Article Figures & Data
Figures
- Fig. 1.
Levels of mPGES and COX-2 protein are increased in NSCLC. A, immunoblot of paired nontumorous (NT) and tumorous (T) lung tissues from six subjects. Equal concentrations of protein (100 μg/lane) were loaded onto a 12% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was sequentially probed for mPGES, COX-2, and β-actin. Cases 1–4 and 6 represent adenocarcinoma. Case 5 is a squamous cell carcinoma. B, this poorly differentiated adenocarcinoma shows diffuse cytoplasmic immunoreactivity with anti-mPGES antibody (×400).
- Fig. 2.
Transformation is associated with increased amounts of mPGES protein and mPGES mRNA. A, cellular lysate protein (100 μg/lane) from BEAS-2B, H2122, and A549 cells was loaded onto a 12% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was sequentially probed with antibodies specific for mPGES and β-actin. B, total cellular RNA was isolated from BEAS-2B, H2122, and A549 cells. Each lane contained 10 μg of RNA. The blot was probed for mPGES mRNA and 18S rRNA. Higher levels of mPGES protein (A) and mPGES mRNA (B) were detected in NSCLC cell lines (H2122 and A549) than in the immortalized nontumorigenic BEAS-2B cell line.
- Fig. 3.
Transformation of lung epithelial cells is associated with increased rates of mPGES transcription. A, nuclei were isolated from BEAS-2B, H2122, and A549 cells. Nuclear run-off analysis was performed as described in “Materials and Methods.” The mPGES and 18S rRNA cDNAs were immobilized onto nitrocellulose membranes and hybridized with labeled RNA transcripts from the different cell lines. B, BEAS-2B cells were transfected with 0.9 μg of human mPGES promoter deletion constructs ligated to luciferase (−651/−20 or −190/−20) and 0.2 μg of pSVβgal. Bars labeled Ras represent cells that also received 0.9 μg of expression vector for Ras. The total amount of DNA in each reaction was kept constant at 2 μg by using a corresponding empty expression vector. Reporter activities were measured in cellular extract 24 h after transfection. Luciferase activity represents data that have been normalized with β-galactosidase. Columns, means; bars, SD; n = 6. ∗, P < 0.001.
- Fig. 4.
TNF-α induces mPGES and COX-2 in NSCLC cell lines. A, H2122 cells were treated with vehicle (CTR) or TNF-α (10 ng/ml) for 6, 12, and 24 h. B, BEAS-2B, H2122, and A549 cells were treated with vehicle or TNF-α (10 ng/ml) for 24 h. In A and B, cellular lysate protein (100 μg/lane) was loaded onto a 12% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed sequentially with antibodies specific for mPGES and COX-2.
Volume 7, Issue 9
