The EIF5A2 promoter luciferase reporter vectors
The EIF5A2 promoter luciferase reporter vectors were constructed by Genechem Co.,
Ltd, Shanghai, China. Briefly, a 2.2-kb cDNA fragment (from -1,584 to +616 of human
EIF5A2 gene) with XhoI and HindIII sites was synthesized and cloned into the Xho I and
Hind III sites of the pGL3 basic luciferase vector to produce pGL3 EIF5A2 containing
seven Gli1 biding sites [GGGGTGGTC (-1563～-1571), ACCCACCTG (-1343～-1351),
GACCATCCA (-962 ～ -970), TGGGGGGTC (-662 ～ -670), CTGGTGGGG (20 ～ -28),
CTGGTGGGG (34～42) and TGGGTGTTC (580～588)]. In the mutagenesis vectors, Gli1
potential binding sites sequences were replaced respectively by GATTCTTAA sequence
that has no known binding sites for any transcription factors [22]. The Gli1 biding site
mutagenesis vectors included pGL3 EIF5A2 Mut 1 [GGGGTGGTC (-1563～ -1571),
GATTCTTAA (-1343～-1351), GATTCTTAA (-962～-970), GATTCTTAA (-662～-670),
GATTCTTAA (20～-28), GATTCTTAA (34～42) and GATTCTTAA (580～588)], pGL3
EIF5A2 Mut 2 [GATTCTTAA (-1563 ～ -1571), ACCCACCTG (-1343 ～ -1351),
GATTCTTAA (-962 ～ -970), GATTCTTAA (-662 ～ -670), GATTCTTAA (20 ～ -28),
GATTCTTAA (34～42) and GATTCTTAA (580～588)], pGL3 EIF5A2 Mut 3 [GATTCTTAA
(-1563～-1571), GATTCTTAA (-1343～-1351), GACCATCCA (-962～-970), GATTCTTAA
(-662～-670), GATTCTTAA (20～-28), GATTCTTAA (34～42) and GATTCTTAA (580～
588)], pGL3 EIF5A2 Mut 4 [GATTCTTAA (-1563～-1571), GATTCTTAA (-1343～-1351),
GATTCTTAA (-962 ～ -970), TGGGGGGTC (-662 ～ -670), GATTCTTAA (20 ～ -28),
GATTCTTAA (34～42) and GATTCTTAA (580～588)], pGL3 EIF5A2 Mut 5 [GATTCTTAA
(-1563～-1571), GATTCTTAA (-1343～-1351), GATTCTTAA (-962～-970), GATTCTTAA
(-662～-670), CTGGTGGGG (20～-28), GATTCTTAA (34～42) and GATTCTTAA (580～
588)], pGL3 EIF5A2 Mut 6 [GATTCTTAA (-1563～-1571), GATTCTTAA (-1343～-1351),
GATTCTTAA (-962 ～ -970), GATTCTTAA (-662 ～ -670), GATTCTTAA (20 ～ -28),
CTGGTGGGG (34 ～ 42) and GATTCTTAA (580 ～ 588)] and pGL3 EIF5A2 Mut 7
[GATTCTTAA (-1563～-1571), GATTCTTAA (-1343～-1351), GATTCTTAA (-962～-970),
GATTCTTAA (-662 ～ -670), GATTCTTAA (20 ～ -28), GATTCTTAA (34 ～ 42) and
TGGGTGTTC (580～588)]. The final vector constructs were verified via DNA sequencing
to ensure accuracy. AsPC-1 cells were transfected with 5μg of either the pGL3 EIF5A2 or
the pGL3 EIF5A2 mutant constructs and 2μg of the renilla luciferase construct (pGL4.75,
Promega) as an internal control for normalization using the Lipofectin reagent method
protocol. 48 hours after transfection, AsPC-1 cells were used for the luciferase assays
according to the protocol from the dual luciferase reporter kit (Promega) and the values
were readed using a luminometer. Each experiment was repeated at least 3 times, each
time in triplicate.