Fig 2: EVO inhibits anti-apoptotic signal proteins through inhibition of Akt, ERK1/2, and p38 in PANC-1 and SW1990 cells. (A) PANC-1 and SW1990 cells were treated with different concentrations of EVO (0-10 µM) for 24 h and then subjected to western blotting. The levels of EVO of Akt, ERK1/2 and p38 were investigated by western blotting with GAPDH as an internal control. (B) Quantitative results of the protein levels of Akt, ERK1/2 and p38, which were adjusted with the GAPDH protein level. (C) PANC-1 and SW1990 cells were pretreated with LY294002 (20 µM), SB203580 (20 µM) or U0126 (10 µM) for 1 h followed by treatment with EVO (2 µM) for 24 h and analyzed using a Cell Counting Kit-8 assay. Results are shown as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. control group; #P<0.05 vs. evodiamine alone group. EVO, evodiamine; ERK, extracellular signal-regulated kinase; P-, phosphorylated; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; CON, control; LY, LY294002; U, U0126; SB, SB203580.

Fig 3: PF-3758309 induces cell death through the ERK signaling pathway. (A) Western blot analysis of phosphorylation of ERK, Akt and p38 in lysates from SH-SY5Y and IMR-32 cells treated with serial concentrations of PF-3758309 for 24 h. The phosphorylation of ERK decreased upon PF-3758309 treatment in both cell lines, but no obvious change in the phosphorylation of Akt was observed. The phosphorylation of p38 was increased in the IMR-32 cells treated with PF-3758309, but no obvious change was noted in SH-SY5Y cells. (B) Western blot analysis of the phosphorylation of ERK, Akt and p38 in lysates from SH-SY5Y and IMR-32 cells treated with 1 µM PF-3758309 at different time points. The phosphorylation of ERK decreased upon PF-3758309 treatment over time in both cell lines, but no obvious change in the phosphorylation of Akt was observed. The phosphorylation of p38 was increased in IMR-32 cells treated with PF-3758309, but no obvious change was noted in the SH-SY5Y cells. All data are representative of 3 independent experiments with n=3–6/group and are means ± SEM.

Fig 4: Expression of p-AKT, p-ERK and p-P38 in xenograft tumor tissues. (A) Expression of p-AKT, p-ERK1/2 and p-P38 as detected by immunohistochemistry (magnification, ×200). (B) Mean optical density of p-AKT, p-ERK1/2 and p-P38 in orthotopic transplanted tumors. *P<0.05 vs. CON; **P<0.01 vs. CON. CON, control; ERK, extracellular signal-regulated kinase; p-, phosphorylated.

Fig 5: Activation of JNK and reduction of p38 and STAT3 activities by TR35 treatment in H1299 and A549 cells. (A,C) A549 and H1299 cells were treated with TR35 (0–4 mg/mL) for 24 and 48 h. STAT3, p38, and JNK steady state and phosphorylated levels were analyzed by western blotting. Tubulin was used as loading controls. (B,D) Quantification of the band density was analyzed by ImageJ software. Data are presented as the mean ± SEM, n = 3 (*p < 0.05, **p < 0.01, ***p < 0.001 vs. CON).