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Figure legends
Figure 1. The proliferation-inhibitory effect of zedoary essential oil on NSCLC 515
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cells. H1299, A549, and H23 cells were treated with various concentrations of zedoary essential oil at 37 oC for 24, 48, and 72 h, respectively. The effect on cell growth was examined by MTT assay, and the percentage of cell proliferation was calculated by defining the absorption of cells without zedoary essential oil as 100%.
This experiment was repeated three times. Bar represents the SEM. Values
significantly were different from the control group. *, P < 0.05.
Figure 2. Effect of zedoary essential oil on cell cycle progression in H1299 cells.
(A) H1299 cells were treated with 110 μg/mL zedoary essential oil for 12, 24, 48, and 72 h, respectively and analyzed for PI-stained DNA content by flow cytometry. (B) The indicated percentages are the mean of three independent experiments, each in
duplicate. The sub-G1 phase H1299 cells increased with time.
Figure 3. Zedoary essential oil induces H1299 cells apoptosis. (A) H1299 cells were treated with 110 μg/ml zedoary essential oil for 24, 48, and 72 h, respectively.
Cell apoptosis percentages were determinate by flow cytometry with annexix V/PI staining. (B) H1299 cells were treated with various concentrations of zedoary essential oil at 37 oC for 48 h. Zedoary essential oil treatment resulted in typical DNA fragmentation as indicated by DNA laddering. M, 100bp DNA marker. D, 1% DMSO
treatment controls.
Figure 4. Effect of zedoary essential oil on caspases activity in H1299 cells. (A) 536
H1299 cells were treated with various concentrations of zedoary essential oil for 48 h.
Cells were then harvested and lysed for the detection of cleaved caspase 3, cleaved PARP and -actin protein expression. (B) H1299 cells were treated with 110 μg/mL zedoary essential oil for 6, 24, 48, and 72 h, respectively. Cells were then harvested and lysed for the detection of pro-caspase 3, cleaved caspase 3, cleaved PARP and -actin protein expression. (C) H1299 cells were treated with various concentrations of zedoary essential oil for 48 h. Cells were then harvested and lysed for the detection of cleaved caspase 9, cleaved caspase 8 and -actin protein expression. (D) H1299 cells were treated with 110 μg/mL zedoary essential oil for 6, 24, 48, and 72 h, respectively. Cells were then harvested and lysed for the detection of pro-caspase 9, cleaved caspase 9, pro-caspase 8, cleaved caspase 8 and -actin protein expression.
Western blot data presented are representative of those obtained in at least three separate experiments. (E) Z-VAD-FMK or the vehicle (DMSO) was added to the medium at 1 h before the 110 μg/mL zedoary essential oil treatment. After the 72 h incubation, the H1299 cell viability was determined using MTT assay. This experiment was repeated three times. Bar represents the SEM. Values significantly
were different from the control group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5. Effect of zedoary essential oil on apoptosis related proteins in H1299 cells. (A) Cells were treated with 110 g/mL of zedoary essential oil for 0, 0.5, 1, 2, 555
and 4 h for the production of ROS. All samples were analyzed by flow cytometric assay as described in Materials and Methods. H1299 cells were treated with various concentrations of zedoary essential oil for 48 h. Cells were then harvested and lysed for the detection of (B) Bcl-2, Bcl-xL and actin (C) Bax and actin (D) p53 and β-actin protein expression. H1299 cells were incubated with 110 g/mL of zedoary essential oil for indicated duration. Levels of AIF, Endo G, and cytochrome c in the (E) cytosolic and (F) mitochondrial fraction were analyzed by immunoblotting.
Western blot data presented are representative of those obtained in at least three
separate experiments.
Figure 6. Effect of zedoary essential oil on the MAPK and AKT/NF-κB signaling pathways. H1299 cells were treated with a vehicle (DMSO) or zedoary essential oil (110 µg/mL) for the indicated time. Cells were then harvested and lysed for the detection of (A) ERK1/2, phospho-ERK1/2, p38, phospho-p38, JNK, phospho-JNK, and β-actin (B) AKT, phospho-AKT, IB, phospho-IB, and β-actin protein expression. Western blot data presented are representative of those obtained in at least three separate experiments.
Figure 7. Effect of zedoary essential oil on anti-tumor activity. H1299 cells were used to establish xenografts in male BALB/c nude mice. Animals (six mices/group) were given control, zedoary essential oil (2.4, 12, 60, 240 mg/kg, respectively) by 574
given i.p. injection 5 times/weekly. (A) tumor volume (mm3), (B) tumor weight (g),
and body weight (g).
Figure 8. The profile of constituents in zedoary essential oil extracts. (A) The GC-MS profile of the zedoary essential oil. (B) Chemical structure of 8,9-dehydro-9-formyl-cycloisolongifolene. (C) Chemical structure of 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-trans-benzofuran.
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