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Table S1. Information of primer sequences

Figure S1. Experimental design. Five-weeks-old female ICR mice were obtained from the BioLASCO Experimental Animal Center (Taipei, Taiwan); 5 mice per group. (A) Mouse model of acute inflammation. (B) Mouse model of chronic inflammation. (C) Mouse model of two-stage skin tumorigenesis. The abbreviations used are as follows: Ac, acetone; Dex, dexamethasone; L-MEFA, 250 μg/mL of MEFA; H-MEFA, 500 μg/mL of MEFA; L-MELA, 250 μg/mL of MELA; H-MELA, 500 μg/mL of MELA.

Figure S2. Effects of MEFA and MELA on mouse tumor incidence and angiogenesis. (A) The change of mice body weight during DMBA-initiated and TPA-promoted skin tumorigenesis. (B) The percent of mice with tumors (tumor incidence) was plotted as a function of the number of test weeks. (C) Tumor morphology of dorsal skin from the indicated groups. (D) The photographs of the distribution of blood vessels on the dermal side of the dorsal skin tissues from the indicated groups. (E) Protein expression of VEGF in dorsal skin tissues from the indicated groups was determined by specific VEGF ELISA. Ac, acetone; Dex, dexamethasone; L-MEFA, 250 μg/mL of MEFA; H-MEFA, 500 μg/mL of MEFA; L-MELA, 250 μg/mL of MELA; H-MELA, 500 μg/mL of MELA. #, p < 0.05 versus vehicle control (Ac/Ac); *, p < 0.05 versus TPA treatment only (Ac/TPA).

Figure S3. Effects of MEFA and MELA on MAPK pathway in mouse skin tumor tissues. The dorsal skins of female ICR mice (5-weeks-old; 5 mice per group) were treated with acetone, dexamethasone, MEFA or MELA (250 μg/mL and 500 μg/mL) 30 min prior to the application of acetone or TPA (5 nmol) twice a week. Total cell extracts of mouse tumor tissues were prepared after mice were sacrificed. Proteins separated by SDS-PAGE were immunoblotted and probed with antibodies of p-p38, p38, p-ERK, ERK, p-JNK, JNK and β-actin. The values under each lane showed relative density of the band of western blot normalized to β-actin. Results shown are representative of the three independent experiments.

Figure S4. Effects of MEFA and MELA on NF-κB translocation in mouse skin tumor tissues. The dorsal skins of female ICR mice (5-weeks-old; 5 mice per group) were treated with acetone, dexamethasone, MEFA or MELA (250 μg/mL and 500 μg/mL) 30 min prior to the application of acetone or TPA (5 nmol) twice a week. Total cell extracts of mouse tumor tissues were prepared after mice were sacrificed, and then were subjected to isolation of nuclear/cytosol fraction respectively. Nuclear/cytosol proteins separated by SDS-PAGE were immunoblotted and probed with antibodies of p50, p65, lamin B1 (internal control for nuclear proteins) and β-actin (internal control for cytosol proteins). The basal p50 and p65 protein levels of nuclear/cytosol extracts of Ac/TPA treatment only were set to 1.0, and the relative changes in which were expressed as multiples of data under each lane. Results shown are representative of the three independent experiments.

Figure S5. Effects of MEFA and MELA on AP-1 activation in mouse skin tumor tissues. The dorsal skins of female ICR mice (5-weeks-old; 5 mice per group) were treated with acetone, dexamethasone, MEFA or MELA (250 μg/mL and 500 μg/mL) 30 min prior to the application of acetone or TPA (5 nmol) twice a week. Total cell extracts of mouse tumor tissues were prepared after mice were sacrificed, and then were subjected to isolation of nuclear fraction. Nuclear proteins separated by SDS-PAGE were immunoblotted and probed with antibodies of p-c-Jun, p-c-Fos and lamin B1 (internal control for nuclear proteins). The basal p-c-Jun and p-c-Fos protein levels of cell nuclear extracts of Ac/TPA treatment only were set to 1.0, and the relative changes in which were expressed as multiples of data under each lane. Results shown are representative of the three independent experiments.

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