Results: 4

1.
Figure 1

Figure 1. Proteomic analyses of cyclin D1-associated proteins. From: Transcriptional role of cyclin D1 in development revealed by a "genetic-proteomic" screen.

a , Silver-stained gels with cyclin D1-containing complexes purified from indicated organs (D1-Ip). Mock-Ip, mock purification from organs of wild-type mice. Cyclin D1 is marked by an asterisk. b, Relative abundance (arbitrary units, au) of Cdks and cell cycle inhibitors in cyclin D1-containing complexes in different compartments. c, Fraction of proteins among high-confidence cyclin D1-intactors and among “mock” purified proteins classified to indicated gene ontology categories.

Frédéric Bienvenu, et al. Nature. ;463(7279):374-378.
2.
Figure 3

Figure 3. Analyses of cyclin D1 transcriptional function in retina. From: Transcriptional role of cyclin D1 in development revealed by a "genetic-proteomic" screen.

a, Expression pattern of genes whose promoter regions were bound by cyclin D1, and which showed altered levels in D1−/− retinas. b, Binding of cyclin D1 to regulatory regions of indicated genes verified by targeted ChIP with anti-FLAG antibodies (to bring down cyclin D1) using postnatal day 0 retinas. c, d, Expression levels of indicated genes quantified in wild-type and D1−/− retinas by RT-PCR. Shown is fold-difference between wild-type versus D1−/− retinas. e, Levels of Notch1 protein in retinas, determined by immunoblotting. f, Levels of Notch1 transcripts in R28 cells, quantified by RT-PCR following knock-down (+shCycD) or overexpression of cyclin D1 (+CycD1). Shown is fold-difference compared to cells infected with control vector. Error bars, SD.

Frédéric Bienvenu, et al. Nature. ;463(7279):374-378.
3.
Figure 2

Figure 2. Analyses of cyclin D1 interaction with the mouse genome. From: Transcriptional role of cyclin D1 in development revealed by a "genetic-proteomic" screen.

a, Scatterplot of chromatin immunoprecipitation (Ip) with anti-FLAG antibody. Log2 intensity values of Ip from knock-in embryos are plotted against values from wild-type embryos. b, Examples of cyclin D1-bound regions. Plots display unprocessed ChIP-enrichment ratios for all probes within a genomic region. c, Location of cyclin D1 binding sites relative to transcription start sites of RefSeq genes. d, Conserved DNA sequence motifs identified among cyclin D1-bound regions. e, Left panel: Unsupervised clustering of cyclin D1-binding events, identified in whole-embryo ChIP-chip. Each horizontal line represents one gene, centered around transcriptional start site. Yellow and blue depict bound and unbound probes. Right panel: Number of tags observed for a given transcript (yellow: ≥4 tags; blue: ≤3 tags).

Frédéric Bienvenu, et al. Nature. ;463(7279):374-378.
4.
Figure 4

Figure 4. In vivo and molecular analyses of cyclin D1 – Notch1 connection. From: Transcriptional role of cyclin D1 in development revealed by a "genetic-proteomic" screen.

a, Whole mounts of D1−/− retinas infected with viruses encoding activated Notch and β-galactosidase (NIN-NICD, see Supplemental Fig. 10) or with vectors encoding only β-galactosidase (NIN), stained with X-gal to visualize clones of infected cells (arrowheads). b, higher magnifications of a. c, percentage of β-galactosidase-positive clones composed of 1, 2, 3 and ≥4 cells. d, Cyclin D1 was immunoprecipitated from P0 retinas and probed with indicated antibodies. e, Targeted ChIP on P0 retinas using anti-FLAG (to bring down cyclin D1) or with anti-CBP antibodies, followed by real-time PCR with Notch1-specific primers. Lower panel: ChIP with anti-FLAG followed by re-ChIP with anti-CBP antibodies and real-time PCR. f, Cyclin D1 was knocked-down (+shCycD1) or overexpressed (+CycD1) in R28 cells. ChIP with anti-cyclin D1, anti-CBP, anti-acetylated histone H4K5 (AcH4K5) or anti-acetylated histone H3K9,14 (AcH3K9,14) antibodies was followed by real-time PCR with Notch1-specific primers. The results show fold-difference compared to cells transduced with empty vectors. g, ChIP using anti-CBP and anti-AcH4K5 antibodies followed by real-time PCR with Notch1-specific primers using wild-type and D1−/− retinas. Error bars, SD.

Frédéric Bienvenu, et al. Nature. ;463(7279):374-378.

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