Journal: Molecular Cancer

Article Title: Downregulation of miR-342 is associated with tamoxifen resistant breast tumors

doi: 10.1186/1476-4598-9-317

Figure Lengend Snippet: Stable miR-342 expression alters gene expression and sensitizes MCF-7/HER2Δ16 cells to tamoxifen . (A) Three independent total RNA samples purified from MCF-7/HER2Δ16 (Parental), stable MCF-7/HER2Δ16 cell lines expressing pCMV-puro-NC (Vector) or pCMV-miR-342 (miR-342) cultured for 48 hr in CS-FBS MEM were analyzed for miR-342 expression by qRT-PCR. (B) Each indicated cell line was cultured for 24 hr in 5% CS-FBS MEM and treated for 96 hr with 100 pM of E2 alone or in combination with 1.0 μM or 5 μM TAM. Each MTT experiment was repeated three times and the data is represented as the mean +/- SE relative to the E2 treated sample. Asterisks indicates significant changes ( p < 0.04). (C) Heat map of microarray expression analysis of genes significantly altered ( p < 0.001) by at least 1.5 fold in stable miR-342 expressing MCF-7/HER2Δ16 cells. (D) Microarray validation by qRT-PCR of three suppressed miR-342 direct target genes (SEMAD, BMP7, GEMIN4) and an upregulated indirect miR-342 target gene (TXNIP), normalized to β-actin, and expressed relative to parental MCF-7/HER2Δ16 cells. Asterisks indicate significant differences ( p < 0.05). (E) MiR-342 inhibition of GEMIN4 and BMP7 3' UTRs. MCF-7 cells were transfected with 20 nM of hsa-miR-342-3p (Ambion) or pre-miR negative control (Ambion) followed by pMir target firefly luciferase reporter plasmid containing 3' UTR sequences from BMP7 or GEMIN4 (Origene) and a renilla luciferase expression vector. At 48 hrs post-transfection cells were analyzed using the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions. Each sample was prepared in duplicate and the entire experiment was repeated three times. Data represents mean +/- SE percent inhibition of luciferase activity of miR-342 transfected cells relative to pre-miR negative control.

Article Snippet: The next day cells were transfected with 1.0 μg of with pMir target firefly luciferase reporter plasmid containing 3' UTR sequences from BMP7 or GEMIN4 (Origene) and 1.0 μg of renilla luciferase expression plasmid pRL-SV40 with Fugene 6.

Techniques: Expressing, Purification, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, Microarray, Inhibition, Transfection, Negative Control, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: Downregulation of miR-342 is associated with tamoxifen resistant breast tumors

doi: 10.1186/1476-4598-9-317

Figure Lengend Snippet: Target genes significantly modulated by miR-342 expression

