Final Supplemental figures from β-Cryptoxanthin Reduced Lung Tumor Multiplicity and Inhibited Lung Cancer Cell Motility by Downregulating Nicotinic Acetylcholine Receptor α7 Signaling

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Supplementary Figure S1. Reduction of mRNA expressions of α7-nachr by BCX supplementation, not α3-, α5-, and β4-nachr, was associated with lung tumor multiplicity. Upper panel: The statistically significant correlation between lung tumor multiplicity (tumor number) and the expression of nachr-α7 mRNA (P = 0.03, r = 0.394, Pearson's correlation) and α7-nAChR protein (P < 0.01, r = 0.355, Pearson's correlation). Bottom panel: There were no significant differences in the nachr-α3, nachr-α5, and nachr-β4 mRNA levels among all the groups. The mRNA levels were measured with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and were normalized to the levels of GAPDH mRNA levels. The columns represent the mean {plus minus} sem. Abbreviation: BCX(1): β-cryptoxanthin 1 mg/kg diet; BCX(10): β-cryptoxanthin 10 mg/kg diet; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Supplementary Figure S2. BCX supplementation was associated with reduced early growth response-1 (Egr1) mRNA in the NNK-treated mice. The mRNA levels of egr1 were measured with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and were normalized to the levels of GAPDH mRNA levels. The columns represent the mean {plus minus} sem. * P = 0.04. Dotted lines indicated significant decreasing levels in a BCX-dose dependent manner (P-linear trend = 0.05). Abbreviation: BCX (1): β-cryptoxanthin 1 mg/kg diet; BCX (10): β-cryptoxanthin 10 mg/kg diet; egr1: early growth response-1; NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Supplementary Figure S3. PI3K downstream Rac1 and ARF6 contribute to lamellipodia suppression by BCX. BEAS-2B cells were transfected with wild type Rac1 or ARF6. Then cells were treated with BCX (1.0, 2.0, 4.0 μM) or MG624 (1.0 μM) for 12 hr. Cells were stained with TRITC-labeled phalloidin and analyzed using fluorescent microscopy. The representative fluorescent images are shown (A, the lamellipodia are indicated by arrowheads). The number of cells with significant lamellipodia was counted in five random fields (C). The representative fluorescent images of BEAS-2B cells were transfected with Rac1-Q61L or ARF6-Q67L are shown (B, the lamellipodia are indicated by arrowheads). The number of cells with significant lamellipodia was counted in five random fields (D). Results of Scratch wound are displayed in (E). *P = 0.04, **P = 0.01 compared with control group.

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