ERα bounds to <i>CLOCK</i> promoter regions in response to E2.

<p>A, Schematic representation of the ERE sites within the <i>CLOCK</i> promoter regions in the CLOCK-WT-Luc constructs. Constructs containing wild-type promoter and mutant promoters (truncation) are shown. Luciferase activity of MCF-7 cells transfected with the indicated constructs together with or without shERα#1 are shown on the right. B, CLOCK luciferase reporter constructs containing wild-type and mutant CLOCK promoters with point mutation in the EREs are shown, together with the luciferase activity of HeLa cells transfected with one of these constructs together with or without ERα. A and B, all experiments were performed in triplicate and repeated at least three times, and the data shown are the means ± SDs. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant). C, ChIP assay showing the recruitment of ERα on <i>CLOCK</i> promoter regions. MCF-7 cells were grown in phenol red-free medium and charcoal striped FCS medium for 2 days and the cells were then treated with vehicle or 1 µM E2 concentrations for 1 h, followed by ChIP assay using antibody against ERα or IgG. Total input DNA at a 1∶10 dilution was used as a positive control for the PCR reaction. Immunoprecipitated DNA was analyzed by PCR with primers specific for <i>CLOCK</i>, the relative positions of which are shown in the right panel of Figure 5C. All experiments were repeated at least of three times.</p

CLOCK promotes MCF-7 cells proliferation.

<p>Cells were transfected with control shRNA (shCon), shCLOCK or shERα#1 in the presence or absence of E2 for six or seven days followed by MTT assay or crystal violet staining. MTT assay of MCF-7 (A) and T47D (B) cells. The cells were treated with E2 for six days. C, Crystal violet staining (MCF-7 cells). The cells were treated with E2 for seven days. Viable colonies were stained with 0.1% crystal violet and photographed. The dye taken up by the colonies were solubilized in 10% SDS and quantified by absorbance at 570 nm. Representative images are shown on the left panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095878#pone-0095878-g006" target="_blank">Figure 6C</a>, and the corresponding quantitative analyses are shown on the right panel. Only representative data from three independent experiments are shown. D, Representative colonies of each experimental group are shown. MCF-7 cells transfected with pcDNA3, pcDNA3-CLOCK or pcDNA3-ERα were selected in the presence of 1 µg/ml G418 for 2 weeks. The cells were then collected and subjected to a soft agar colony culture. Photographs of the colonies were taken one week after seeding. All experiments were repeated at least three times. A-C, Data are the means ± SDs. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05).</p

Western blot analyses of CLOCK and ERα expression in cells treated with E2 or ICI.

<p>A, ERα and ERβ expression in MCF-7, T47D, MDA-MB-231, and MCF10A cells. B, CLOCK expression in MCF-7, T47D, MDA-MB-231, and MCF10A cells that had been treated with vehicle (control) or 1 µM E2 for 24 h. Cells were cultured in 5% charcoal striped FCS and phenol red free medium for 2 days before stimulated with E2. C, CLOCK and ERα expression in MCF-7, T47D and MDA-MB-231 cells that had been treated with vehicle, 1 µM E2 or 0.1 µM ICI alone or in combination for 24 h. Cells were cultured for 2 days in 5% charcoal striped FCS and phenol red free medium for two days before they were treated with ER ligands. D, CLOCK and ERα expression in T47D cells transfected with empty vector pcDNA3 or pcDNA3-Flag-ERβ. E, CLOCK and ERα expression in MCF-7 cells transfected with control shCon or two different shERα (shERα#1 and shERα#2). F, CLOCK expression in MCF-7 cells transfected with pcDNA3 or pcDNA3-Flag-ERα. B-F, 24 h after transfection, the cells were harvested and subjected to western blot analysis. In all experiments (A-F), β-actin expression was used as a reference. The blot shown is the representative result from three independent experiments. Image of the blot is shown in the top panel of each figure, with the quantitative analysis of the bands in the blot shown in the plot below. The levels of CLOCK or ERα signal obtained from control cells were set to 1. All experiments were repeated at least three times. Data shown in the graphs are the means ± SDs of three experiments. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant).</p

Proposed model showing the crosstalk between E2-ERα signaling and circadian rhythm.

<p>Proposed model showing the crosstalk between E2-ERα signaling and circadian rhythm.</p

Correlation between ERα and CLOCK expression in human breast tumor tissue samples.

<p>A, Representative results showing the immunohistochemical staining of ERα and CLOCK in serial sections of the breast tumor tissues. Each sample was incubated with antibody against ERα or CLOCK. Positive staining and negative staining are indicated by brown and blue staining, respectively (×200 Magnification). B, Correlation between ERα and CLOCK expression suggested by the 32 breast tumor samples. <i>χ</i>² test was used for statistical analysis. <i>P</i> values less than 0.05 were considered to indicate statistical significance.</p

ERα regulates <i>CLOCK</i> promoter activity.

<p>A, Schematic illustration of estrogen response elements in <i>CLOCK</i> promoter containing <i>CLOCK</i> sequence from −884 to +992 fused to luciferase (CLOCK-WT-Luc). B, Luciferase activity of HeLa cells transfected with CLOCK-WT-Luc and increasing amounts of REV-ERBα expression plasmid. C, Luciferase activity of HeLa cells transfected with CLOCK-WT-Luc plus REV-ERBα or ERα expression plasmids or both. D, Luciferase activity of HeLa cells transfected with CLOCK-WT-Luc and different amounts of ERα expression plasmid. E, Luciferase activity of HeLa cells transfected with CLOCK-WT-Luc and increasing amounts of ERβ expression plasmid. MCF-7 (F), T47D (G) and MDA-MB-231 (H) cells were grown in steroid-depleted media for 2 days, and then transfected with CLOCK-WT-Luc, followed by treatment with E2 or ICI alone or in combination. F-H, For control, cells were transfected with pGL3. pGL3 containing no <i>CLOCK</i> sequence was used as a mock DNA constructs. I, MCF-7 cells grown in normal media were transfected with the indicated shRNA (ERα; Con as a negative control) and CLOCK-WT-Luc. B-I, The graph depicts the normalized luciferase activity for each condition. Each experiment was performed in triplicate and repeated at least of three times. Data shown are the means ± SDs. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant).</p

AIB1 regulates the morphologies of breast cancer cells.

