Journal: PLoS Genetics

Article Title: A Mechanism Misregulating p27 in Tumors Discovered in a Functional Genomic Screen

doi: 10.1371/journal.pgen.0030219

Figure Lengend Snippet: (A) Western analysis of p27 protein levels in p27 wild-type and p27 − / − thymus tissues and 20 of the 44 p27 +/− lymphomas. The blots were probed with α-tubulin as a loading control. (B) A schematic of the region on Chromosome 4 flanking the retroviral insertion sites is shown. The relative positions of Id3 , E2F2 , Ddefl1 , Tcea3 , Zfp46 , and Hnrpr are indicated. (C) Id3 protein levels in a subset of the lymphomas from (A) p27 +/− lymphomas. α-tubulin is shown as a loading control. The tumors with Id3 retroviral insertions near Id3 are indicated with an asterisk. The lymphoma with M-MuLv insertion closest to Id3 has the highest level of Id3 protein. doi:10.1371/journal.pgen.0030219.g001

Article Snippet: The cDNAs were diluted 1:10, and 5 μl added to each reaction containing Taqman master mix at 1× concentration and the p27 Mm00438167_g1, Hprt1 Mm00446968_m1, Id3 Mm00492575_m1, or Id3 Mm01188138_g1 Assay on Demand primers and probe (Applied Biosystems).

Techniques: Western Blot, Control, Retroviral

Journal: PLoS Genetics

Article Title: A Mechanism Misregulating p27 in Tumors Discovered in a Functional Genomic Screen

doi: 10.1371/journal.pgen.0030219

Figure Lengend Snippet: (A) p27 transcript levels in p27 +/− thymus and p27 +/− lymphomas were determined by quantitative RT-PCR using probe and primer sequences at the border of exons 1 and 2. The amount of RNA in each sample was normalized to HPRT1 gene expression. The fold change observed is relative to the p27 transcript levels in the p27 +/− normal thymus indicated by the striped bar. The p27 low lymphomas from A are shown in black, and lymphomas with higher p27 protein are shown in gray. An asterisk marks lymphomas with retroviral insertions near Id3. (B) p27 protein levels increase dependent on the presence of the Id3 siRNA. α-tubulin expression is shown as a loading control. (C) Quantitative RT- PCR indicates Id3 siRNA decreased Id3 mRNA and increased p27 mRNA abundance. HPRT1 mRNA was used to normalize RNA amounts in each sample. All primer-probe sets are located at exon borders. doi:10.1371/journal.pgen.0030219.g002

Article Snippet: The cDNAs were diluted 1:10, and 5 μl added to each reaction containing Taqman master mix at 1× concentration and the p27 Mm00438167_g1, Hprt1 Mm00446968_m1, Id3 Mm00492575_m1, or Id3 Mm01188138_g1 Assay on Demand primers and probe (Applied Biosystems).

Techniques: Quantitative RT-PCR, Gene Expression, Retroviral, Expressing, Control

Journal: PLoS Genetics

Article Title: A Mechanism Misregulating p27 in Tumors Discovered in a Functional Genomic Screen

doi: 10.1371/journal.pgen.0030219

Figure Lengend Snippet: (A) A schematic of the full-length p27 promoter and a minimal p27 promoter are shown. The E-box sequences CANNTG are marked by asterisks. (B) The E12/NeuroD2 E-protein heterodimers activate the full-length and minimal p27 promoters. (C) The percent induction of the mutant p27 minimal promoter, with three of the four conserved bases in the E boxes mutated (CANNTG to CGNNAT), is shown relative to the wild-type promoter. (D) Id3 represses E-protein activation of the p27 minimal promoter. The SV40-β-galactosidase or CMV-β-galactosidase plasmids were used as an internal control for each sample. doi:10.1371/journal.pgen.0030219.g003

Article Snippet: The cDNAs were diluted 1:10, and 5 μl added to each reaction containing Taqman master mix at 1× concentration and the p27 Mm00438167_g1, Hprt1 Mm00446968_m1, Id3 Mm00492575_m1, or Id3 Mm01188138_g1 Assay on Demand primers and probe (Applied Biosystems).

