Fig 2: DCP acts on Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR compensatory signaling pathwaysHLE cells were pretreated with PD98059 (80 μM) for 1 h and then treated with DCP (80 ng/ml) and/or Sorafenib (20 μM) for 3 h. Western blot analysis was performed to evaluate the expression of pERK, p-MEK1/2Ser217/221, p-c-RafSer259, p-AktSer473, p-mTORSer2481, PI3K p110α, Ras, Bcl-2, Mcl-1, and Bax. Gels were run under the same experimental conditions. The cropping lines in the figure represent full-length gels. A. 80 μM PD98059 for 1 h completely inhibited the expression of p-ERK in HLE cells. B. The changes in the Ras/Raf/MEK/ERK cascade in HLE cells exposed to DCP when ERK was inhibited. Bars indicate means ± S.D. (n = 3). *p < 0.05, **p < 0.01 vs. the vehicle control. C. The changes in the Ras/PI3K/Akt/mTOR cascade in HLE cells exposed to DCP and Sorafenib after ERK was inhibited. The expressions of p-MEK1/2 and p-c-Raf were decreased and Ras was activated (C, right panel). Bars indicate means ± S.D. (n = 3). *p < 0.05, **p < 0.01 vs. the cells exposed to DCP.

Fig 3: BRAF and RAF1 siRNA knockdowns downregulate cyclin D1 and viability in ESS1 cells.A Immunoblotting evaluations were performed in ESS1 cells 4 days after RAF1 or BRAF siRNA transfections (each at 150 nM). The actin stain is a loading control. B Cell viability was determined in ESS1 cells 6 days after RAF1 or BRAF siRNA transfections (each at 150 nM) using a Cell-titer Glo® luminescence assay. Data were normalized to the scramble siRNA control and represent the mean values (±s.d.) from quadruplicate cultures, averaged from two independent experiments. Statistically significant differences between scramble siRNA control and RAF1 siRNA or BRAF siRNA treatments are indicated as **p < 0.01, ***p < 0.001.

Fig 4: Effects of GRh2 on (A) LPS-induced antioxidative enzymes (catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx)); heme oxygenase-1 (HO-1), Trx-1, NF-E2-related factor 2 (Nrf2)/Keap1 and KAP1; (B) TLR4, PI3K, Akt, and mTOR; and (C) Raf-1, p-Raf-1, Mek and p-Mek protein expression in the lungs. Protein levels of catalase, SOD, GPx, HO-1, Trx-1, Nrf2/Keap1, KAP1, TLR4, PI3K, Akt, mTOR, Raf-1, p-Raf-1, Mek and p-Mek protein expression in lung homogenates were evaluated by western blot analysis after LPS challenge 6 h hours later. Densitometric analysis of the relevant bands was performed. Data are represented as mean ± S.E.M. (n = 2). ## p < 0.05 and ### p < 0.001 compared with sample of control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.

Fig 5: (A) GRh2 and Raf-1 inhibitor (GW-5074) reduced NO; (B) TNF-α; (C) IL-1β; (D) IL-6; and (E) IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-6, and IL-10 were detected at 6 h after LPS challenge by ELISA. Data are represented as mean ± S.E.M. (n = 5). ### p < 0.001 compared with sample of control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.