1. Relative luciferase activity Relative luciferase activity
A. FOXF2 rs A>C
B. HEYL rs G>A
Relative luciferase activity
Relative luciferase activity
P = 0.01
P = 0.004

2. Supplementary Figure 1. Functional analysis of FOXF2 rs A>C (A), HEYL rs784621G>A (B) by luciferase reporter assay. The promoter fragments of FOXF2 and HEYL, containing rs A>C and rs784621G>A respectively, were synthesized by PCR. Each PCR primer was shown in Supplementary Table 1. The PCR products were cloned into the pGL3-basic plasmid (Promega, Madison, WI, USA). The correct sequence of all the clones was verified by DNA sequencing. The pRL-SV40 Renilla luciferase control reporter vector (Promega), which provides constitutive expression of Renilla luciferase, was used in combination with pGL3-basic-FOXF2 or pGL3-basic-HEYL constructs to cotransfect H1299 cells using Effectene transfection reagent (Qiagen, Hilden, Germany). Expression of Renilla luciferase provides an internal control value to which expression of the Firefly luciferase reporter gene may be normalized. The cells were collected 48 hr after transfection, and the cell lysates were prepared according to Promega’s instruction manual. The luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega), and the results were normalized by using the activity of Renilla luciferase. All experiments were performed in triplicate. Each bar represents mean ± SEM from three independent experiments carried out in triplicate. P value, Student’s t-test.