To measure viability of dividing cells in response to AZC (Sigma-Aldrich), cultures were grown in the presence of varying concentrations of AZC. Two days after plating, cells were harvested and cell numbers were calculated. To measure cell viability, the total number of cells was compared with cells with no AZC added. To measure viability of quiescent cells in response to AZC, cells were treated with varying concentrations of AZC and after 5 days of treatment, viability was measured using trypan blue (Thermo Fisher) exclusion. To measure exclusion, 100 μl of a 0.4% trypan blue solution was added to 100 μl of cells, and the resulting mixture was loaded onto a hemacytometer. To calculate cell viability, the number of blue cells was divided by the total number of cells. For each species, the experiment was conducted for three biological replicates. For each biological replicate, measurements were made for four technical replicates and the average cell viability was calculated.
To measure viability of quiescent cells in response to AZC and MG115 treatment, mouse and naked mole rat cells were cultured as described above and treated with 5 μM of MG115 (Abcam) for 24 h before treating with varying concentrations of AZC. The media was changed every day with fresh MG115 and AZC. After 5 days of AZC treatment, cells were harvested and viability was determined as described above.
To measure viability of quiescent cells in response to AZC and MG115 treatment, mouse and naked mole rat cells were cultured as described above and treated with 5 μM of MG115 (Abcam) for 24 h before treating with varying concentrations of AZC. The media was changed every day with fresh MG115 and AZC. After 5 days of AZC treatment, cells were harvested and viability was determined as described above.