Sp3 is a ubiquitous transcription factor closely related to Sp1. Here we show that Sp3 is a target for SUMO modi®cation in vivo and in vitro. SUMO modi®cation of Sp3 occurs at a single lysine located between the second glutamine-rich activation domain and the DNA-binding domain. Mutational analyses identi®ed the sequence IKXE as essential for SUMO conjugation to Sp3. We identi®ed the protein inhibitor of activated STAT1 (PIAS1) as an interaction partner of Sp3 and Ubc9. Moreover, PIAS1 strongly stimulated SUMO conjugation to Sp3, thus acting as an E3 ligase for SUMO conjugation to Sp3. All mutations that prevented SUMO modi®cation in vitro strongly enhanced the transcriptional activity of Sp3, showing that SUMO modi®cation silences Sp3 activity. SUMOmodi®ed Sp3 bound to DNA with similar speci®city and af®nity as unmodi®ed Sp3. However, DNA-bound Sp3 did not act as a substrate for SUMO modi®cation.

Sp3 is a ubiquitous transcription factor closely related to Sp1. Both proteins contain a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich domains in the N-terminal moiety. Immunoblot analyses of Sp3 reveal a striking complex protein pattern of up to eight distinct species. This pattern is not observed in Sp3-deficient cell lines showing that all signals reflect Sp3 antigen. In this study, we have unraveled the complexity of Sp3 expression. We show that four isoforms of Sp3 that retain different parts of the N terminus are expressed in vivo. The four isoforms derive from alternative translational start sites at positions 1, 37, 856, and 907. An upstream open reading frame located at position ؊47 to ؊18 regulates expression of the two long isoforms. Unlike Sp1, none of the Sp3 isoforms is glycosylated. However, all four isoforms become SUMO-modified in vivo and in vitro specifically and exclusively at lysine residue 551. The transcriptional activity of the two long isoforms strongly depends on the promoter settings, whereas the small isoforms appear to be inactive. The transcriptional activity of all the Sp3 isoforms is regulated by SUMO modification. Our results demonstrate that Sp3 has many unique features and is not simply a functional equivalent of Sp1.The transcription factor Sp3 is a ubiquitously expressed member of the Sp family of transcription factor that is involved in the expression and regulation of many genes, including housekeeping genes, tissue-specifically expressed genes, viral genes, and cell cycle-regulated genes (1, 2). Sp3 contains a highly conserved DNA-binding domain close to the C terminus and two glutamine-rich activation domains in the N-terminal moiety. The expression pattern, the structure, and the DNAbinding properties of Sp3 are very similar to Sp1, which suggested originally that these two proteins exert similar functions. The physiological roles of Sp1 and Sp3, however, appear to be significantly different. Sp1 knock-out mouse embryos are severely retarded in growth, and die after day 10 of embryonic development (3). Sp3-deficient embryos develop until birth, but die invariably of respiratory failure immediately after birth (4). In addition, late tooth and bone developmental processes are impaired in Sp3Ϫ/Ϫ mice (4, 5).Functional analyses of the transcriptional properties of Sp1 and Sp3 also revealed significant differences between these two transcription factors (6). On many reporter constructs containing multiple Sp-binding sites Sp3 is, unlike Sp1, inactive or acts only as a weak activator (7). The molecular basis for the inactivity of Sp3 under these conditions has been mapped to an inhibitory domain located between the second glutamine-rich activation domain and the zinc finger region (8). More recently, it was shown that Sp3 is post-translationally modified by the small ubiquitin-like modifier (SUMO) 1 within its inhibitory domain and that SUMO modification leads to inactivation (9, 10).All previously published studies with Sp3 (more than 500 ...

The use of stem cells to support tissue repair is facilitated by loading of the therapeutic cells with magnetic nanoparticles (MNPs) enabling magnetic tracking and targeting. Current methods for magnetizing cells use artificial MNPs and have disadvantages of variable uptake, cellular cytotoxicity and loss of nanoparticles on cell division. Here we demonstrate a transgenic approach to magnetize human mesenchymal stem cells (MSCs). MSCs are genetically modified by transfection with the mms6 gene derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesises single-magnetic domain crystals which are incorporated into magnetosomes. Following transfection of MSCs with the mms6 gene there is bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by MR and which have no deleterious effects on cell proliferation, migration or differentiation. The assimilation of magnetic nanoparticle synthesis into mammalian cells creates a real and compelling, cytocompatible, alternative to exogenous administration of MNPs.

A multimodal nonlinear optical microscope that combines coherent anti-Stokes Raman scattering (CARS), two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and sum-frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF-7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO 2 ) − allowed identification of tumors. Label-free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer.

BackgroundThe constant increase of the use of nanomaterials in consumer products is making increasingly urgent that standardized and reliable in vitro test methods for toxicity screening be made available to the scientific community. For this purpose, the determination of the cellular dose, i.e. the amount of nanomaterials effectively in contact with the cells is fundamental for a trustworthy determination of nanomaterial dose responses. This has often been overlooked in the literature making it difficult to undertake a comparison of datasets from different studies. Characterization of the mechanisms involved in nanomaterial transport and the determination of the cellular dose is essential for the development of predictive numerical models and reliable in vitro screening methods.ResultsThis work aims to relate key physico-chemical properties of gold nanoparticles (NPs) to the kinetics of their deposition on the cellular monolayer. Firstly, an extensive characterization of NPs in complete culture cell medium was performed to determine the diameter and the apparent mass density of the formed NP-serum protein complexes. Subsequently, the kinetics of deposition were studied by UV-vis absorbance measurements in the presence or absence of cells. The fraction of NPs deposited on the cellular layer was found to be highly dependent on NP size and apparent density because these two parameters influence the NP transport. The NP deposition occurred in two phases: phase 1, which consists of cellular uptake driven by the NP-cell affinity, and phase 2 consisting mainly of NP deposition onto the cellular membrane.ConclusionThe fraction of deposited NPs is very different from the initial concentration applied in the in vitro assay, and is highly dependent of the size and density of the NPs, on the associated transport rate and on the exposure duration. This study shows that an accurate characterization is needed and suitable experimental conditions such as initial concentration of NPs and liquid height in the wells has to be considered since they strongly influence the cellular dose and the nature of interactions of NPs with the cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0157-1) contains supplementary material, which is available to authorized users.

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We report on the construction of a highly flexible system for advanced biological imaging, where all the following imaging techniques are integrated into the same microscope: Coherent anti-Stokes Raman scattering (CARS), two photon excitation fluorescence (TPEF), second harmonic generation (SGH), sum frequency generation (SFG), fluorescence lifetime imaging (FLIM) and differential interference contrast (DIC). The system employs a Nd:YVO 4 laser as pump (7 ps, 1064 nm), and two tunable OPOs (6 ps, 700 -1000 nm). Our microscope comprises a heater stage and perfusion cell for imaging of live cells, and features an atomic force microscope (AFM) which enables optical imaging at 10 nm resolution. Multimodal imaging of breast cancer cells and tissue will be demonstrated as well as imaging of anticancer drugs in living cells.

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