<p>(A) Light microscopic images showing the morphology of different breast cancer cells. (B) Expression levels of AIB1, ERα and E-cadherin in different breast cancer cells as detected by western blot analysis. Cell extracts were prepared from the different cell lines and probed with specific antibody against AIB1, ERα and E-cadherin or β-actin. (C) Light microscopic images showing EMT morphological changes induced in T47D cells after treatment with EGF (50 ng/ml) or E2 (20 nM) for 24 h. (D) Western blot analysis showing the levels of AIB1, ERα, E-cadherin and β-actin expressions in T47D cells after treatment with EGF or E2. (E) Coimmunoprecipitation showing AIB1 and ERα complex increased after treatment with EGF or E2. T47D cells treated without or with EGF (50 ng/ml) or E2 (20 nM) for 24 h were subjected to immunoprecipitation with anti-AIB1 or control IgG antibodies, followed by western blot analysis with anti-ERα and anti-AIB1 antibodies. (F) Light microscopic images showing T47D cells without or with AIB1 knockdown (shAIB1#1 and shAIB1#2), ERα knockdown (shERα) or both AIB1 and ERα knockdown. Cells were treated with the corresponding iRNA and then plated out in 50-mm dishes and incubated for 3 days before observing. (G) The cells from (F) were counted and plotted as percentage of clustered or scattered cells relative to total number of cells (400–500). (H) Western blot analysis showing the levels of AIB1, ERα, E-cadherin and β-actin proteins in T47D cells from (F).</p

Identification of SUMOylation sites in DEC1.

<p>(A) Putative SUMOylation sites of DEC1. (B) COS-7 cells were transfected with HA-tagged wild-type DEC1 or its mutants K159R, K279R or K159R/K279R (2K/2R) and Myc-tagged SUMO1, and then subjected to western blot with anti-HA antibody. (C) COS-7 cells were transfected with HA-tagged wild-type DEC1 or its mutant K159R/K279R (2K/2R) with or without Flag-tagged SENP1 or SENP1 mutant (mu) and then subjected to western blot with anti-HA antibody. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

AIB1 regulates ERα-mediated SNAI1 expression.

<p>(A) Schematic illustration of ERα-binding elements in SNAI1 promoter. Fragments A to C were chosen for PCR amplification in ChIP assays. Three truncated versions of the SNAI1 promoter were made, and the length of each is shown in the illustration. (B) ChIP assays showing AIB1- and ERα-SNAI1 promoter interaction in T47D cells. Cross-linked chromatin was extracted from T47D cells and subjected to immunoprecipitation with anti-AIB1, anti-ERα or control IgG, and the resulting precipitated DNA was used as template for PCR-ampαlification of SNAI1 promoter using specific primer covering region A, B or C of the promoter region. (C) Reporter gene assays of different truncated versions of SNAI1 promoter in the presence of AIB1 or ERαoverexpression. Each of the truncated SNAI1 promoters was fused to luciferase gene in pGL3 and the resulting construct was introduced into T47D cells along with AIB1 or ERα construct or both. The levels of luciferase activity in these cells were determined 48 h after transfection. Luciferase activity was normalized to β-galactosidase activity, used to evaluate transfection efficiency. Each experiment was performed in triplicates and repeated at least of three times. Data are the means ± SDs. Statistically significant differences (<i>P</i><0.05) in paired Student’s t-test are marked with an asterisk.</p

ERα ligands regulate the expression of CLOCK at the transcription level.

<p>Analysis of CLOCK mRNA levels in MCF-7 cells by real-time PCR. MCF-7 cells were cultured in phenol red free medium and charcoal striped FCS medium for 2 days before being treated with E2 or ICI and the expression of <i>CLOCK</i> was then analyzed by real-time PCR. Expression of <i>CLOCK</i> was normalized against <i>GAPDH</i> mRNA level (internal control). A, Cells treated with different concentrations of E2 (10⁻¹⁰ to 10⁻⁶ M) for 8 h. B, Cells treated with 1 µM E2 for different periods of time. C, Cells treated with 1 µM E2 or 0.1 µM ICI for 12 h. D, MCF-7 cells transfected with empty vector for ERα (pcDNA3), ERα, shCon (control for shERα) or shERα#1 construct. E, MCF-7 cells were cultured in phenol red-free medium and charcoal-striped FCS medium for 2 days before being treated with E2, Act D or CHX and the expression of <i>CLOCK</i> was then analyzed by real-time PCR. Cells treated with 0.5 µg/ml Act D, 10 µg/ml CHX alone or in combination with 1 µM E2 for 12 h. A-E, Relative levels were calculated by giving an arbitrary value of 1 to the control. <i>CLOCK</i> transcript levels were normalized to <i>GAPDH</i> transcript level and expressed as arbitrary units relative to the vehicle control (set as 1). Each experiment was performed in triplicate and repeated at least three times. Data shown are the means ± SDs. <i>P</i> value was determined by ANOVA with Bonferroni test (*, <i>P</i><0.05. ns, not significant).</p