Hydroxyurea, aphidicolin and thymidine all cause S phase arrest.

<p>Cells were treated with different drugs for 24 h in all the panels. The distribution of cell cycle was assessed by flow cytometry. The experiments were performed in triplicate. Error bars represent standard deviation. *<i>P</i><0.05, #<i>P</i><0.01, when compared with control group.</p

Growth retardation of <i>Cul4b</i> heterozygous mice during embryonic development.

<p>(A) Bodyweights of <i>Cul4b</i> heterozygous mice and littermate wild-type females after birth. Data were presented as mean±SD. N = 8, *: p<0.05; **: p<0.01; ***: p<0.001. (B–E) Representative photographs of <i>Cul4b</i> heterozygous embryos and littermate wild-type controls at 9.5 (B), 10.5 (C), 12.5 (D) and 14.5 (E) dpc. The bar represents 1 mm in (B–C) and 2 mm in (D) and (E), respectively.</p

Morphology and histology of placentas of wild-type, <i>Cul4b</i> heterozygous and absorbed embryos at 14.5 dpc.

<p>(A) Representative photographs of placentas of wild-type, <i>Cul4b</i> heterozygous and absorbed embryos at 14.5 dpc. (B) H&E staining of radial sections of placentas. sp, spongiotrophoblast layer; la, labyrinthine layer. Lower panels are the higher magnification of the upper panels. (C) Immunohistochemisty of radial sections of placentas with an antibody to PECAM, an angiogenesis marker. Middle panels are the higher magnification of the upper panels, and lower panels are the higher magnification of the middle panels.</p

Distribution of <i>Cul4b</i> genotypes in progeny of <i>Cul4b</i>^+/flox/<i>Cul4b</i>^+/null;<i>EIIa-Cre</i>^+/− females.

<p>Litters were dissected at the times shown and genotyped by PCR as described in Materials and Methods.</p>a<p>ND indicates that the <i>Cul4b</i> genotype could not be determined by PCR.</p

Characterization of X chromosome inactivation by Cul4b expression in heterozygous mice.

<p>(A–C) Percentages of cells positive for Cul4b of <i>Cul4b</i> heterozygous mice and littermate wild-type female controls at 4 months (A), 3 weeks (B) and newborn (C). More than 2,000 cells of each tissue were scored. Hi, hippocampus; Ki, kidney; Li, liver; Lu, lung. Data were presented as mean±SD. *: p<0.05; **: p<0.01; ***: p<0.001. (D–E) Representative images of liver (D) and hippocampus (E) at 3 weeks stained with an antibody against Cul4b. Sections were counterstained with haematoxylin. Lower panels are the higher magnification of the upper panels. (F) Immunohistochemistry of paraffin sections of <i>Cul4b</i> heterozygous embryos at 7.5 dpc with an anti-Cul4b antibody. Embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. Middle and right panels are the higher magnification of the left panel.</p

Supplementary Figures 1 - 2 from MicroRNA-26a/b Regulate DNA Replication Licensing, Tumorigenesis, and Prognosis by Targeting CDC6 in Lung Cancer

Supplementary Fig. 1 Confirmation of cell synchronisation at the G1/S boundary. H1299 cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs after the final transfection, cells were synchronised at the G1/S boundary with mimosine, and the cell synchrony was determined by flow cytometry. Supplementary Fig. 2 MicroRNA-26a/b inhibit pre-replicative complex formation and cell cycle progression by suppressing CDC6 in human lung cancer cells. (A) SPAC cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs post transfection, cells were synchronized at the G1/S boundary with mimosine, and chromatin binding assay was performed with an aliquot of each cell sample. (B) The remaining cells were then released into fresh medium and harvested at different time points for FACS analysis.</p

Generation of <i>Cul4b</i> flox mice.

<p>(A) Strategy for generation of <i>Cul4b</i> floxed targeting vector. On the top was shown the wild-type allele of <i>Cul4b</i> gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. <i>BamHI</i> (labeled B) and <i>XbaI</i> (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. <i>BamHI</i> digested DNA was hybridized with 5′ probe and <i>XbaI</i> digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of <i>Cul4b</i> gene from the brain tissue of brain-specific knockout mice (<i>Cul4b</i>^flox/Y;<i>Nesin-Cre</i>). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of <i>Cul4b</i> mRNA isolated from brain tissues of wild-type males (<i>Cul4b</i>^+/Y;<i>Nestin-Cre</i>^+/−), wild-type females (<i>Cul4b^+/+</i>;<i>Nestin-Cre</i>^+/−), heterozygous females (<i>Cul4b</i>^+/flox;<i>Nestin-Cre</i>^+/−), and conditional knock-out males (<i>Cul4b</i>^flox/Y;<i>Nestin-Cre</i>^+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.</p

Morphology and histology of <i>Cul4b</i> null embryos.

<p>(A) Uterus excised from pregnant female at 12.5 dpc. Arrows indicate absorbed embryos. (B) Photomicrographs of E7.5 embryos dissected from surrounding deciduas tissue of the same uterus. The two embryos on the left appear normal in size and morphology and the two on the right were much smaller and were partially deteriorated. The bar represents 200 µm. (C) H&E staining of paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc. Littermate embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. The genotype of each embryo was determined by immunohistochemistry using an anti-Cul4b antibody, as shown below. (D) Immunohistochemistry of paraffin sections of wild-type and <i>Cul4b</i> null embryos at 7.5 dpc with an anti-Cul4b antibody. (E–F) Photomicrographs with higher magnification of the stained section shown in (D).</p

Increased MN formation in shRPA1 cells.

<p>A. Quantification of <i>RPA1</i> mRNA levels shRPA1 cells. RNA was extracted from shNeg MCF-7 cells and shRPA1 MCF-7 cells. The expression of RPA1 mRNA was quantified by real-time quantitative RT-PCR assay. The assay was performed in triplicate and relative means ± s.d. were shown. B. RPA1 protein level in shRPA1 cells. Equal amounts of protein lysates were subjected to SDS-PAGE (12%) and then detected using the antibodies against RPA1 and β-actin, respectively. C. Effect of RPA1 RNAi on cell cycle distribution of MCF-7 cells. The percentage of cells in S phase increased about 15% in shRPA1 cells when compared with shNeg cells (*<i>P</i> = 0.002). The experiments were performed in triplicate. Error bars represent standard deviation. For synchronization of cells, cells were starved in DMEM with 0.2% FBS for 48 h and then stimulated to initiate a new cell cycle in fresh media containing 10% FBS for 20 h. Afterward, cells were used for BrdU incorporation assay and cell cycle analysis. D. Reduction in cell proliferation as measured by BrdU incorporation assay. There was a marked reduction in BRDU-positive shRPA1 cells when compared with shNeg MCF-7 cells (*<i>P</i> = 0.004). The experiments were performed in triplicate. Error bars represent standard deviation. E. Increased MN-γ-H2AX (+) in shRPA1 cells. The assay was performed in triplicate and relative means ± s.d. were shown. *<i>P</i> = 0.008, when compared with shNeg MCF-7 cells.</p

Elevation of MN-γ–H2AX (+) in miCUL4B cells.

<p>*<i>P</i><0.05,</p><p>**<i>P</i><0.01.</p