Antibodies and reagents
Antibodies plasmids and chemicals used in the study were listed: GAPDH (sc-47724, Santa cruz biotechnology, for western blotting), YAP (ab205270, abcam, for western blotting; sc-376830, Santa cruz biotechnology, for Immunofluorescence and Immunohistochemistry), p-YAP (ab76252, abcam, for western blotting), CYR61 (26689-1-AP, proteintch, for western blotting), c-Myc (ab32072, abcam, for western blotting), CyclinD1 (ab16663, abcam, for western blotting), LaminB1 (PB9611, Boster, for western blotting), RhoA (#2117, CST, for western blotting), ROCK1 (ab134181, abcam, for western blotting), PKA C-ɑ（#4782，CST, for western blotting），TGR5 (ab72608, abcam, for western blotting，for Immunofluorescence and Immunohistochemistry)， Ki67 (#9449, CST, for Immunohistochemistry). Ursodeoxycholic acid was obtained from Target Mol (Shanghai, China), while INT-777 was obtained from Medchemexpress. TGR5 Receptor Agonist or CCDC, SBI-115, H89, and SQ22536 were purchased from Selleck, while KT5720 was purchased from Sigma.
Cell culture and reagents
The human colon cells HCT116 and SW480 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science, Shanghai, China, with mycoplasma contamination detection and STR profiling. The cells were cultured in Dulbecco, s modified Eagle medium containing 10% foetal bovine serum (FBS, Gibio, USA) containing penicillin/streptomycin at 37℃ with a 5% CO2 humidiﬁed atmosphere.
The cells were seeded into 6-well plates with complete media and then used for the clonogenic assays. After adhering overnight, the cells were treated with the indicated drugs or transfected with plasmids or siRNA before treatment with the indicated drugs. The cells were treated with the indicated drugs for three to four days, followed by replacement of growth media with or without drugs every 2 days. After 7-14 days of culture under this condition, the media was discarded, the cells were fixed with 4% paraformaldehyde and then washed with phosphate buffered saline (PBS) after staining using 0.5% crystal violet for 15 min, followed by photographing.
Cell viability assays and EdU incorporation assays
MTT assay was used to determine the cell viability. About 5×103 cells were seeded per well in 96-well plates, and treated with the indicated drugs after adhering for 24h in complete medium. Then, the cells were incubated in MTT solution (5mg/ml) for 4 h, the medium was discarded, and the formazan crystals were dissolved in 150ul of DMSO followed by measuring of the absorbance at 490nM. The EdU incorporation assay was then performed using an EdU cell proliferation kit (RiboBio. Guangzhou, China).
Western blot (WB)analysis
For Western blot analysis, cells were lysed on ice with RIPA buffer (Beyotime) containing protease and phosphatase inhibitors (Roche). The proteins sample (30μg) were then separated using SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA) with the BioRad wet transfer system., followed by immunoblotting.
Small interfering RNA (siRNA) and plasmid transfection
The cells were trypsinised and transferred to 6-well pates at approximately 70-80% confluence. The siRNAs targeting TGR5, PKA Cα and the negative control were purchased from RiboBio (Guangzhou, China), while the pcDNA3.1, pcDNA3.1YAP, and pcDNARhoA-V14 plasmids used in this study were generated in our laboratory. Transfection of the plasmids and siRNA was performed using Lipofectamine 2000 (Invitrogen/Life Science) according to the manufacturer’s instructions.
Extraction of nucleoprotein and cytoplasmic protein
The nuclear and cytoplasmic proteins were isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech, C510001). The extracted nuclear and cytosolic proteins were then separated using SDS-PAGE and transferred to a PVDF membrane, followed by immunoblotting.
