Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Motor neuron and pancreas homeobox 1/HLXB9 promotes sustained proliferation in bladder cancer by upregulating CCNE1/2

doi: 10.1186/s13046-018-0829-9

Figure Lengend Snippet: MNX1 directly upregulates CCNE1 and CCNE2 promoter activity in bladder cancer cells. a Real-time PCR of cell cycle–related gene mRNA expression. Gene expression levels were normalized to GAPDH . Pseudo-colors represent the intensity scale of MNX1 versus vector or MNX1 shRNA#1/2 versus Scramble, generated by log2 transformation. b Western blotting of CCNE1, CCNE2, phosphorylated Rb (p-Rb), and total Rb protein expression; α-tubulin was used as the loading control. c The scatter diagram of CCNE1 and CCNE2 from cBioportal program. d and e Left: Luciferase activity assays of T24 and 5637 cells showed transactivation and repression of the CCNE1 ( d ) and CCNE2 ( e ) promoters by MNX1 overexpression and MNX1 knockdown, respectively. Right: Schematic illustration of ChIP PCR fragments for the indicated nucleotide regions of the CCNE1 ( d ) and CCNE2 ( e ) promoters (top). The ChIP enrichment assay confirmed that MNX1 binds to the P4 and P5 promoter of CCNE1 ( d ) and the P6 and P7 promoters of CCNE2 ( e ); immunoglobulin G (IgG) was used as the negative control. The results from three independent experiments were evaluated. * p < 0.05; ** p < 0.01. shRNA, short hairpin RNA

Article Snippet: An anti-MNX1 rabbit polyclonal antibody (1:500 dilution; Sigma Aldrich), an anti-CCNE1 Rabbit polyclonal antibody (1:1000 dilution;Proteintech), an anti-CCNE2 Rabbit polyclonal antibody (1:1000 dilution;Proteintech), an anti-α-tubulin mouse monoclonal antibody (1:4000 dilution; Sigma-Aldrich), an anti-Rb rabbit polyclonal antibody (1:1000 dilution; Cell Signaling Technology), an anti-p-Rb Rabbit polyclonal antibody (1:1000 dilution; Cell Signaling Technology), were used in this study.

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Plasmid Preparation, shRNA, Generated, Transformation Assay, Western Blot, Control, Luciferase, Over Expression, Knockdown, Negative Control

Journal: The Journal of Cell Biology

Article Title: Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry

doi: 10.1083/jcb.200201025

Figure Lengend Snippet: Role of Np95 in the reentry in the cell cycle of TD myotubes. (A) TD myotubes were infected with dl520 (MOI, 400 pfu/cell), dl312 (MOI, 400 pfu/cell), or adenoviral vectors encoding E2F-1 (MOI, 400 pfu/cell), cycE (MOI, 100 pfu/cell)/cdk2 (MOI, 400 pfu/cell), or stimulated with 5% FCS, as indicated on top. 48 h after infection, total cellular lysates were extracted and immunoblotted (40 μg) with the indicated antibodies. Lanes myoblasts and myotubes are as in C. (B) TD myotubes were infected with adenoviral vectors, as indicated on the right. MOIs were as follows: cycE, 100 pfu/cell; cdk2, 400 pfu/cell; and Np95, 60 pfu/cell. 24 h later, cells were treated with BrdU. 48 h after infection, cells were fixed and stained with anti-BrdU (red), and either anti–myosin heavy chain (green, top two rows, MHC), or anti-cycE (green, middle row) antibodies, or detected by epifluorescence (green, bottom two rows, to detect GFP-Np95). Nuclear counterstaining was performed with DAPI (blue). For each condition (horizontally paired panels), two pictures of the same microscopic field are shown. All left panels display the blue (DAPI) and green (either MHC, or cycE, or GFP) channels. All right panels display the green (either MHC, or cycE, or GFP) and red (BrdU) channels. Stainings are also indicated by a color code (squares).

Article Snippet: Antibodies used were as follows: rabbit antiserum to cycE (Santa Cruz Biotechnology, Inc.), mAb clone G3–245 to pRb (BD PharMingen; pRb in all figures), rabbit antiserum to pRb phosphorylated on serine 807/811 (Cell Signaling; ph-pRb in all figures), mAb against E2F-1 (a gift of K. Helin, European Institute of Oncology, Milan Italy), goat antiserum against lamin B (Santa Cruz Biotechnology, Inc.), rat Th-10a mAb to Np95 , mouse mAb to BrdU (Becton Dickinson), and rabbit antiserum to muscle-specific myosin heavy chain (a gift of G. Cossu, Stem Cell Research Institute, Rome, Italy).

Techniques: Infection, Staining