Fig 2: Effect of ophiobolins 1–11 on COX 2 and iNOS protein levels in LPS-stimulated murine macrophage RAW 264.7 cells. Cells were pretreated with LPS (100 ng/mL) and then treated with compounds (10 µM) or curcumin (20 µM) for 24 h. The total protein levels were analyzed by western blot. (A) The expression of COX 2 and iNOS protein levels. (B) The quantification histogram of COX 2 and iNOS (C). ### p < 0.001 vs. control group; * p < 0.05 vs.LPS group.

Fig 3: Cell cytotoxicity, NO, TNF-a and their-related molecules. BV2 and HT22 cells were treated with PSE for 24 h, or pretreated with PSE for 2 h before exposure to LPS (1 µg/ml) for 24 h (only BV2). The cytotoxicity of PSE was assessed using a WST-8 assay in BV2 (A) and HT22 (D) cells. The levels of NO (B) and TNF-a (E), the protein expression of iNOS and COX2 (C), and the cytosolic and nuclear levels of NF-?B (F) were measured. The data are expressed as the mean ± SD. ##p < 0.01 compared with the vehicle-treated cells; **p < 0.01 compared with the LPS-stimulated cells.

Fig 4: Cisplatin induced PTGS2 via reactive oxygen species (ROS) mediated ERK1/2-NF?B pathway. (A) Cells were treated with cisplatin and subjected to analysis of PTGS2 expression by Western blotting, as well as PGE2 generation by ELISA as described in the Materials and Methods. (B) Cells seeded in 6-well plates were treated with cisplatin for the indicated times, and subjected to detection of the levels of reactive oxygen species (ROS) by flow cytometry. Values were expressed as the means ± SEM of three separate experiments. (C) Cells were plated at a density of 5 × 105/well in 6-well plates and treated with cisplatin for 24 and 48 h prior to immunoblot analysis of ERK1/2 activation. (D–F) Cells were treated with 10 mM NAC for 6 h before stimulation with cisplatin, and subjected to detection of the levels of reactive oxygen species (ROS) by flow cytometry, expressions of PTGS2 and the activation of ERK1/2 by Western blot analysis, and PGE2 generation by ELISA. (G and H) A549 cells were treated with 10 µM U0126 for 12 h before stimulation with 250 ng/ml cisplatin, followed by detection of PTGS2 expression and ERK1/2 activation by Western blotting, as well as PGE2 generation by ELISA. (I and J) Cells were treated with 10 mM NAC for 6 h prior to stimulation with 250 ng/ml cisplatin and subjected to detection of the PTGS2 promoter activity and NF-?B translocation by flow cytometry and Western blot analysis, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 and “n.s.” means “no significance.” The loading of each compartment in immunoblot analysis was indicated by GAPDH (cytoplasmic) and PCNA (nucleus).

Fig 5: The effects of PTGS2 on cisplatin-induced apoptosis. (A and B) Cells were plated at a density of 2.5 × 105/well in 12-well plates. After 12 h, cells were treated with 1 µg/ml or 4 µg/ml cisplatin for 48 h. For groups of co-treatment, cells were pre-treated with celecoxib for 6 h prior to the treatment of cisplatin. Cell apoptosis was quantified by annexin-V/APC and 7-AAD staining. *P < 0.05; **P < 0.01; ***P < 0.001.