The role of the novel transcription factor ZBED6 for the adhesion/clustering of insulin-producing mouse MIN6 and TC6 cells was investigated. contacts are known to be important for beta-cell embryogenesis, differentiation, proliferation and survival6. In our previous study we observed that culture, indicating that ZBED6 affects beta-cell PF-05241328 adhesion and cell-to-cell contacts. We have also observed that direct cell-to-cell contacts between beta-cells and neural crest stem cells (NCSCs) promote beta-cell survival7 and co-transplantation of islets with NCSCs increases beta-cell proliferation8. Therefore, the aim of the present study was to further investigate the role of in insulin-producing cell adhesion/contact events, using mouse MIN6 and TC6 cells, and to evaluate the effects of knockdown on the ability of beta-cells to interact with mouse NCSCs. Results Stable in TC6 and MIN6 cells by using lentiviral vectors that express shRNA sequences (sh1 and sh2) were used. Furthermore, we recently observed that the effects of sh1- and sh2-mediated knockdown could be reversed by reconstitution of expression, which strongly indicates that sh1/sh2-induced phenotype occurs via specific knockdown1. A mock lentiviral vector made up of a scrambled shRNA sequence was used to generate a negative control cell collection (shMock). silencing was confirmed by Western blotting as efficient suppression of ZBED6 protein expression was observed in both cell lines (Fig. 1A+B). Open in a separate windows Physique 1 Stable knockdown-induced morphological changes in TC6 and MIN6 cells.TC6 and MIN6 cells were transduced with either (sh1 and sh2) or mock shRNA lentiviral vectors. ZBED6 protein expression in TC6 (A) and MIN6 (B) cells was examined by immunoblot; amidoblack staining for total protein was used as loading control. (C) Morphology of TC6 and MIN6 cells after 3 days of culture; equivalent numbers of TC6 or MIN6 cells were seeded to NUNC plastic culture plates without any covering. Arrowheads point to the three-dimensional cell clusters observed in sh1 and sh2 cells, but not in shMock cells. Pictures were taken with a 20X objective. knockdown in TC6 cells.(A) Equivalent numbers of shMock, sh1 and sh2 TC6 cells were seeded onto mouse laminin (10?g/ml) coated 24-well plates and incubated for 24?hours. The expression of total FAK was determined by immunoblot and normalized to amidoblack staining of total protein. Results are means??S.E.M for 6 indie experiments. (B) The phosphorylation of FAK was examined by immunoblot using the phospho-FAK (Y397) antibody. Ratios of pY397/total FAK were quantified and results are means??S.E.M for 6 indie experiments. (C) Equal numbers of shMock, sh1 and sh2 TC6 cells were seeded onto mouse laminin (10?g/ml) coated cover slips and incubated for 24 hours. Cells were stained with a phospho-FAK (Y397) antibody. Images were generated from confocal Z-stack scanning using Imaris Easy 3D model. Left panel: upper XY layer of cells not in ART1 direct contact with cover slip. Note the low number of FAK-activation sites in shMock cells, as compared to sh1 or sh2 cells. Right panel: Bottom XY layer of cells close to the cover slip. Note that shMock cells have strong FAK phosphorylation sites whereas sh1 or sh2 cells have weaker and fewer. Results are representative for 3 impartial experiments. Scale bar: 20?m. (D) Area of all phospho-FAK sites on the bottom XY layer was quantified by Image J. The results were normalized to the total cell number in each specific image. Results were summarized from 3 impartial experiments. *denotes P? ?0.05, #denotes P? ?0.01 using Students t-test. knockdown on beta-cell junctions. Using a pan-cadherin antibody cell-to-cell junctions were visualized three-dimensionally, but no difference in total cadherins between shMock and sh1 or sh2 cells on a plastic support could be observed PF-05241328 (Fig. 4). Insulin generating cells are known to express both E-cadherin and N-cadherin11. We therefore stained TC6 cells with an E-cadherin specific antibody. Using this antibody beta-cell junctions were less intensely stained in sh1 or sh2 cells as compared to shMock cells (Fig. 4). Also when produced on a laminin-coated support sh1 or sh2 cells exhibited weaker E-cadherin junctions (Fig. 4). Open in a separate window Physique 4 Staining of shMock, sh1 and sh2 TC6 cells with PF-05241328 ZBED6, pan-cadherin and E-cadherin antibodies.Equal numbers of cells were seeded onto cover slips with or without 10?g/ml mouse laminin (LA) covering. After 3 days culture, cells were fixed and stained. Images were PF-05241328 generated from confocal.