Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Software, Transfection

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 1. miR-206 targets cyclin D1. (A) Sequence alignment between miR-206 and the 3′UTRs of cyclin D1 from different species. In brackets the 3′UTR size. (B) Diagram of the luciferase reporter construct with the putative miR-206 binding site (WT 3′UTR) and mutations (3′UTR MUT). (C) Relative lucif- erase activity was measured in HeLa cells after transfection of reporter constructs along with pSP65-U1 (CTR) or pSP65–206 (miR-206). Relative Firefly luciferase values were determined by a ratio of Firefly to Renilla luciferase with the control set to 1.00. Values are the means ± SD of 3 separate experi- ments. **A Student t test performed between control and miR-206 transfected cells yielded P values < 0.01.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Sequencing, Luciferase, Construct, Binding Assay, Activity Assay, Transfection, Control

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 2. Expression kinetics of miR-206 and cyclin D1 in differentiating C2C12 cells. C2C12 myoblasts were seeded in GM at 1.5 × 104/cm2. Cells were shifted in DM 24 h after plating and left to differentiate for further 72 h. (A) Northern blot analysis of miR-206 expression in C2C12 cells after 24 h in GM (0) and at different time points upon shift to DM. (B) Western blot analysis of cyclin D1 and MyHC expression in C2C12 cells cultured as in (A). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) MyHC immunofluorescence staining (green) of C2C12 cells after 24 h in GM (DM 0 h) and after 72 h in DM (DM 72 h). Nuclei were counterstained in blue (DAPI) and individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 20 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Western Blot, Cell Culture, Immunofluorescence, Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 3. miR-206 controls cyclin D1 accumulation in C2C12 cells. C2C12 myoblasts were seeded in GM at 2.5 × 103/cm2. Cells were transfected 24 h after plaiting. (A) Northern blot analysis of miR-206 expression (upper) and western blot analysis of cyclin D1 expression (lower) in C2C12 cells 48 h after transfection with a control vector (CTR) or with a miR-206 expression vector (miR-206). Cells were kept in GM throughout the experiment. (B) The effect of miR-206 overexpression on C2C12 cell proliferation and differentiation was evaluated 48 h after transfection by 1 h BrdU incorporation and MyHC staining, respectively. Results are represented relative to the BrdU+ nuclei or nuclei in MyHC+ cells in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. (C) Immunofluorescence staining of cyclin D1 (pink) and MyHC (green) 48 h after transfection. Nuclei were counterstained in blue with DAPI. Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. To obtain cyclin D1 images, before merging, individual pictures were pseudocol- ored using a LEICA Microsystems Imaging software. Bar = 10 μm. (D) C2C12 myoblasts were seeded at low (LD) and high (HD) density in GM. Cells were shifted to DM the day after plating and analyzed after further 3 d. The panels show a northern blot analysis of miR-206 expression (left panel) and a western blot analysis of cyclin D1 and differentiation associ- ated marker expression (right panel) after 24 h in GM and 72 h after shift- ing to DM. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Northern Blot, Expressing, Western Blot, Control, Plasmid Preparation, Over Expression, BrdU Incorporation Assay, Staining, Immunofluorescence, Imaging, Software, Marker

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 4. Inhibition of miR-206 rescues cyclin D1 in myotubes (A) Experimental scheme. C2C12 myoblasts were induced to differ- entiate in DM in the presence of AraC. After 3 d, AraC was washed out and cells left to recover in DM for further 24 h. Finally, pure myotubes were transfected with LNA against miR-206 and analyzed 48 h later. (B) Northern blot analysis of miR-206 and miR-1 expression (left panel) and western blot analysis of cyclin D1 expression (right panel) in pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Cyclin D1 expression in proliferating myoblasts is also shown (GM). Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (C) Double immunofluorescence staining of MyHC and cyclin D1 of pure myotubes transfected with a control LNA (LNA C) or anti-miR-206 LNA (LNA 206). Individual pictures of the same field, taken with a DC camera, were merged using a LEICA Microsystems Imaging Equipment. Bar = 10 μm.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Inhibition, Transfection, Northern Blot, Expressing, Western Blot, Control, Double Immunofluorescence Staining, Imaging

