Fig. 3: CCM modulation of BMAL1–CLOCK target gene expressions.
From: Pharmacological targeting of BMAL1 modulates circadian and immune pathways
a, Effect of CCM (100 μM) on target genes compared to BMAL1 knockdown. The RNA levels of BMAL1 target genes were quantified by RT–qPCR in unsynchronized U2OS cells. Six and 12 biological replicates were used for knockdown and CCM treatment, respectively. b, Dose-dependent effects of CCM on BMAL1 target genes in unsynchronized U2OS cells (n = 3, biological replicates). c, The effects of CCM (100 μM) are impaired when BMAL1, CLOCK or NPAS2 is knocked down in unsynchronized U2OS cells (n = 5, biological replicates). d, Validation of BMAL1 and CLOCK siRNAs by confirming reduction in their protein levels in U2OS cells. This experiment was performed independently two times with similar results. e, CCM dose-dependent modulation of real-time circadian rhythm observed in peritoneal macrophages directly isolated from Per2–Luc (PER2::Luc) mice (synchronized, n = 3–6). f, CCM demonstrates no significant cytotoxicity on U2OS cells (n = 3, biological replicates) at concentrations of 100 µM and 200 µM for 48 h. The lysis buffer provided in the CyQUANT LDH Cytotoxicity Assay Kit was used as a positive control. g, Monitoring of the circadian rhythm in synchronized U2OS cells (n = 3, biological replicates) using RT–qPCR, showing differences between DMSO vehicle (black) and 100 μM CCM treatment (red). All error bars (a–c and e–g) denote the mean ± s.d. Statistical tests were performed using unpaired two-tailed t-test. Amp., amplitude; Conc., concentration; NS, not significant; Veh, vehicle; RLU, relative luminescence.
