Fig. 2: Glioblastoma cell lines show distinct time-dependent treatment responses to TMZ and clock modulators.

a TMZ was shown to methylate the DNA leading to DNA damage and apoptosis. MGMT is able to remove methyl-adducts thus preventing DNA damage and apoptosis. BMAL1 and MGMT were shown to be antiphase and TMZ treatment sensitivity highest and the peak of BMAL1 expression. KL001 was shown to stabilize CRY. SR9011 was described as an REV-ERB (NR1D1 and NR1D2) agonist. Both drugs were chosen as they showed promising and specific effects in GBM cell lines and mouse models before. b Timepoints for time-dependent treatment were chosen based on distinct points in the BMAL1 expression profile of T98G cells which showed the most robust rhythms. This was done based on previous reports suggesting a link between BMAL1 rhythm and TMZ sensitivity. The experimental scheme regarding synchronization at different timepoints prior to treatment is depicted. c Shows normalized proliferation curves for TMZ for the different synchronization times and d Shows the normalized proliferation curves for the different treatments at 0 h synchronization. Shown is the mean over nine measurements (three biological replicates with three technical replicates each). Cells were synchronized by medium change and treated with the drugs at the indicated concentrations. Proliferation measurements were performed as percent confluence in the IncuCyte S5. e The heatmap of treatment effects for all performed treatments. AUC was calculated and normalized to the DMSO control of the respective plate. Treatment effect was calculated as 1-AUC. Therefore, DMSO had a treatment effect of 0, cells that proliferated less than DMSO had a treatment effect >0 and cells that proliferated more than DMSO had a treatment effect<0. Statistical analysis was performed using One-way ANOVA. Error bars represent standard deviation. Icons and the scheme in panel a were generated in Biorender.com.