Supplementary Figure 1. Methylation level of 4 CpG sites located in the CYP24A1 promoter and gene in 33 nonmalignant lung samples and 153 lung AC included in the CURELUNG study. Non-malignant samples had a significantly higher β-value (p < 0.05, Wilcoxon rank sum test) for these CpG sites as compared to lung AC.

0 1

0.06

0.08

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0.14

0 1

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0.65

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Non-malignant Tumors samples

Non-malignant Tumors samples

Non-malignant Tumors samples

Non-malignant Tumors samples

DN

A m

eth

ylat

ion

lev

els

(β)

cg02673418 cg02604290 cg24582168p < 0.0001 p = 0.005 p = 0.0006

cg24849555

p = 0.01

TSA(nM)

1,25-D3

5-Aza(M)

A B

1,25-D3

A549H460

SKLU-1

DNMT1

DNMT3A

GAPDH

C

Supplementary Figure 2. (A) Quantitative RT-PCR of CYP24A1 expression in H460 and SK-LU-1 cells treated with increasing concentrations of 5-Aza followed by 100 nM of 1a,25-dihydroxyvitamin D3 (1,25-D3). (B) Quantitative RT-PCR of CYP24A1 expression in H460 and SK-LU-1 cells treated with increasing concentrations of TSA followed by 100 nM of 1,25-D3. Barplots and error bars represent mean and standard deviation of triplicate data points. (C) Basal protein expression of DNMT1 and DNMT3A in A549, NCI-H460 and SK-LU-1 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.