RNA extraction, reverse transcription and real-time PCR
Total RNA was extracted using a Cell Total RNA Isolation Kit (Foregene CO.LTD. Chengdu, China) according to the manufacturer’s protocol. Retrotranscription was performed using the Reverse Transcriptase M-MLV (Takara, Japan), while real-time PCR was performed with a SYBR Premix Ex Taq™ kit (Takara, Japan) on the iQ5 Real-Time PCR detection system (Bio Rad, Hercules, USA). primers used in this study were provided in Supplementary Table S1. The expression levels for target genes were normalized to GAPDH and calculated as previous demonstrated.
RhoA activity detection
RhoA activity in the cells was determined using RhoA Pulldown Activation Assay Biochem Kit (Cytoskeleton, Inc, Denver, Colorado, USA). The methods used have been described in a previously published protocol.
Immunofluorescence (IF) staining
Cells grown on coverslips were fixed with paraformaldehyde/PBS (4%) for 15 min, and permeabilized in Triton X-100/PBS (0.5%) for 20 min at room temperature. Then, they were blocked with BSA/PBS (3%) for 30 min, and incubated with primary antibodies at 4℃ overnight followed by incubating with Alexa Fluor 488-conjugated secondary antibodies (1:1000 dilution) at room temperature for 1 h. After the nuclei were stained with 5μg/mL DAPI (Invitrogen) for 5 min, the images were captured with a fluorescence microscope (Eclipse 80i, Nikon, Japan) at ×400 magnifications.
Immunohistochemistry (IHC) was performed using a commercially available immunohistochemical assay kit (Zhongshan Golden Bridge Biotechnology Co., Ltd, Beijing, China). The methods used have been described in a previously published protocol .
Rhodamine-labeled phalloidin staining
HCT116 and SW480 cells were stained using Rhodamine-labeled phalloidin in order to analyze the F-actin cytoskeleton. The cells grown on coverslips were fixed using 3.7% formaldehyde solution for 10 min on ice, and permeabilized with 0.5% Triton X-100 at room temperature for 10 min. They were then stained using Rhodamine-labeled phalloidin (US EVERBRIGHT® INC., Suzhou, China) for 20 min at room temperature, followed by staining with 5μg/mL DAPI (Invitrogen) for 5 min. The cells were then detected using a fluorescence microscope (Eclipse 80i, Nikon, Japan) at × 400 magnifications.
The whole cell cyclic adenosine monophosphate (cAMP) levels were detected using a Human cAMP ELISA kit (j&l Biological, Shanghai, China) according to the manufacturer's instructions.
In vivo experiments
5-6-week-old male C57BL/6 mice obtained from Beijing HFK Bioscience. The mice were supplied with food and water ad libitum and maintained under constant temperature and humidity in a 12-h dark/light cycle. All animal experiments were approved by the Experimental Animal Care and Use Committee of University of Tokyo and Tokyo Metropolitan Institute of Gerontology and were performed in accordance with the ARRIVE guidelines.
The mice were divided into five groups: Blank group (1), AOM/DSS group (2), AOM/DSS+0.05% UDCA group (3), AOM/DSS+0.1% UDCA group (4), and AOM/DSS+0.2% UDCA group (5) (n=10/per group). Groups two, three, four, and five were administered with a single i.p injection of the mutagen azoxymethane (AOM, Sigma-Aldrich) (10mg/kg body weight) in combination with three cycles of 1% DSS in drinking water for seven days, followed by regular drinking water for 14 days. The blank group (group one) were fed with a normal diet, while groups three, four, and five were fed with a diet containing 0.05%, 0.1%, and 0.2% UDCA, respectively. All the in vivo experiments were performed according to the institute guidelines and approved by the Animal Ethics Committee of the China Institute of Science.
The qualification of western blotting assay and clonogenic assay was performed using the Image J. All the statistical calculations were performed using the GraphPad Prism 5. The data were expressed as mean ± S.D, and analyzed with a two-tailed Student’s t-test. Statistical significance was defined as P-value of < 0.05 (*), < 0.01 (**), and < 0.001 (***). No statistical methods were used to predetermine sample size, and all experiments were performed using at least three biological replicates.