Techniques: Mutagenesis, Activation Assay, Control

Journal: PLoS Genetics

Article Title: A Mechanism Misregulating p27 in Tumors Discovered in a Functional Genomic Screen

doi: 10.1371/journal.pgen.0030219

Figure Lengend Snippet: Quantitative RT-PCR detected transcript levels for Id3 and p27 in PP1 inhibited (A) Lck-expressing cells LGY-6871, (B) SV40–180 large T-protein transformed cells, (C) LGY-6871 cells infected with an Id3 retrovirus, and (D) LGY-6871 cells infected with the control retrovirus are shown in the graph. PP1 was added immediately after the time-0 timepoint was collected, and later aliquots were collected at the specified times. The Id3 probe and primers detect endogenous and retroviral expressed Id3 . HPRT1 gene expression was used to normalize each sample. The fold change in transcript levels is relative to the 0 timepoint. (E) Representative autoradiograph of nuclear run-on assays from PP1 inhibited Lck-expressing cells LGY-10442–2 is shown and quantitative data from three experiments. The p27 transcript levels were normalized to EF1α transcript levels. PP1 was added to the culture after the 0 timepoint and washed out after 6 h as indicated by the arrow. (F) FACS analysis of PP1-inhibited Lck-transformed lymphoma cells LGY-10442–2. The cells were treated as described for (E). The arrow indicates depletion of early S-phase cells doi:10.1371/journal.pgen.0030219.g004

Article Snippet: The cDNAs were diluted 1:10, and 5 μl added to each reaction containing Taqman master mix at 1× concentration and the p27 Mm00438167_g1, Hprt1 Mm00446968_m1, Id3 Mm00492575_m1, or Id3 Mm01188138_g1 Assay on Demand primers and probe (Applied Biosystems).

Techniques: Quantitative RT-PCR, Expressing, Transformation Assay, Infection, Control, Retroviral, Gene Expression, Autoradiography

Journal: PLoS Genetics

Article Title: A Mechanism Misregulating p27 in Tumors Discovered in a Functional Genomic Screen

doi: 10.1371/journal.pgen.0030219

Figure Lengend Snippet: (A) Representative FACS analysis of thymi from lck − / − p27 +/+ , lck − / − p27 +/− , and lck − / − p27 − / − mice indicating loss of one copy of the p27 gene rescues the lck − / − phenotype. (B) The total cellularity for thymi and DP cells from all three genotypes are shown in the graph. Data were collected from three lck − / − p27 +/+ , seven lck − / − p27 +/− , and seven lck − / − p27 − / − mice. (C) Quantitative RT-PCR detected transcript levels for Id3 and p27 in wild-type small DN3 and DN4 cells. The fold change in the mRNA levels in the DN4 cells is relative to the DN3 cells. doi:10.1371/journal.pgen.0030219.g005

Article Snippet: The cDNAs were diluted 1:10, and 5 μl added to each reaction containing Taqman master mix at 1× concentration and the p27 Mm00438167_g1, Hprt1 Mm00446968_m1, Id3 Mm00492575_m1, or Id3 Mm01188138_g1 Assay on Demand primers and probe (Applied Biosystems).

Techniques: Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Dynamic changes in E-protein activity regulate T reg cell development

doi: 10.1084/jem.20132681

Figure Lengend Snippet: E-proteins regulate thymic foxp3 + T reg cell development. (a and b) Foxp3 + and CD25 + or HelioE + thymocytes from tamoxifen-treated WT Cre + (WT), E2Af/fHEBf/+ER-Cre (f/f f/+); E2Af/+HEBf/f ER-Cre (f/+f/f); E2Af/fHEBf/f ER-Cre(f/f f/f) mice analyzed by flow cytometry. Numbers in each quadrant indicate percentage. Data are representative of at least three independent experiments. (c) The total number of thymocytes, total number of CD4 + cells, the percentage and absolute number of Foxp3 + CD4SP thymocytes from each tamoxifen-treated genotyped group of mice were calculated from flow cytometry data. Each dot represents data from one mouse (7–10 mice total in each group); means are shown. **, P < 0.01. Data are pooled from at least three independent experiments. (d) Tamoxifen-treated 3–4-mo-old WT Cre and E2Af/fHEBf/f ER-Cre Foxp3-GFP-KI mice were analyzed for Foxp3 + cells in spleen, pLN, and mLN by flow cytometry; numbers in each quadrant indicate percentages. (e) The percentage of Foxp3 + cells among CD4 + cells in spleen, pLN, and mLN. Each dot represents data from 1 mouse (6–9 mice total in each group); graph shows means. *, P < 0.05. Data are representative of at three independent experiments (f and g) Thymocytes from age-matched (6–8 wk) WT Id3 −/− , Id3 −/− IL-4 −/− , Id2f/fId3f/f CD4-Cre mice were analyzed by flow cytometry. (f) Representative flow cytometry plots showing the percentage of CD4 + SPFoxp3 + cells in one mouse. (g) Combined data; each dot represents data from one mouse (5 mice total in each group). **, P < 0.01. Data are pooled from three independent experiments.

Article Snippet: For human probe: ID3, Hs00954037_g1; E2A, Hs00413032_m1; HEB, Hs00918966_m1; Foxp3, Hs01085834_m1; Hprt, Hs02800695_m1.

