Measurement of cell viability and clonogenic assay

The viability of LV control and miR-320c HCT116 cells was determined using alamarBlue assay as previously described [10]. All assays were carried out with appropriate controls. Briefly, 5000 cells were cultured in a 96-well plate and cell viability was measured at the indicated time points by adding 10% volume alamarBlue assay reagent and measuring absorbance at 570λ. The colony forming ability of HCT116 cells transduced with miR-320c was determined using clonogenic assay as previously described [35, 36]. Briefly, LV control or miR-320c HCT116 cells were seeded in 12-well plates in different serial dilution (1:2 to 1:64). Initial seeding density was 0.015 × 10⁶ cells per well, and incubated at 37°C under 5% CO2 for 10 days. The plates were then washed and stained with Diff-Quik stain set (Siemens), and the plates were scanned and number of colonies was observed under microscope. The fraction of surviving cells was estimated by comparison of miR-320c to LV control cells. The experiment was done twice in duplicate. Furthermore, the clonogenic assay was conducted to examine the effect of 5-Fluorouracil on colony formation in both cells. A total of 1 × 10⁶ cells were seeded in T25 flask. After 48 hours of exposure to 1.5 μM of 5-Fluorouracil, the cells were trypsinized and reseeded in 12-well plates as described above to observe the effect of the drug.