Fig. 5

Bmal1 knockdown prevents dexamethasone (DEX)-induced circadian rhythms and effects on tumor growth in vivo. a, b Bmal1 knockdown efficiency was evaluated for gene expression by real-time PCR (n = 3–7, Mann-Whitney rank sum test: p < 0.05) (a) and flow cytometry for protein expression (n = 6, Mann-Whitney rank sum test: p < 0.01) (b). c–g Clock gene expression in DEX- or phosphate-buffered saline (PBS)-treated tumors generated by subcutaneous (s.c.) injection of Scrambled shRNA- or Bmal1 shRNA-transfected B16 cells treated every 2 days for 11–13 days. Significant rhythms are illustrated with fitted cosine curves, otherwise data are connected by straight lines between data points, indicating no significant circadian rhythms. (n = 15–16, 3–4 mice/time point; two-way ANOVA: Bmal1, Per2 and Cry1: group: p < 0.0001, time: p < 0.0001; Per1: group: p < 0.0001, time: p > 0.05; Nr1d1: group: p < 0.0001, time: p < 0.001). h–m Cell cycle gene expression in DEX- or PBS-treated tumors generated by s.c. injection of Scrambled shRNA- or Bmal1 shRNA-transfected B16 cells. Significant rhythms are illustrated with fitted cosine curves, otherwise data are connected by straight lines between data points, indicating no significant circadian rhythms (n = 14–16, 3–4 mice/time point; two-way ANOVA: group: all genes: p < 0.0001, time: Cdk1 and Cyclin E: p > 0.05, Cdk2 and Wee1: p < 0.05, p21 and c-Myc: p < 0.01). n Relative tumor volume of Scrambled shRNA or Bmal1 shRNA s.c. tumors injected intra-tumorally with DEX or PBS at day 0, 2, 4 and 6. Scrambled shRNA tumors: n = 7–22, two-way ANOVA, p < 0.0001; Bmal1 shRNA tumors: n = 10–20, two-way ANOVA: p > 0.05 (no significant difference between DEX and PBS throughout the experiment). Data are represented as mean ± standard error of the mean. For details of statistics, see Additional file 1