Techniques: Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Dynamic changes in E-protein activity regulate T reg cell development

doi: 10.1084/jem.20132681

Figure Lengend Snippet: E-protein activity controls NF-κB activation. (a and b) 16610D9 cells were transiently transfected with NF-κB luciferase reporter (PGL4.32) and CMV-Renilla along with E47 and Id protein expression plasmids individually (a) or in combination (b). Transfected cells were cultured overnight, and then stimulated with 2 µg/ml anti-CD3 and 1 µg/ml anti-CD28 for 2 h. Luciferase activity was measured and normalized by Renilla luciferase activity. Data shown are derived from mean ± SD of duplicate transfections in each experiment and are representative of at least three independent experiments. (c) 16610D9 cells were retrovirally transduced with empty vector, Id1-, Id2- or Id3-expressing vectors, and were stimulated with 2 µg/ml plate-bound anti-CD3 and anti-CD28 for 2h. Whole-cell lysates obtained were immunoblotted with antibodies against the indicated proteins. Data are representative of three independent experiments.

Article Snippet: For human probe: ID3, Hs00954037_g1; E2A, Hs00413032_m1; HEB, Hs00918966_m1; Foxp3, Hs01085834_m1; Hprt, Hs02800695_m1.

Techniques: Activity Assay, Activation Assay, Transfection, Luciferase, Expressing, Cell Culture, Derivative Assay, Transduction, Plasmid Preparation

Journal: Scientific Reports

Article Title: Dies1/VISTA expression loss is a recurrent event in gastric cancer due to epigenetic regulation

doi: 10.1038/srep34860

Figure Lengend Snippet: ( a) Expression of Dies1 , ID2 and ID3 in E-, M- and RE-cells. Asterisks stand for significantly distinct comparisons ( p < 5.00E-02). ( b) Expression of Dies1 , ID2 and ID3 in E-cells after exogenous stimulation with BMP4 ( Dies1 expression fold change from 2.0x to 16.8x, ID2 expression fold change from 11.3x to 17.2x, and ID3 expression fold change from 1.5x to 7,1x). ( c) Brightfield images of E-cells transfected with Non-Targeting siRNA (NT siRNA) and Dies1 siRNA and expression of Dies1 , ID2 and ID3 .

Article Snippet: Quantitative-Real-Time-PCR (qRT-PCR) was performed using RNA from 3 biological replicas (E, M, RE-cells and cancer cell lines described), in a ABI Prism 7000 Sequence Detection System and using TaqMan Gene Expression Assays (Applied Biosystems) or PrimeTime qPCR Assays (Integrated DNA Technologies), for the following genes: mouse CDH1 (Mm00486909_g1), Vim (Mm01333430_m1), ID2 (Mm00711781_m1), ID3 (Mm00492575_m1), Dies1 (Mm00472312_m1) and; human Dies1 (Hs00735289_m1), ID2 (Hs00171409_m1), ID3 (Hs00747379_m1) and miR-125a-5p (hsa-miR-125a-5p).

Techniques: Expressing, Transfection

Journal: Scientific Reports

Article Title: Dies1/VISTA expression loss is a recurrent event in gastric cancer due to epigenetic regulation

doi: 10.1038/srep34860

Figure Lengend Snippet: ( a) Conservation of the murine and human Dies1 seed region for miR-125a-5p. Asterisks stand for conserved nucleotides, dashes for complementary regions. ( b) Dies1 and miR-125a-5p expression in gastric cancer cell lines in comparison with normal stomach mucosa (in log scale). Also included is a summary of the Dies1 promoter methylation status (same legend as in applies), for comparison and correlation purposes. Asterisks stand for significantly distinct comparisons ( p < 5.00E-02). ( c) RNA expression analysis confirming miR-125a-5p inhibition (from 0,016x to 0,001x vs. negative control) and concomitance increased expression of Dies1 (from 1,58x to 1,94x vs. negative control), ID2 (from 0,87x to 2,19x vs. negative control) and ID3 (from 0,92x to 1,87x vs. negative control. ( d) Representation of the RNA expression analysis results correlating miR-125a-5p expression with Dies1 expression for each biological replicate performed.

Article Snippet: Quantitative-Real-Time-PCR (qRT-PCR) was performed using RNA from 3 biological replicas (E, M, RE-cells and cancer cell lines described), in a ABI Prism 7000 Sequence Detection System and using TaqMan Gene Expression Assays (Applied Biosystems) or PrimeTime qPCR Assays (Integrated DNA Technologies), for the following genes: mouse CDH1 (Mm00486909_g1), Vim (Mm01333430_m1), ID2 (Mm00711781_m1), ID3 (Mm00492575_m1), Dies1 (Mm00472312_m1) and; human Dies1 (Hs00735289_m1), ID2 (Hs00171409_m1), ID3 (Hs00747379_m1) and miR-125a-5p (hsa-miR-125a-5p).

Techniques: Expressing, Comparison, Methylation, RNA Expression, Inhibition, Negative Control