Ix71 fluorescence microscopy

The wound healing assay was performed to evaluate the effect of DNAzyme transfection on the migration of PC-3 cells. Briefly, the cells were seeded into a 24-well plate at a density of 2.0×10⁴ cells/well in 2 mL DMEM containing 10% FBS and cultured to 90% confluence. The cell mono-layer was subjected to a mechanical scratch wound using a sterile pipette tip, and the cells were washed with PBS twice. The cells were then treated with N-Ac-l-Leu-PEI/Dz nanocomplex in serum-free DMEM for 6 hours and further incubated in 10% FBS-containing DMEM for different time. The digitized images of wound area were captured with IX71 fluorescence microscopy (Olympus, Tokyo, Japan). Three representative zones were chosen to measure the average length of cell migration at each time point through Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA).

The sublines CNE3-GX6 and CNE3-GX11, with increased tumorigenicity compared with other sublines, as demonstrated by soft agar colony formation assay, animal experiments and analysis of Zbtb7a expression levels, were selected. The pRNAT-U6.1/Neo (GenScript, Piscataway, NJ, USA) vector was used for all transfection experiments, and these were performed as previously described (13 (link)). The cells were plated in 6-well plates at a density of 105 cells/well, and transient transfection was performed with 4 µg plasmid and 10 µl Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) when the cells covered >50% of the area. A transfection efficiency of 70–80% was observed by IX71 fluorescence microscopy (Olympus Corporation) after 48 h, and the transfected cells were used for subsequent assays. The untreated cells and cells transfected with the empty vector and shRNA were named as blank control (BC), negative control (NC) and shRNA, respectively.

The HeLa cells were seeded into 6-well plates at a density of 2.2×10⁵ cells/well and cultured at 37°C overnight. After treating with 0.1% Triton X-100, PEI25K, RNase A and RGP for 48 hrs (RNase A concentration of 4 μg/mL), the cells were washed with PBS twice and fixed with 75% ethanol for 30 mins. Followed by rinsing with PBS three times, the cells were suspended in PBS containing 0.3% Triton X-100 and incubated at room temperature for 5 mins. According to the manufacturer’s protocol, the cells were incubated with TUNEL detection solution in the kit for 1 hr, washed with PBS three times and detected using an IX71 fluorescence microscopy (Olympus).

The PC-3 cells were obtained from the Shanghai Institute of Cell Bank (Shanghai, People’s Republic of China) and cultured in DMEM supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin and 10% FBS at 37°C in a humidified atmosphere of 5% CO₂. For cellular uptake assay, the cells were cultured in six-well plates at 37°C for 24 h at an initial density of 3.0×10⁵ cells/well, treated with PEI/DOX-Duplex/siRNA for 6 h and observed through IX71 fluorescence microscopy (Olympus Corporation, Tokyo, Japan). In addition, the intracellular DOX release was monitored at different incubation times.

At 28 days post-administration, the HepG2-xenografted mice were sacrificed and dissected. Tumor tissues as well as major organs (heart, lung, liver, spleen and kidney) were harvested and fixed in 4% paraformaldehyde at 4 °C for 2 days. Then, the paraffin-embedded tissues were sectioned into slices of 20-μm thickness, stained with hematoxylin and eosin (H&E), and observed on an Olympus IX71 fluorescence microscopy (Tokyo, Japan). The apoptotic cells in tumor tissues were determined by in situ TUNEL detection kit according to the manufacturer’s protocol. To determine the Ki67 and ABCG2 expression in tumor tissues, the slices were treated with 0.2% Triton X-100 in PBS for 5 min and blocked with 5% goat serum for 2 h. Subsequently, the samples were incubated with primary antibodies at 4 °C overnight, and the HRP-conjugated secondary antibody was added into the samples. After the incubation at 25 °C for 1 h, the samples were observed through fluorescence microscopy. In addition, the expression of ABCG2 was analyzed at the mRNA level using qPCR as described in the section “Determination of ABCG2 mRNA expression”, in which 1 g of tumor tissues were homogenized in TRAzol Universal reagent at 4 °C (1000 rpm, 5 min) and then centrifuged at 120,000 rpm for 10 min (4 °C).

The cell culture and further treatment with release supernatants were performed as described in the section Wound healing assay, and the cell migration assay was conducted using Transwell chamber (Costar, Corning, NY, USA). The serum-free DMEM (200 µL) containing 2.0×10⁴ cells and 1% bovine serum albumin were added into the upper chamber of transwell with 8 µm pores, whereas 600 µL of 10% FBS-containing DMEM was added in the lower chamber. After the cell migration at 37°C for 24 h, nonmigrating cells on the top of membrane were carefully removed by mechanical wiping. The cells that have migrated to the lower surface of membrane were fixed with 75% ethanol at 4°C for 20 min and stained with 0.2% crystal violet for 15 min. After washing with PBS three times, the cells were detected in a random objective field (200× magnification) using an Olympus IX71 fluorescence microscopy.