Fig 1: Rhythmic effect of HSP90 inhibitors is associated with circadian expression of HSP90 genes.(A) Line graphs are replotted on the basis of the primary screening results in Fig. 1 to show that several HSP90 inhibitors are more effective at inhibiting cell proliferation at a specific time of day. The x axis depicts circadian phase as determined by the timing of dex addition (see Materials and Methods). (B) Temporal mRNA expression of the indicated HSP90 isoforms of dex-synchronized U2OS cells across circadian time (15). PJTKCycle < 0.05 indicates significant rhythms of HSP90AA1, HSP90AB1, and HSP90B1 (details are in data file S3). (C) Western blot analysis of the indicated HSP90 isoforms and BMAL1 in control (CTL) and BMAL1 knockout U2OS (BMAL1 KO) cells collected every 6 hours for 24 hours after dex stimulation. (D) Statistical analysis of the Western blot data in (C) showing protein abundance over time in CTL (gray circle) and BMAL1 KO (red brown circle) cells. JTKCycle identified a significant 24-hour rhythm in the expression of HSP90AA1, HSP90AB1, and HSP90B1 in CTL cells. Data were normalized to actin and are represented as means ± SEM from n = 3 independent experiments. (E) mRNA expression of the indicated HSP90 isoforms in control (CTL) and REV-ERB a/ß double-knockout U2OS cells (REV-ERBa/ß DKO). **P < 0.005; two-way ANOVA with Bonferroni’s multiple comparisons test. Data are shown with the means ± SD; n = 3 to 6 in each genes. (F) Line graphs, depicting means based on the data in fig. S6B, show expression profiles of the indicated HSP90 isoforms in CTL (gray) and REV-ERBa/ß DKO (light brown) cells. (G) Bar graphs depict cell viability assays performed 48 hours after 17-AAG treatment in CTL (gray bar) and REV-ERBa/ß DKO (light brown bar) cells. ***P < 0.005. Data show means ± SD; n = 6 for all doses.