Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 1. Sox2 and VRK1 expression in normal epithelium and tumor cell lines. (a) Expression of Sox2 and VRK1 in stratified epithelium from a normal human tonsil. VRK1 was detected with rabbit polyclonal VC1 antibody, and Sox2 was detected with murine mAb, by immunohistochemistry. (b) Sox2 and VRK1 in tonsil epithelium were detected by confocal immunofluorescence. (c) Expression of Sox2 and VRK1 protein was detected by immunoblot analysis. In MDA-MB-231 cells Sox 2 was determined after immunoprecipitation of endogenous Sox2 (bottom) with a monoclonal antibody and detected with a polyclonal antibody. (d) Colocalization of Sox2 and VRK1 in nuclei of NT2 cells by confocal microscopy. To the right is shown the profile of the signal in the plane. Similar co-localization of Sox2 and VRK1 was detected in human MCF7 and MDA-MB-231cell lines (Supplementary Fig. S1a). F: lymphoid follicle.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Western Blot, Immunoprecipitation, Confocal Microscopy

Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 2. Sox2-VRK1 protein interaction. (a) Human endogenous Sox2 interacts with human endogenous VRK1. The VRK1-Sox2 intracellular complex was detected in MDA-MB-231(top), NT2 (center) and HEK293T (bottom) cells. Cell lysates were immunoprecipitated with a mAb anti-Sox2 mAb ( E-4) or anti-VRK1 (1B5)64. Human endogenous VRK1 was detected in immunoblots with anti-VRK1 (VC1) antibody64 and Sox2 was detected with Y-17 antibody. Control immunoprecipitations were done with a nonspecific antibody. The Sox2-VRK1 interaction was confirmed in transfection experiments with tagged HA-VRK1 and Flag-Sox2 (Supplementary Fig. S1b) and in pulldown assays using GST-VRK1 (Supplementary Fig. S1c). (b) In vitro kinase assay using VRK1 or kinase-dead VRK1 (K179E) with immunoprecipitated myc-Sox2 as substrate. (c) Lack of interaction between VRK1 and Sox4. Tagged Sox4 wt and mutants (S395X or A350P) were co-transfected with either VRK1-myc (left) or GST-VRK1 (right) for either immunoprecipitation (IP) or pull down assays (PD) respectively. (d) Lack of interaction between endogenous VRK1 and c-myc. Endogenous VRK1 was immunoprecipitated with anti-VRK1 (1B5)64.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Immunoprecipitation, Western Blot, Control, Transfection, In Vitro, Kinase Assay

Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 3. Sox2 knockdown results in loss of VRK1 and of cell proliferation. (a) Depletion of Sox2 in NT2 cells. Sox2 was knocked down and the level of Sox2 protein determined by immunofluorescence and western blot. One hundred cells were used for immunofluorescence quantification with the LAS Lite program. Sox2 was detected with a mouse monoclonal anti- Sox2 (E-4, 1:100). VRK1 was detected using a rabbit polyclonal anti-VRK1 antibody (1:200). (Student’s Test: * < 0.05; ** < 0.005; *** < 0.0005). siC: si-control. The western blot with the knockdown of Sox2 is shown to the right. (b) Effect of silencing Sox2 or VRK1 on the rate of cell proliferation in MDA-MB-231 cells. An equal number of cells (200 000) were seeded and counted every twenty-four hours (left). The results show the mean of three experiments, in which each point was determined in triplicate. (c) Expression of proteins associated to cell cycle progression. Cells were also used to determine the expression of different proteins associated with proliferation (center). Because of the low number of cells the level of Sox2 was determined by qRT-PCR (right). siC: si-control, siS: si-Sox2, siV: si-VRK1-02.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Knockdown, Immunofluorescence, Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 4. Sox2 activates endogenous VRK1 gene expression. (a) Effect of Sox2 overexpression in MDA-MB-231 cells infected with the retroviral plasmid pBabe-Sox2. Cells were studied by confocal immunofluorescence and the quantification of the fluorescence is shown in the graph (right). (b) Effect of Sox2 overexpression on endogenous VRK1 protein in cell lysates of MDA-MB-231 cells determined by immunoblot. (c) Levels of endogenous VRK1 RNA from control or infected MDA-MB-231 cells with pBabe- Sox2. Sox2 and VRK1 RNA levels were determined by qRT-PCR. (d) Effect of overexpression of Sox2 on the proximal VRK1 promoter (−1028 + 52) in a luciferase reporter construct. At the bottom is shown the expression of transfected Sox2. Reporter gene experiments were performed three times, and each individual luciferase assay was determined in triplicate.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Gene Expression, Over Expression, Infection, Retroviral, Plasmid Preparation, Immunofluorescence, Fluorescence, Western Blot, Control, Quantitative RT-PCR, Luciferase, Construct, Expressing, Transfection

Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 6. VRK1 depletion upregulates SOX2 gene expression. (a) In MDA-MB-231 cells, human endogenous Sox2 levels increase, when VRK1 levels decrease. 72 hours after siControl, si-VRK1-02 and si-VRK1-09 treatment, was performed the fixation and permeabilization of MDA-MB-231 cells. By immunofluorescence, human endogenous Sox2 was detected with a mouse monoclonal anti-Sox2 (E-4, 1:100). Human endogenous VRK1 was detected using a rabbit polyclonal anti-VRK1 antibody (1:200). DAPI (1:1000). In western blots, human endogenous VRK1 was detected with anti-VRK1 (1B5, 1:1000). Human endogenous Sox2 was detected using a goat polyclonal anti-Sox2 (Y-17, 1:500) and human β-actin was detected using a mAb anti-β-actin (1:5000). (b) VRK1 depletion caused a reduction of Sox2 mRNA detected by qRT-PCR. The experiment was determined three times in triplicate determinations. siCt: siControl; siV-02: siVRK1-02; siV-09: siVRK1-09.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Gene Expression, Immunofluorescence, Western Blot, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 7. VRK1 overexpression downregulates SOX2 gene expression. (a) MDA-MB-231 cells were transduced with pQCXIP (empty vector) or pQCXIP-VRK1 and selected with puromycin. The presence of Sox2 and VRK1 proteins in nuclei was determined by confocal microscopy (left) and the cell florescence was quantified (right). Sox2 was detected with a mouse monoclonal anti-Sox2 (E-4, 1:100). VRK1 was detected using a rabbit polyclonal anti-VRK1 antibody (1:200). VRK1 was detected, using a mouse monoclonal anti- VRK1 (1B5, 1:1000). β-actin was detected using a mouse monoclonal anti-β-actin antibody (1:5000). (b) The levels of Sox2 and VRK1 RNA were determined by qRT-PCR in control (pQCXIP) and VRK1 transduced cells (pQCXIP-VRK1) cells. The effect occurs also at mRNA level.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Over Expression, Gene Expression, Transduction, Plasmid Preparation, Confocal Microscopy, Quantitative RT-PCR, Control

Journal: Scientific reports

Article Title: Oncogenic Sox2 regulates and cooperates with VRK1 in cell cycle progression and differentiation.

doi: 10.1038/srep28532

Figure Lengend Snippet: Figure 8. Sox2 and VRK1 levels are downregulated during NT2 cell differentiation induced by retinoic acid. (a) Time course of the expression of different proteins in NT2 cells induced to differentiate with retinoic acid (RA). At each time point cells were lysed and the extracts were loaded in 10% acrylamide gel and analyzed by immunoblots. The quantification of the proteins in the immunoblots is shown in Supplementary Fig. S9. (b) The expression of Sox2 and VRK1 proteins in NT2 cells was detected by immunofluorescence before and after induction of differentiation with retinoic acid (RA). Sox2 was detected using a mouse mAb anti-Sox2 (E-4, 1:100). VRK1 was detected using a rabbit polyclonal anti-VRK1 (1:200) antibody. (c) Effect of RA-induced differentiation of NT2 cells on the gene expression levels Sox2, Oct4, VRK1 and macro H2A2. Total RNA was extracted at the indicated times and the RT-PCR was performed using specific primers indicated in the methods section.

Article Snippet: Sox2: pCMV6-myc-Flag-Sox2 (Origene; Rockville, MD) or pBabe-Sox2.

Techniques: Cell Differentiation, Expressing, Acrylamide Gel Assay, Western Blot, Immunofluorescence, Gene Expression, Reverse Transcription Polymerase Chain Reaction