Fig. 3.

TMZ sensitivity depends on MGMT activity and correlates with daily rhythms in MGMT mRNA abundance and promoter methylation in vitro. a) Circadian rhythms in MGMT expression peak approximately 10 h after peak Bmal1 expression in human LN229 GBM cells. Bioluminescence of cells transduced with either MGMT-luciferase (MGMTL) or Bmal1-luciferase (B1L) was recorded for 48 h in vitro (n=5 cultures each, mean ± SEM). b) LN229 cells collected every 4 h over 48 h show daily rhythms in MGMT mRNA (n=6 cultures each, mean ± SEM, JTK cycle p = 0.05) and c) MGMT promoter methylation (n=3 cultures each, mean ± SEM, JTK cycle p = 0.002). d) LN229-B1L and e) GL261-B1L cells show decreased cell number when treated with 100μM TMZ at either the peak or trough of Bmal1, with highest sensitivity at the peak. This time-of-day-dependent sensitivity is abrogated when co-treating with the MGMT inhibitor O⁶-BG (n=6 per group; mean±SEM; ****p < 0.0001, ns indicates p > 0.05)