The reagents used in this study are listed in Table S1.
Two human cholangiocarcinoma cell lines (RBE and HUCCT1) were obtained from the American type culture collection (ATCC, Manassas, VA, USA) and were authenticated by monitoring cell vitality, mycoplasma contamination, and short tandem repeat profiling. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37 ◦C.
Molecular cloning was performed by standard protocols. All construct sequences were verified by DNA sequencing. The detailed information concerning expression constructs and the primers used for molecular cloning is provided in Supplementary Tables S2 and S3.
Reverse Transcription-Quantitative PCR
Total RNA was extracted using TRIzol agent (Invitrogen, Waltham, MA, USA) and subjected to cDNA synthesis using PrimeScript RT Master Mix (Takara, Shiga, Japan). qPCR was performed using iQ SYBR Green Master kit (Roche, Shanghai, China) following the manufacturer’s instructions. All data were normalized to the housekeeping gene β-actin, and quantitative measures were obtained using the comparative CT method.
Colony formation survival and CCK-8 assays
A total of 1 × 104 cells were seeded into 6-well in triplicates for plate colony formation survival assay or 5 × 103 cells were seeded into 96-well in triplicates for CCK-8 assay. For colony formation assays, cells were fixed after two weeks by methanol, stained with 0.2% crystal violet solution then photographing. Colonies consisting of > 50 cells were counted. For CCK-8 assays, 10 µL CCK-8 solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well every 7 days after seeding. The plates were incubated in an incubator for 3 h, and then absorbance at 450 nm was determined.
Co-Immunoprecipitation Assay and Immunoblotting
For immunoblotting analysis, modified RIPA buffer (50 mM Tris-HCl, pH7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) was used to lyse cells. BCA protein assay reagent (Yeasen, Shanghai, China) was used to detect protein concentrations. Cellular extracts were resolved through SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica, USA), then incubated with the indicated primary antibodies. Enhanced chemiluminescent substrate kit (Yeasen) was used to analyze corresponding antibody specific signals. Table S4 lists the antibodies used. To immunoprecipitate endogenous proteins, cell extracts were incubated with primary antibodies or control IgG in a rotating incubator overnight at 4 °C, followed by incubation with protein A/G magnetic beads (Sigma-Aldrich, St. Louis, MO, USA) for another 3 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting.
Xenograft Tumorigenicity Assay
A total of 3 × 106 cells in 300 µl PBS were injected subcutaneously into the flank region of 6-week-old BALB/c female nude mice (Shanghai SLAC Laboratory Animal, Shanghai, China) for subcutaneous inoculation. The tumors were measured every 7 days after injection and the tumor volume was calculated by the formula (length × width2)/2. The mice were sacrificed 5.5 weeks after inoculation
Immunohistochemistry (IHC) staining was carried out using EnVision Detection Systems Peroxidase/DAB (DAKO, Shanghai, China) following the manufacturer’s recommendations, using an antibody against RNF216 (ab25961, 1:500, Abcam), DIAPH3 (ab227276, 1:500, Abcam) and Ki67 (ab15580, 1:500, Abcam). Interpretation of the IHC results was performed by two independent pathologists who were blinded to the clinicopathological information. By recording the percentage of positive staining (0 = negative, 1 ≤ 10%, 2 = 10–50%, 3 ≥ 50%) and staining intensity (0 = no, 1 = weak, 2 = moderate, 3 = strong) for each sample, immunoreactivity score (IRS) (0–9) was calculated by multiplying positive staining percentage with staining intensity. Low and high expression were defined according to the median IRS.
Wound Healing Assay
Cell were seeded in culture inserts (SPL Life Science, Gyeonggi-do, Korea) at 2 × 104 on 6-well plates. After 24 h of incubation, the wound was created by 1 ml tips. Images were taken at the indicated time points and the wound closure ratios were calculated.
Migration and invasion assays
Transwell chambers were used to perform migration and invasion assays in the absence (migration) and presence (invasion) of growth factor- reduced Matrigel (Corning, NY, USA). Briefly, 2.5 × 104 cells in FBS-free medium were plated in the top chamber. Growth medium containing 10% FBS was used as a chemoattractant in the lower chamber. After indicated times, migrated and invaded cells were fixed and stained with 0.1% crystal violet. Cells were
counted under an inverted microscope at 100 × magnification.
Cells were treated with 100 g/mL cycloheximide (CHX) and then harvested at indicated time points for immunoblotting analysis. The densitometry of Western blots was quantified using ImageJ software.
All data are presented as the mean ± standard deviation from at least three independent experiments. Overall survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. All statistical analyses were performed using SPSS software. A value of p < 0.05 was considered statistically significant.