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 5. miR-206 inhibits cell proliferation in Ras-transformed fibro- blasts. (A) Expression levels of cyclin D1 in NIH3T3(Ras) cells as compared with NIH3T3(BN) cells. (B) Real-time PCR analysis of miR-206 expres- sion in NIH3T3(Ras) cells. Results are shown relative to untransformed NIH3T3(BN) cells set to value 1.00. Each sample was analyzed in tripli- cate, and values are the means ± SD of 3 independent experiments. **A Student t test performed between untransformed and transformed cells yielded P values < 0.01. (C) NIH3T3(Ras) cells were transfected with a control vector (CTR) or with a miR-206 expression vector (miR-206) and analyzed 24 h later. Upper, northern blot analysis of miR-206 expression; lower, western blot analysis of cyclin D1 expression. (D) Effect of miR-206 forced expression on cell proliferation as determined by 1 h BrdU incor- poration. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values < 0.05. Equal RNA and protein loading was confirmed by detecting, snRNA U2, and β-tubulin, respectively.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 6. Relationship between miR-206 downregulation and cyclin D1 expression in NSCLCs. (A) Northern blot analysis of miR-206 in different murine tissues. snRNA U2 levels were used as a loading control. (B) Real-time PCR analysis of miR-206 expression in human NSCLC tissues. Results are shown relative to the matched normal lung tissues set to value 1.00. Each sample was analyzed in triplicate, and values are the means ± SD of three independent experiments. **A Student t test performed between normal and tumor tissues yielded P values < 0.01. (C) Western blot analysis of cyclin D1 expression in normal and neoplastic lung tissues. Equal protein loading was confirmed by detecting actin. n, normal tissue; t = tumor tissue

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Expressing, Northern Blot, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Cyclin D1 is a major target of miR-206 in cell differentiation and transformation.

doi: 10.4161/cc.26674

Figure Lengend Snippet: Figure 7. miR-206 inhibits cancer cell proliferation through repression of cyclin D1. (A) A549 and HeLa cells were transfected with a control vec- tor (CTR) or with a miR-206 expression vector (miR-206) and analyzed 72 h later. Top panel: northern blot analysis of miR-206 expression; lower panel, western blot analysis of cyclin D1 expression. Equal RNA and protein loading was confirmed by detecting, snRNA U2 and β-tubulin, respectively. (B) Effect of miR-206 forced expression on cell prolifera- tion as determined by 1 h BrdU incorporation and immunofluorescence staining. Data are reported relative to BrdU+ nuclei in CTR (set to 1.00), as individually assessed in each independent experiment. Values are the means ± SD of 3 separate experiments. *A Student t test performed between control and miR-206 transfected cells yielded P values <0.05.

Article Snippet: The mouse monoclonal antibody 72–13G (Santa Cruz Biotechnology) was used to stain cyclin D1-positive cells.

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Northern Blot, Western Blot, BrdU Incorporation Assay, Immunofluorescence, Staining

Journal: Biochimica et biophysica acta

Article Title: Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein.

doi: 10.1016/j.bbamcr.2014.01.027

Figure Lengend Snippet: Fig. 6. Dp71d interacts with IMPα2/IMPβ1. (A) Pull down assays were performed by incubating glutathione-sepharose beads preincubated with bacterially expressed GST (negative con- trol) or GST-Dp71d (full length) with nuclear extracts from C2C12 cells. Beads were recovered by centrifugation and eluted proteins were analysed by Western blot using specific anti- bodies against IMPα2 and IMPβ1 (upper panel). Inputs correspond to 5% of nuclear extract prior to pull down. Lower panel shows purified GST and GST-Dp71d proteins that were visualized by SDS-PAGE followed by Coomassie brilliant blue staining. (B) Complexation of the ZZ domain with IMPs in living cells. C2C12 cells transiently expressing TetraGFP or TetraGFP-ZZ proteins were immunoprecipitated using GFP-trap and immunoprecipitated proteins subjected to Western blot analysis using the indicated anti-IMP antibodies. Inputs correspond to 5% of nuclear extract prior to immunoprecipitation. Un, unbound fraction; B, bound fraction. (C) ALPHAScreen binding assays [43] were performed by incubating the His-tagged ZZ domain of Dp71d with increasing concentrations (0–30 nM) of bacterially expressed GST alone or GST-IMPβ1, -IMPα2, or predimerized GST-IMPα2/β1 (where GST- IMPβ1 was biotinylated and GST-IMPα2 unlabelled) — see Section 2. Triplicate data points from a single typical experiment are representative of three independent experiments fitted using SigmaPlot software to determine the apparent dissociation constants (Kd). ND, not able to be determined due to low binding.

