CCD alters rhythmic RB phosphorylation in a cyclin D1–dependent manner.

(A) Western blot analysis of phosphorylated RB at multiple sites (pRB-S807/811, pRB-S795, pRB-S780, pRB-S612), total RB, CCNB1, cyclin D1, and CDK4 with the specific antibodies as indicated in the control (CTL) and jet lag (JL) cells collected every 6 hours (24 hours, 30 hours, 36 hours, 42 hours, 48 hours) for 24 hours after the final dex stimulation, as depicted with black arrowheads in Fig 1A. GAPDH (αGAPDH) is loading control. Representative images were taken from n = 3 independent experiments. (B) Statistical analysis of the WB data in (A) showing time-dependent variation of protein abundance as indicated in CTL (grey circle) and JL (brown circle) cells. GAPDH was used to normalize protein levels. *p p pMetaCycle p = 0.0481) and CCND1 protein (p = 0.006) in CTL cells (grey circle). Data normalized are represented as mean ± SEM from n = 3 independent experiments. (C) Schematic representation of the elements of the human cyclin D1 promoter (upper). Elements of human cyclin D1 promoter are represented by different colors. The transcriptional start site is identified by a black line and arrow. The data show log2 fold-change values for gene expression of signaling-dependent transcriptional activators or repressors that directly target enhancer elements in the cyclin D1 promoter, as indicated by the corresponding color codes (See S15 Table). (D) Carcinogen (MCA)-induced tumor bearing mice were exposed to a chronic jet-lag schedule (see Materials and methods), following which tumor and liver tissues were harvested from Jet-lag or Control mice at the indicated time points (ZT3, ZT9, ZT15) and subjected to western blot analysis using the indicated antibodies. Representative images from n = 3 independent experiments are shown. (E) Immunoblot analysis using liver extracts prepared from mouse liver tissues collected at 4-hour intervals as indicated for 24 hours in constant darkness. Specific antibodies were used for detecting endogenous pRB-S807/811, cyclin D1, CDK4, and CCNB1 proteins, as indicated. Anti-GAPDH (αGAPDH) was used for loading control. Similar results were obtained in two independent experiments. (F) HEK293T cells were transiently transfected with a cyclin D1-Luc reporter construct (pcyclin D1(−1748)-Luc) alone or co-transfected with plasmids expressing CLOCK, BMAL1, and CRY1, as indicated. After 24 hours, the cells were lysed and cyclin D1 promoter-driven luciferase activity was measured and normalized with pRL-TK activity. Representative results from three independent experiments performed are shown with the means ± SEM; n = 3. ***p S3 Data. AP-1, activator protein 1; BMAL1, brain and muscle Arnt-like protein-1; CCD, chronic circadian desynchrony; CCNB1, cyclin B1; CCND1, cyclin D1; CDK, cyclin dependent kinase; CLOCK, circadian locomotor output cycles protein kaput; CREB/ATF, cAMP response element-binding protein/activating transcription factor; CRY1, Cryptochrome1; CSL, CBF1, Suppressor of Hairless, Lag-1; CT, circadian time; dex, dexamethasone; E-box, enhancer box; Ets, E26 transformation-specific transcription factor; E2F, E2 transcription factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Jmj, jumonji and AT-rich interaction domain containing 2 (Jarid2); LEF/TCF, lymphoid enhancer-binding factor/T-cell factor; Luc, luciferase; MAR, matrix-associated region; MCA, methylcholanthrene; NFKB, nuclear factor kappa-light-chain-enhancer of activated B cells; Oct-1, POU domain, class 2, transcription factor 1; pRB, phosphorylated RB; pRL-TK, renilla luciferase reporter of the HSV-thymidine kinase promoter; RB, retinoblastoma; SP1/EgR, specificity protein 1 (SP1)/early growth response (EgR) transcription factor; STAT3/5B, signal transducer and activator of transcription 3/5B; WB, western blot; YY-1, yin yang 1 transcription factor; Z-box, Z-DNA forming sequence; ZT, zeitgeber time.