G box f3 imaging system

Protein was extracted using urea buffer. Western blots were performed using the Invitrogen NuPAGE^® Novex^® Gel System and reagents (ThermoFisher Scientific). Antibodies used for blotting were anti-RhoB (Proteintech, Manchester, UK) and anti-HIF-1α (Sigma), with anti-α-Actin (Sigma) or anti-GAPDH (Proteintech) as loading control, with overnight rocking at 4 °C. Membranes were blocked in tris-buffered saline with 5% Marvel (Premier Foods, Hertfordshire, UK) and 0.05% Tween 20 (Sigma) for 1 h at room temperature followed by incubation in the appropriate secondary antibody (goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (1:10,000) (both Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. Luminescence was revealed by incubation with enhanced chemiluminescent reagent (Life Technologies) and signal detected on a G:BOX F3 imaging system (Syngene, Cambridge, UK).

The zeta potential and size of the complex were determined using ZetaPALS (Brookhaven, USA) at 25 °C with a 90° scattering angle by dynamic light scattering (DLS). The complexation of siMMP-9 to GT was also confirmed by agarose gel electrophoresis. GT/siMMP-9 complexes was suspended in nuclease-free water, 3 µL 6× gel loading buffer was added to each sample and 15 µL of the mixture was loaded onto each well in 4 % agarose gel with 1× GoldView II nuclear staining dye (Solarbio, Beijing, China). Electrophoresis was run in TBE buffer (pH 8.3) at 80 V for 20 min and the gel was observed by G:Box F3 imaging system (Syngene, Cambridge, UK). The morphologies of GT/siMMP-9 complexes were observed on an S-4800 scanning electron microscope (SEM, HI-9056-0003, Hitachi, Japan).