Article Snippet: GFP fusion proteins were immunoprecipitated using the GFPTrap® bead system (Chromotek, Germany) in accordance with the manufacturer's instructions.

Techniques: Centrifugation, Western Blot, SDS Page, Staining, Expressing, Immunoprecipitation, Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Software

Journal: Biochimica et biophysica acta

Article Title: Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein.

doi: 10.1016/j.bbamcr.2014.01.027

Figure Lengend Snippet: Fig. 7. Identification of a CRM1-recognised nuclear export signal (NES) in the carboxy-terminal domain of Dp71d. (A) C2C12 cells seeded on glass coverslips were incubated for 24 h with the CRM1-specific inhibitor Leptomycin B (LMB) diluted in methanol or with methanol alone (control). Cells were immunostained with primary anti-Dp71d antibody and a fluorescein- conjugated secondary antibody (green) and counterstained with DAPI (nuclei, blue). (B–D) C2C12 cells seeded on glass coverslips were transfected to express GFP-Dp71d, TetraGFP- Amino or TetraGFP-ZZCarboxyl (green) and 8 h post-transfection incubated for additional 12 h without (control) or with LMB. Schematics of the N- and C-terminal portions of Dp71d expressed in the TetraGFP-Amino and TetraGFP-ZZCarboxyl constructs respectively. The putative CRM1-recognised NES sequences located in each portion of Dp71d are shown. Cells were fixed, counterstained with DAPI (nuclei, blue), imaged by CLSM, and images such as those shown were analysed as per the legend to Fig. 1 for endogenous Dp71d (A) and GFP- based fusion proteins (B–D), as described in Section 2. Results represent the mean +/−SEM (n = 50), with significant differences in the absence or presence of LMB denoted by the p values. (E) Interaction between the C-terminal domain of Dp71d and CRM1 as determined by immunoprecipitation. Lysates from C2C12 cells transiently transfected to express TetraGFP, TetraGFP-Dp71d, TetraGFP-Amino or TetraGFP-ZZCarboxyl were immunoprecipitated using GFP-Trap and immunoprecipitated proteins subjected to Western analysis with antibodies against CRM1 and GFP. Inputs correspond to 5% of nuclear extract prior to immunoprecipitation; Un, unbound proteins; B, bound proteins. (F) C2C12 cells transiently transfected to express GFP fused to full-length Dp71d (GFP-Dp71d-NES WT; NES sequence within amino acids 505–515 is shown) or its mutant variant (GFP-Dp71d-NES mut; mutated residues within the NES are denoted in red) (green) were cultured on glass coverslips, fixed 24 h post-transfection, stained with DAPI (nuclei, blue) and subjected to CLSM analysis; typical single optical Z-sections are shown (scale bar, 10 μm). Fn/c values were determined as above, with significant differences between cells expressing WT or Mut NES denoted by the p values.

Article Snippet: GFP fusion proteins were immunoprecipitated using the GFPTrap® bead system (Chromotek, Germany) in accordance with the manufacturer's instructions.

Techniques: Incubation, Control, Transfection, Construct, Immunoprecipitation, Western Blot, Sequencing, Mutagenesis, Variant Assay, Cell Culture, Staining, Expressing

Journal: Molecular Cell

Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

doi: 10.1016/j.molcel.2017.03.005

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Phospho-Chk1 (Ser296) , Cell Signaling , Cat# 2349.

Techniques: Transduction, Recombinant, Protease Inhibitor, SYBR Green Assay, Mutagenesis, Purification, Gel Extraction, Imaging, Sequencing, Negative Control, Real-time Polymerase Chain Reaction, Software, Expressing