N MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5diphenyltetrazolium bromide reduction) assay. In short, stable transfected HT29 and HCT116 cells had been seeded at a density of 5 104 cells/well in 96-well plates. Subsequently, cells have been irradiated having a single dose of 0, two, four, six, or eight Gy. Just after 72 h, the culture medium was removed and replaced with 0.5 mg/mL MTT and allowed 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 using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). 2.eight. Colony Formation Assay For the clonogenic formation assay, transfected cells had been seeded in 6-well plates at a density of six 103 cells/well and exposed to 2 Gy of irradiation on day two. Immediately after 10 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 space temperature. Subsequent, the staining remedy was very carefully removed from every effectively and rinsed with water. Ultimately, the amount of cell colonies having a size 1 mm was counted making use of ImageJ application (Java 1.eight.0_172). two.9. Cell Cycle and Apoptosis Analysis by Flow Cytometry Just after synchronization with serum starvation for 24 h, cells had been irradiated at a dose of 4 Gy. Following 4 days of incubation, floating and adherent cells have been harvested for cell cycle and apoptosis evaluation. For cell cycle evaluation, cells have been fixed with 75 ethanol at four C overnight. Immediately after cells were washed twice with PBS, they have been resuspended with PI/Triton X-100 (20 /mL PI, 0.1 Triton X-100, and 0.two 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 accordance with the manufacturer’s protocol (cat no. 559763; BD Biosciences, San Diego, CA, USA). The signals of 1 105 stained cells in every single sample were detected via flow cytometry (Beckman Coulter, Fullerton, CA, USA). two.10. Cyclic-di-GMP (sodium) Data Sheet Western Blotting c-Met, caspase-3, poly (ADP-ribose) polymerase (PARP), and GAPDH have been quantified making use of Western blotting. Immediately after 72 h of irradiation, the whole-cell extract was isolated applying RIPA buffer (1 mM EDTA [pH eight.0], one hundred mM NaCl, 20 mM Tris [pH eight.0], 0.five Nonidet P-40, and 0.five Triton X-100). In brief, equal amounts of protein have been separated by SDSPAGE and transferred to polyvinylidene difluoride membranes. Membranes were then incubated with Trident Universal Protein Blocking Reagents (GTX30963; GeneTex, o-3M3FBS Protocol Irvine, CA, USA) for 30 min at room temperature. This was followed by incubation with primary antibodies at 4 C overnight. Target proteins have been probed together 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 utilized as a loading manage for the whole-cell lysates. Subsequently, the membranes have been incubated having a 1:5000 dilution of an HRP-conjugated antibody for 1 h at room temperature. Protein bands had been created using an enhanced chemiluminescence detection reagent, and signals had been captured employing the ChemiDoc MP Imaging Method (Bio-Rad Laboratories, Hercules, CA, USA). ImageJ software was made use of for protein quantification. two.11. Luciferase Reporter Assay The predicted miRNA-148a binding web page of the Met three UTR sequence (five -AGGCCACAAAAACACUGCACUGU-3 ) (cat. no. CW306396) or mutant three -UTR sequence (5 -AGGCCACAAAAACACACGUGACU-3 ) (ca.
