br a fluorescence spectrophotometer using nm Ex and nm Em
a fluorescence spectrophotometer using 488 nm Ex and 525 nm Em filter settings.
2.9. siRNA or plasmid transfection
1 × 104 HCT-116 cells were seeded in 6-well plate and transfected with siRNA and plasmids after the cell density reached 60%. siRNA and plasmid were transfected using Lipofectamin 3000 (Shanghai, China, Invitrogen) according to the manufacturer's instructions. The transfec-tion of After 8 h transfection, Bou (25 μM) was added and incubated for another 16 h, subsequently cells were collected and subjected to further functional evaluation.
ChIP experiments were performed using a Pierce Agarose ChIP kit according to the manufacturer's protocol. After treatment, antibody against the UCP2 epitope (5 μg) was used to immunoprecipitate chro-matin in HCT-116 cells. Normal rabbit IgG was used as the negative control. ChIP was performed overnight at 4 °C, and immune complexes were collected using the Protein A/G Plus Agarose in the kit and wa-shed extensively. The DNA was extracted and sheared to fragments of 200–1000 bp by several sonications. The immunoprecipitated DNA was amplified by real-time PCR using primers specific for the mouse UCP2 promoter region. Relative PGC-1α at the UCP2 promoter was de-termined and normalized to the input DNA.
2.11. CoIP and Western blot analysis
Cells were lysate with ice-cold RIPA extract buﬀer, and supple-mented with a complete protease inhibitor mixture (Roche, China). For CoIP assay, Approximately 500 μg of cell extracted proteins were used for immunoprecipitation by incubation with 20 μL Protein A/G and 10 μg primary 30931-67-0 for 2 h at 4 °C under constant shaking ac-cording to the manufacturer's instructions. The immunoprecipitates were washed three to four times with a cold PBS, and the im-munoprecipitated beads were washed with immunoprecipitation (IP) lysis buﬀer and then eluted with 20 μl neutralized buﬀer; supernatants were separated and subjected to immunoblotting as reported (Rao
et al., 2017). Non-specific IgG was viewed as a control. Primary anti-bodies used are listed at Table 1.
2.12. Luciferase reporter activity assays
HEK293 cells were transfected with corresponding plasmids for a reporter assay using lipofectamin 3000 (Invitrogen). The plasmids for PGC-1α-luciferase and the UCP2- luciferase were generated based on the study. Twenty-four hours after transfection, the cells were stimu-lated with Bou (25 μM) as indicated. The cell lysates were harvested after drug administration, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, China). Firefly luciferase reporter gene measurements were normalized to Renilla luciferase activity.
2.13. Animal experiment
Four-week-old BALB/c male nude mice were bred in a pathogen-free environment at the Laboratory Animal Centre, Sun Yat-sen University (Guangzhou, China). All animal procedures were approved by the Sun Yat-sen University Committee on Ethics for the Use of Laboratory Animals in accordance with the Animal Welfare Legislation of China. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). HCT-116 cells (1 × 107 cells per mouse) were subcutaneously inoculated into the dorsal flank of nude mice. When tumor dimensions reached ∼75 mm3, the mice were randomly divided into two groups (ten mice per group) for intraperitoneal treatment with either: vehicle control (1% Tween-80 in saline) (Group 1); Bou, 50.0 mg/kg every other day by i.p. (Group 2). The body weight and tumor sizes of all mice were recorded every other day. Tumor sizes were determined by Vernier caliper measurements, and tumor volumes were calculated according to the formula: [(shortest diameter)2 × (longest diameter)]/2. After 16 days treatment, the mice were killed, and their tumors and interested organs were removed, weighed, photographed, and fixed in 10% par-aformaldehyde (PFA) and stored in liquid nitrogen for im-munohistochemistry assays and mechanism studies.
2.14. H&E staining and immunohistochemistry
The tissues fixed in 4% formaldehyde solution were embedded in paraﬃn after dehydration in a graded ethanol series (70%–100%). Embedded samples were sectioned (4 μm thick) with a rotary micro-tome and stained with hematoxylin and eosin (H&E) for microscopy examination. Sections were viewed with a light microscope (Japan, Olympus) and photographed at × 100 magnification. The Section of immunohistochemistry image was first labeled by an identification number code without the information of the grouping. The numbers and sizes of adipocytes of each slide were calculated with Image J Software (Califlorinia, USA) by Servicebio Company (BeiJing, China). Quantification analysis was performed in six randomly selected fields per sample in blind.
2.15. Statistical analysis
All results were expressed as mean ± S.E.M. and analyzed using GraphPad Prism 5 Software from 4 independent experiments. Comparisons between two groups were assessed by Student's t-tests for independents samples. Statistical significance was set at P < 0.05.