Article Snippet: The next day cells were transfected with 1.0 μg of with pMir target firefly luciferase reporter plasmid containing 3' UTR sequences from BMP7 or GEMIN4 (Origene) and 1.0 μg of renilla luciferase expression plasmid pRL-SV40 with Fugene 6.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 1. Identification of a novel protein-protein interaction; Gemin2 self-interaction. A, gel-filtration analysis of Gemin2. The chromatogram demon- strates the elution of purified recombinant Gemin2 (*, the major elution peak of the protein). The arrows indicate the elution point of each molecular standard protein, and numbers correspond to the proteins shown at the inset graph. The inset graph indicates the semi-log plots for the molecular mass of the standard proteins and Gemin2 against the elution volume of these proteins in gel-filtration chromatography. B, the result of the mammalian two-hybrid assay. This experiment was independently conducted three times, and the errors bars represent standard deviation. Gal4-Gemin2 and VP16-SMN, Gemin2, and Gemin4 were expressed in CHO-K1 cells. The combination of Gal4-Gemin2 and VP16-Gemin4 was examined as a negative control. Protein-protein interaction was determined by measurement of luciferase reporter activity. C, the result of in vitro pull-down assay. In vitro translated 35S-labeled Gemin2 was incubated with in vitro translated biotinylated SMN, Gemin2, and Luc (luciferase as a negative control) and formed complexes that were captured with streptavidin beads. Proteins that remained bound to the beads were analyzed by SDS-PAGE and visualized by autoradiography. 10% of 35S-labeled protein used in the assay was loaded as an input. D, Gemin2 self-association using the purified recombinant Gemin2. Purified GST, GST-Gemin2, and Gemin2-His were resolved by SDS-PAGE and visualized by Coomassie staining (left panel). Right panels show the results of the GST pull-down assay. Purified GST or GST-Gemin2 were immobilized on glutathione-Sepharose and incubated with purified Gemin2-His. After resolution by SDS-PAGE, the bound proteins were visualized by Coomassie staining or Western blot using an anti-Gemin2 or an anti-His antibody. 30% of the Gemin2-His used in each reaction were loaded as an input.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Filtration, Purification, Recombinant, Chromatography, Two Hybrid Assay, Standard Deviation, Negative Control, Luciferase, Activity Assay, In Vitro, Pull Down Assay, Labeling, Incubation, SDS Page, Autoradiography, Staining, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 2. Interaction domain mapping for Gemin2 self-association and Gemin2-SMN association. A, schematic representation of the full-length and deletion mutants of Gemin2 (left panel). The interaction was examined using a mammalian two-hybrid assay. Gal4-fused proteins for full-length Gemin2 or Gemin2-deletion mutants were expressed in CHO-K1 cells with VP16-SMN (middle panel) or VP16-Gemin2 or VP16-Gemin2 mutants (right panel). Protein- protein interactions were determined with the same procedures as in Fig. 1B. The experiment was independently conducted three times, and the errors bars represent the standard deviation. B, confirmation of the properties of the Gemin2 self-association and the Gemin2-SMN association in the mutant Gemin290 –269. The interaction assay was performed by the same method as in Fig. 1C.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Two Hybrid Assay, Protein-Protein interactions, Standard Deviation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 5. Comparison of SMN oligomerization between untreated HeLa cells and Gemin2 knockdown cells. A, evaluation of gene expression of SMN complex components in HeLa cells following Gemin2 knock- down. The relative expression levels of the target genes were determined by qRT-PCR using total RNA from cells 44 h after transfection with Gemin2 siRNA. The black and white bars show the results of qRT-PCR analysis using two different primer sets against each target gene, and the errors bars represent the standard deviation. B, Gemin2 gene silencing with siRNA. Whole cell extracts were prepared from untreated, Gemin2 siRNA-trans- fected (siGemin2), negative control siRNA transfected (siControl), and mock transfected (Mock) HeLa cells. The cell extracts were analyzed by Western blotting using anti-SMN, anti-Gemin2, and anti-PRMT5 polyclonal antibodies. PRMT5 was used as loading control. C, sedimentation analysis of the cell extracts of native HeLa cells (WT, top panel), Gemin2 knockdown cells with siRNA (siGemin2, middle panel), and cells transfected with negative control siRNA (siControl, bottom panel). The cell extracts were sedimented on a 6–38% (w/v) sucrose density gradient. Fractions, indicated by numbers 1–13 from top to bottom of the centrifuge tube, were sub- jected to SDS-PAGE followed by Western blotting using anti-SMN polyclonal antibody. D, cytoplasmic soluble fraction from the siRNAs (siGemin2 or siControl) treated and untreated (WT) HeLa cells were immunoprecipi- tated with anti-SMN antibody. 2% of the input (left panel) and immunoprecipitates (right panel) were analyzed by Western blotting using anti-SMN, Gemin2, Gemin3, Gemin7, and SmB/B antibodies.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Comparison, Knockdown, Gene Expression, Expressing, Quantitative RT-PCR, Transfection, Standard Deviation, Negative Control, Western Blot, Control, Sedimentation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

doi: 10.1074/jbc.m609297200

Figure Lengend Snippet: FIGURE 6. The SMA-derived mutant SMN(D44V) caused a reduction in amino-terminal self-association and Gemin2 binding. Assay samples for the mutant SMNs were constructed from wild-type human SMN cDNA and were subjected to in vivo (A and B) and in vitro (C and D) binding assays as described above. The in vivo binding assay was independently conducted four times, and the errors bars represent standard deviation. The lower numerical values (C and D) indicate the percentage of the pull-downed 35S-labeled proteins against 10% of 35S-labeled protein used in the assay.

Article Snippet: Aswell asGemin2, the roles and themechanisms of the other Gemin proteins in the SMN complex are not yet fully characterized, although recent analysis reveals that, in addition to the SMN protein, Gemin3, -5, -6, and -7 all associate directly with the Sm proteins (24), and Gemin2, -3, -4, -6, and -8 and SMN are important for the U1 snRNP assembly activity of the SMN complex (20, 25, 26).

Techniques: Derivative Assay, Mutagenesis, Binding Assay, Construct, In Vivo, In Vitro, Standard Deviation, Labeling

Journal: FEBS letters

Article Title: Gemin5 plays a role in unassembled-U1 snRNA disposal in SMN-deficient cells.

doi: 10.1002/1873-3468.13031

Figure Lengend Snippet: Figure 5. Gemin5 directs the unassembled U1 snRNP to P bodies for unassembled U1

Article Snippet: The primary antibodies used in this study include antibodies against U1A (Santa Cruz, sc- 101149), Sm B (Santa Cruz, sc-25372), SMN (Santa Cruz, sc-15320), Gemin3 (ProteinTech Group, 11324-1-AP), Gemin4 (Santa Cruz, sc-166418), Gemin5 (ProteinTech Group, 24897-1- AP), DCP1A (ProteinTech Group, 22373-1-AP), EDC4 (ProteinTech Group, 17737-1-AP), Gemin1 (Abcam, ab5831), GAPDH (ProteinTech Group, 60004-1g), tubulin (ProteinTech Group, 66031-1-Ig), and actin (ProteinTech Group, 23660-1-AP).

Techniques: