained as follows: mouse monoclonal anti-p53 antibody, mouse monoclonal anti-p16 antibody, mouse monoclonal anti-cyclooygenase-1 (COX-1) antibody, mouse monoclonal anti-COX-2 antibody, mouse monoclonal anti- -actin antibody, and mouse monoclonal anti-lamin A/C antibody were from Santa Cruz Biotechnology (Dallas, TX); rabbit polyclonal anti-p21 antibody, rabbit polyclonal anti-phosphorylated (p)-retinoblastoma (pRB) antibody and rabbit polyclonal anti-p-p53 antibody were from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antisterol 27-hydroxylase antibody and 27-OHC were from Avanti Polar Lipids (Alabaster, AL); mouse anti-CD68 antibody was from BioLegend (San Diego, CA); fibronectin enzyme-linked immunosorbent assay (ELISA) kit, goat fluorescein isothiocyanate polyclonal secondary antibody, and rabbit Dylight 650 polyclonal secondary antibody were from Abcam (Cambridge, UK); indomethacin, a nonselective COX inhibitor, and celecoxib, a selective 27-OHC-2 inhibitor, were from Sigma (St. Louis, MO); manganese (III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a peroxynitrite scavenger, was from Calbiochem (La Jolla, CA); diaminofluorescein-2, a fluorescent indicator, was from Sekisui Medical (Tokyo, Japan); PF-04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist and EIA prostaglandin kit were from Cayman Chemical (San Diego, CA); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), and antibiotic-antimycotic were purchased from Invitrogen Life Technologies (Grand Island, NY).

Techniques: Immunostaining, Marker, Translocation Assay, Western Blot, Control

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: 27-Hydroxycholesterol accelerates cellular senescence in human lung resident cells.

doi: 10.1152/ajplung.00351.2015

Figure Lengend Snippet: Fig. 5. Effects of 27-OHC on senescence- associated protein production, senescence- associated -galactosidase (SA--gal) activ- ity, and cell growth in human lung fibro- blasts. Human fetal lung fibroblasts (HFL-1) were exposed to various concentrations of 27-OHC for 24 h, and cell lysates were harvested. Expression of senescence-associ- ated protein was evaluated by Western blot- ting. Band intensity for each senescence- associated protein, including p53 (A), p21 (B), p16 (C), and phosphorylated retinoblas- toma (p-pRB) (D), was assessed by densi- tometry and is expressed relative to the -ac- tin band intensity. SA--gal activity (E and F) and cell growth (G) were determined in 20th passage cells after chronic exposure to 27-OHC for 2 wk. Arrows indicate SA-- gal-positive cells. Values are means SE (n 4). *P 0.05 and **P 0.01 vs. control group. ns, Not significant.

Article Snippet: Commercially available reagents were obtained as follows: mouse monoclonal anti-p53 antibody, mouse monoclonal anti-p16 antibody, mouse monoclonal anti-cyclooygenase-1 (COX-1) antibody, mouse monoclonal anti-COX-2 antibody, mouse monoclonal anti- -actin antibody, and mouse monoclonal anti-lamin A/C antibody were from Santa Cruz Biotechnology (Dallas, TX); rabbit polyclonal anti-p21 antibody, rabbit polyclonal anti-phosphorylated (p)-retinoblastoma (pRB) antibody and rabbit polyclonal anti-p-p53 antibody were from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antisterol 27-hydroxylase antibody and 27-OHC were from Avanti Polar Lipids (Alabaster, AL); mouse anti-CD68 antibody was from BioLegend (San Diego, CA); fibronectin enzyme-linked immunosorbent assay (ELISA) kit, goat fluorescein isothiocyanate polyclonal secondary antibody, and rabbit Dylight 650 polyclonal secondary antibody were from Abcam (Cambridge, UK); indomethacin, a nonselective COX inhibitor, and celecoxib, a selective 27-OHC-2 inhibitor, were from Sigma (St. Louis, MO); manganese (III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a peroxynitrite scavenger, was from Calbiochem (La Jolla, CA); diaminofluorescein-2, a fluorescent indicator, was from Sekisui Medical (Tokyo, Japan); PF-04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist and EIA prostaglandin kit were from Cayman Chemical (San Diego, CA); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), and antibiotic-antimycotic were purchased from Invitrogen Life Technologies (Grand Island, NY).

Techniques: Expressing, Western Blot, Activity Assay, Control

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: 27-Hydroxycholesterol accelerates cellular senescence in human lung resident cells.

doi: 10.1152/ajplung.00351.2015

Figure Lengend Snippet: Fig. 6. Effects of 27-OHC on senescence-associated protein production, SA--gal activity, and cell growth in adult bronchial epithelial cells. Adult bronchial epi- thelial cells were obtained from healthy subjects, and the cells were exposed to various concentrations of 27-OHC for 24 h. Expression of senescence-associated proteins was evaluated by Western blotting. Band in- tensity for each senescence-associated protein, includ- ing p53 (A) and p16 (B), was assessed by densitometry and is expressed relative to the -actin band intensity. SA--gal activity (C) and cell growth (D) were deter- mined in 6th passage cells after chronic exposure to 27-OHC for 2 wk. Values are means SE (n 4). *P 0.05 and **P 0.01 vs. control group.

Article Snippet: Commercially available reagents were obtained as follows: mouse monoclonal anti-p53 antibody, mouse monoclonal anti-p16 antibody, mouse monoclonal anti-cyclooygenase-1 (COX-1) antibody, mouse monoclonal anti-COX-2 antibody, mouse monoclonal anti- -actin antibody, and mouse monoclonal anti-lamin A/C antibody were from Santa Cruz Biotechnology (Dallas, TX); rabbit polyclonal anti-p21 antibody, rabbit polyclonal anti-phosphorylated (p)-retinoblastoma (pRB) antibody and rabbit polyclonal anti-p-p53 antibody were from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antisterol 27-hydroxylase antibody and 27-OHC were from Avanti Polar Lipids (Alabaster, AL); mouse anti-CD68 antibody was from BioLegend (San Diego, CA); fibronectin enzyme-linked immunosorbent assay (ELISA) kit, goat fluorescein isothiocyanate polyclonal secondary antibody, and rabbit Dylight 650 polyclonal secondary antibody were from Abcam (Cambridge, UK); indomethacin, a nonselective COX inhibitor, and celecoxib, a selective 27-OHC-2 inhibitor, were from Sigma (St. Louis, MO); manganese (III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a peroxynitrite scavenger, was from Calbiochem (La Jolla, CA); diaminofluorescein-2, a fluorescent indicator, was from Sekisui Medical (Tokyo, Japan); PF-04418948, a selective prostaglandin E2 receptor 2 (EP2) antagonist and EIA prostaglandin kit were from Cayman Chemical (San Diego, CA); Dulbecco’s Modified Eagle’s Medium (DMEM), fetal calf serum (FCS), and antibiotic-antimycotic were purchased from Invitrogen Life Technologies (Grand Island, NY).

Techniques: Activity Assay, Expressing, Western Blot, Control