N MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5diphenyltetrazolium bromide reduction) assay. In brief, stable transfected HT29 and HCT116 cells have been seeded at a density of five 104 cells/well in 96-well plates. Subsequently, cells have been irradiated having a single dose of 0, two, four, 6, or 8 Gy. Following 72 h, the culture medium was removed and replaced with 0.5 mg/mL MTT and permitted to stand for 1 h at 37 C for the formation of purple formazan. The precipitated formazan was dissolved with one hundred ofBiomedicines 2021, 9,four ofDMSO, and absorbance was measured at 570 nm having a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). 2.8. Colony Formation Assay For the clonogenic formation assay, transfected cells had been seeded in 6-well plates at a density of 6 103 cells/well and exposed to 2 Gy of irradiation on day 2. Soon after ten days of incubation, the colonies were fixed with methanol/acetic acid (3:1) and stained with 0.five crystal violet in 50/50 methanol/water for 20 min at room temperature. Subsequent, the staining resolution was carefully removed from every single well and rinsed with water. Ultimately, the number of cell colonies having a size 1 mm was counted using ImageJ software (Java 1.8.0_172). 2.9. Cell Cycle and Apoptosis Analysis by Flow Cytometry Just after synchronization with serum starvation for 24 h, cells were irradiated at a dose of four Gy. Following four days of incubation, floating and adherent cells had been harvested for cell cycle and apoptosis evaluation. For cell cycle analysis, cells had been fixed with 75 ethanol at four C overnight. Soon after cells have been washed twice with PBS, they were resuspended with PI/Triton X-100 (20 /mL PI, 0.1 Triton X-100, and 0.2 mg/mL RNase A) and incubated within the dark for 30 min. To detect apoptosis, we stained the harvested cells with PE-labeled Annexin-V/7-AAD, in line with the manufacturer’s protocol (cat no. Lacto-N-biose I medchemexpress 559763; BD Biosciences, San Diego, CA, USA). The signals of 1 105 stained cells in every sample had been detected by way of flow cytometry (Beckman Coulter, Fullerton, CA, USA). two.ten. Western Blotting c-Met, caspase-3, poly (ADP-ribose) polymerase (PARP), and GAPDH were quantified employing Western blotting. Soon after 72 h of irradiation, the whole-cell extract was isolated making use of RIPA buffer (1 mM EDTA [pH eight.0], 100 mM NaCl, 20 mM Tris [pH 8.0], 0.5 Nonidet P-40, and 0.five Triton X-100). In brief, equal amounts of protein were separated by SDSPAGE and transferred to polyvinylidene difluoride membranes. Membranes had been then incubated with Trident Universal Protein Blocking Reagents (GTX30963; GeneTex, Irvine, CA, USA) for 30 min at space temperature. This was followed by incubation with major antibodies at 4 C overnight. Target proteins have been probed with the following antibodies: anti-phospho-c-Met, -c-Met, -caspase-3, -PARP (1:1000; Cell Signaling Technology, Danvers, MA, USA). Anti-GAPDH (1:1000; Abcam, Cambridge, MA, USA) was applied as a loading manage for the whole-cell lysates. Subsequently, the membranes had been incubated using a 1:5000 dilution of an HRP-conjugated antibody for 1 h at space temperature. Protein bands had been created employing an enhanced chemiluminescence detection reagent, and signals were Hexazinone Technical Information captured employing the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). ImageJ software was utilised for protein quantification. two.11. Luciferase Reporter Assay The predicted miRNA-148a binding web page on the Met three UTR sequence (five -AGGCCACAAAAACACUGCACUGU-3 ) (cat. no. CW306396) or mutant three -UTR sequence (5 -AGGCCACAAAAACACACGUGACU-3 ) (ca.