Gel imaging system

HIF-1α, Twist 1, and VE-cadherin expression was analyzed using western blotting. Lysates were prepared using a buffer containing 1% SDS, 10 Mm Tris–HCl, pH 7.6, 20-μg/ml aprotinin, 20-μg/ml leupeptin, and 1-mM AEBSF. The protein concentration of lysates was measured using the Bradford method. Approximately 20 μg of protein was separated on an 8% SDS-PAGE gel and electroblotted onto a PVDF membrane. After blocking with 5% fat-free milk in TBS-Tween overnight, the membrane was incubated with primary antibodies overnight at 4°C. After washing with TBS-Tween three times, the membrane was labeled with horseradish peroxidase-conjugated anti-goat IgG (1:1,000) for 1 h at room temperature (RT). Blots were developed using a DAB kit, GAPHD was used as an internal control, and the bands were analyzed using a gel imaging system (Kodak).

pENTRY-PstCTE1 was used as the
plasmid DNA material with a size of approximately 2.5 kb.^{59 (link)} The plasmid isolation from the E. coli Top10/pENTRY-PstCTE1 was achieved using the
AnalyticJena plasmid isolation kit (innuPREP Plasmid Mini Kit 2.0)
by following the manufacturer’s protocol. The drug candidates
(10, 20, and 50 μM) were mixed with 40 μg of plasmid DNA
in sterile PBS buffer. The mixture was transferred to the 96-well
plate. NIR (630 nm) light (or dark) was exposed from 10 cm of distance
for 1 h at room temperature. Then, the mixture was separated by performing
agarose gel electrophoresis (1%, 60–70 V, 50–60 min)
and visualized under UV light (Kodak, Gel Imaging System).
Inhibition
of the photocleavage activity was tested by the addition of NaN₃ for singlet oxygen trapping and KI for OH radical trapping
as previously reported by Wang et al.^{30 (link)} The drug candidates (final concentration, 50 μM), plasmid
DNA (40 μg), and inhibitors alone or mixed (2 or 10 μM)
were merged in PBS buffer. NIR (630 nm) light (or dark) was applied
from 10 cm of distance for 1 h at room temperature. The results were
recorded by agarose gel electrophoresis.