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: 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.

Fig 4: IGF-1R signaling drives terminal differentiation of BMP6 stimulated C2C12 cells in conditions permissive for adipogenic differentiation.(A) A schematic highlighting relevant time points in the differentiation protocol utilized to generate data shown in this figure. (B) Heat map depiction and unsupervised clustering analyses of 4255 significantly changing qualifiers (p≤0.01, fold change≥1.5) in BMP6 stimulated cells from “48 hours post BMP6 stimulation” to “post 3 days in induction media” time points. IGF-1 levels are highlighted in the heat map. Each gene is represented by a single row and each sample in three independent biological replicates for each time point by a column. Two distinct clusters indicate genes induced (red) and repressed (green). (C) Independent Q-PCR validation of IGF-1 and IGF-1R transcripts at indicated time points in the BMP6 stimulated cells. The expression at the 48 hour time point was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), *p<0.05. (D) Schematic representation of the differentiation protocol for cells with or without IGF-1 or IGF-1R knockdown is illustrated. (E) Knockdown validation of IGF-1 and IGF-1R transcripts as indicated in panel D. The expression in the IGF-1 or IGF-1R proficient cells was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), *p<0.05. (F) Q-PCR validation for Ucp-1 transcript levels in cells transduced with or without short hairpin constructs against indicated genes (IGF-1 or IGF-1R) post two days of BMP6 pretreatment followed by adipogenic differentiation and 4 hours forskolin stimulation. The expression in IGF-1 and IGF-1R proficient cells in absence of forskolin stimulation was set to 1. (G) Q-PCR validation for BAT markers (Cidea, Cox7a1, Ntrk3) in indicated cells pretreated with BMP6 followed by adipogenic differentiation. Expression in IGF-1 or IGF-1R proficient cells was set to 1 and Gapdh served as the endogenous control. Results represent triplicate analyses of three independent biological replicates (mean ± SD), *p<0.05. (H) Representative bright field images (magnification: 40X) and Oil-Red-O staining (magnification: 10X) in cells with attenuated IGF-1 or IGF-1R expression. (I) Q-PCR assessment of IGF-1 transcript levels in Optn and Cox2 proficient and knockdown cells. Results represent triplicate analyses of three independent biological replicates (mean ± SD) and similar results were obtained in at least two independent analyses. Analyses for panels G and H was performed at the end of differentiation cascade illustrated in Figure 1A following two days of BMP6 (250 ng/mL) stimulation.

Fig 5: Cotreatment with Aspirin and Cisplatin suppressed NF-κB/COX-2 signaling pathway in colon cancer cells. (A) The expression level of COX-2 in the whole cell lysate of human colon cancer cells RKO and LoVo treated with Cisplatin (15μM /5μM) and/or Aspirin(10mM /5mM) for 48 hours was analyzed by Western blot (n=3). (B) Human colon cancer cells RKO and LoVo were treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 h. The amount of PGE2 in cell culture media was detected by Prostaglandin E2 High Sensitivity in vitro competitive ELISA Kit (n=4). (C) Human colon cancer cells RKO and LoVo were incubated with combination of Aspirin and Cisplatin at indicated dose for 48 h after pretreatment with COX-2-selective inhibitor celecoxib (CB) at indicated dose for 8 h, and then cell viability was determined by MTT analysis (n=6). (D) Human colon cancer cells RKO and LoVo were treated with Cisplatin alone (15μM /5μM) or Aspirin alone (10mM /5mM) or their combination for 48 h. The expression levels of p65, p50, COX-2 and c-myc in nucleus, p65, p50, p-p65, IKKα, IKKβ, p-IKKα/β, p-IκBα and IκBα in cytoplasm were respectively detected by western blot assay (n=3). (E) Immunofluorescence assay of human colon cancer cell RKO treated with Cisplatin alone (15μM) or Aspirin alone (10mM) or their combination for 48 h was implemented to observe the subcellular localization of p65 and p50. The representative images were taken by laser scanning confocal microscope. Scale bars, 25 μm (n=3). (F) The streptavidin-biotin pulldown assay was performed to test the binding of p65 and p50 at COX-2 promoter region in human colon cancer cells RKO and LoVo treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 h (n=3). (G) Human colon cancer cells RKO and LoVo were transfected with COX-2 promoter (-892/+9 fragments) driven-luciferase plasmids and pRL-TK Renilla luciferase construct (Promega). After 24 hours, the cells were treated with Cisplatin (15μM /5μM) and/or Aspirin (10mM /5mM) for 48 hours. Then luciferase activities were measured according to the Dual-Luciferase Assay System protocol (Promega) (n=4). (H) Human colon cancer cells RKO and LoVo were pretreated with NF-κB activator LPS or NF-κB inhibitor QNZ at indicated dose for 8 h, and then incubated with combination of Aspirin and Cisplatin at indicated dose. After 48 h, cell viability was determined by MTT analysis (n=6). Data were presented as means ± SD, *P<0.05, **P<0.01, ***P<0.001.