br Cell cycle analysis br Assessment of
2.6. Cell cycle analysis
Assessment of cell proliferation was evaluated using classic PI staining. Cells were seeded in complete medium in 6-cm culture plates at a density of 1 × 106 cells per well, and controls were incubated in DMSO. Twenty-four hours after plating, the cells were washed three times with PBS, then treated with the drug-loaded patches for 72 h in complete medium. Each well was then collected into separate flow tubes. Cells were fixed with 70% cold ethanol (for at least 1 h at −20 °C) overnight at 4 °C, then stained with 500 μl of PI solution (containing RNA enzyme) for 10 min at room temperature (in the dark). All samples were analyzed by flow cytometry (FACSCalibur TM, Becton Dickinson). Experiments were repeated three times.
2.7. Western blotting
Western blotting was performed using an SDS-PAGE Electrophoresis System. Cells were treated with patch infusion solution for 96 h (con-trols were treated with DMSO), then collected by plate-scraping on ice. Whole cell lysates were prepared using cell lysis buffer supplemented with proteinase inhibitors and phosphatase inhibitors. Protein con-centrations were determined using the Bradford Protein Assay Kit. Equal amounts of protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked using TBST (Tris buffered saline with 0.05% Tween-20) with 5% non-fat dry milk for 60 min at room temperature. The membranes were then incubated with primary Erastin overnight at 4 °C. Primary anti-bodies against CDK1, cyclin D1, cyclin E, P21, survivin, caspase-3, caspase-9, and GAPDH were used at dilutions of 1:1000, 1:1000, 1:5000, respectively. Membranes were washed in TBST three times, then incubated for 1 h at room temperature with HRP-conjugated sec-ondary antibodies. The membranes were washed three times and vi-sualized using enhanced chemiluminescence substrate (MILLIPORE). Band signals were quantified using Image J software.
2.8. Real-time PCR analysis
Cells were treated with the range of concentrations of drug-loaded patches for 2 days. Total RNA was extracted from sw480 and LoVo using TRIzol reagent. One microgram of total RNA was used as a template to synthesize first-strand complementary DNA using oligo-dT primer with the Omniscript RT kit (Qiagen, USA). QPCR were per-formed using SYBR Green PCR master mix reagent (Takara, Japan) on an ABI Prism 7500 qPCR system (Advanced Biosystems, USA). The human gene-specific primers for cDNA synthesis are summarized in Table 1.
After sacrificing the mice, the tumors were removed and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into sections. Tissue sections were deparaffinized, and antigens were retrieved using an antigen retrieval solution. Then, paraffinized tissue sections were
Sequences of RT-PCR primers.
Human Genes Primers Sequences
GAPDH F 5'-GCACCGTCAAGGCTGAGAAC-3'
Cyclin D1 R 5'-TGGTGAAGACGCCAGTGGA-3'
Cyclin E R 5'-CAGGTTCCACTTGAGCTTGTTC-3'
CDK1 R 5'-TGCTCTGCTTCTTACCGCTC-3'
P21 R 5'-CATGTACTGACCAGGAGGGATAG-3'
Caspase 3 R 5'-GGCTCAACGTTAGTGCCAGGA-3'
Caspase 9 R 5'-CTGGCAGCATCATCCACACATAC-3'
survivin R 5'-AGAGTGAGCCCACTGCTCAAAGA-3'
incubated with primary antibodies overnight at 4 °C, then with bioti-nylated secondary antibody on the next day. The dilution ratios of the primary antibodies were 1:100. Antigens were retrieved under high pressure for 2 min in citrate buffer (0.01 M sodium citrate, pH 6.0). All sections were examined and scored independently by two investigators without any knowledge of the clinicopathological data associated with the patients. At least five fields were randomly chosen. The expression of Ki67 was determined using the ratio of positive cells to staining in-tensity per field. A score of 0 was assigned for staining less than 5%, 1 for staining of 5–10%, 2 for staining of 10–50%, and 3 for staining > 50%. Intensity was graded as follows: 1, weak; and 2, strong. A total
2.10. Animal xenograft model
Male BALB/c nude mice (5 weeks old and weighing 20 − 23 g) were purchased from the Animal Centre of Beijing Vital River Laboratory Animal Technology Co. All animals received care in accordance with the guidelines of the animal welfare committee at Capital Medical University. To establish human colon cancer xenografts, sw480 cells (5 × 106 cells/100 μl) were subcutaneously injected into the right flanks of the nude mice after 1 week of acclimatization to housing conditions. When tumors had reached a certain volume, the animals were subjected to various drug treatments as described below.