Colorimetric thiazolyl blue tetrazolium bromide

Stably selected cells with confirmed knockdown or overexpression efficiency were plated in five 96-well plates at a density of 1000–2000 cells per well. The number of viable cells was followed up for 5 days. Colorimetric thiazolyl blue tetrazolium bromide (Sigma-Aldrich, Oakville, ON, Canada) was added to each well (final concentration 0.5 mg/mL). For each plate, the cells were repeatedly plated in six wells and 570 nm absorbance was measured (with reference to 650 nm) using an EMax plate reader (Molecular Devices, Sunnyvale, CA). The mean of the absorbance was plotted against time and standard error of the mean was plotted as error bars. Statistical analysis was carried out using Student's t-test on day 5 and P < 0.05 was used as a cutpoint for statistical significance.

Stably infected miR-142-3p OE cells and their controls were plated in 6 wells of five 96 well plates at a density of 1000 cells for DOK infected with either miR-142 OE, OE control, or combined miR-142 OE/TGFBR1 ORF, or miR-142 OE/Control OFF. 500 Cal27 cells infected with the same vectors were plated with 500 cells per well. Cell viability was measured once a day for 5 days using Colorimetric thiazolyl blue tetrazolium bromide (Sigma-Aldrich) as previously described [77 (link)] with the highest and lowest value from each condition being discarded. Statistical significance was determined using Student's t-test on day 5 with a cutoff value of P < 0.05.