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Additional file 3. Supplementary Materials and Methods.

Additional file 2: Supplementary Figure S1. Effects of C28 on microtubule organization in BC and non-transformed breast cell lines and schematic model depicting the proposed mechanism of action of C28 inhibitor. (a) MCF7, T47D, MDA-MB-231 and MCF12A cells were treated with 10 μM of C28 for 48 h. Cells were fixed and stained with anti-α-tubulin antibody (green) while the nuclear DNA was stained by DAPI (blue). Representative confocal microscopy images of mitotic phase cells are shown. Scale bar, 5 μm. (b) C28 binds to LMTK3 promoting its proteasome-mediated degradation. Downregulation of LMTK3 leads to a decrease in NUSAP1 and downstream proteins CDK1 and phospho-β III tubulin (S172), resulting in cell cycle arrest, abnormal spindles and microtubules instability.

Polynucleobacter asymbioticus strain QLW-P1DMWA-1T represents a group of highly successful heterotrophic ultramicrobacteria that is frequently very abundant (up to 70% of total bacterioplankton) in freshwater habitats across all seven continents. This strain was originally isolated from a shallow Alpine pond characterized by rapid changes in water temperature and elevated UV radiation due to its location at an altitude of 1300 m. To elucidate the strain’s adjustment to fluctuating environmental conditions, we recorded changes occurring in its transcriptomic and proteomic profiles under contrasting experimental conditions by simulating thermal conditions in winter and summer as well as high UV irradiation. To analyze the potential connection between gene expression and regulation via methyl group modification of the genome, we also analyzed its methylome. The methylation pattern differed between the three treatments, pointing to its potential role in differential gene expression. An adaptive process due to evolutionary pressure in the genus was deduced by calculating the ratios of non-synonymous to synonymous substitution rates for 20 Polynucleobacter spp. genomes obtained from geographically diverse isolates. The results indicate purifying selection., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 1011

Figure S1. MALDI imaging intensity box plots of identified peptides for UBE2N (m/z 1043.635 & m/z 1203.593; top) and SPTBN1 (m/z 959.513 & m/z 1203.593 bottom). Intensity plots of the two identified peptides UBE2N and SPTBN1 detected differentially regulated by MALDI imaging. Figure S2. Tissue-microarray-based immunohistochemical evaluation of UBE2N by means of image scope comparing normal mucosa vs. CRC and euploid vs. aneuploid CRCs after combining data of the training and validation TMA set. Bar plots and ROC curve of immunhistochemical data of UBE2N after combining data of the training and validation TMA set. Figure S3. Exemplary immunohistochemical UBE2N stainings of the TMA validation set. Images are presented as an overview (A) as well as an image section (B). Overview of exemplary immuohistochemical images of UBE2N. The images present results from the TMA validation set. Figure S4. Tissue-microarray-based immunohistochemical evaluation of SPTBN1 by means of image scope comparing normal mucosa vs. CRC and euploid vs. aneuploid CRCs in the training and validation set. Bar plots of immunhistochemical data of SPTBN1. The images present results from the TMA validation set. (DOCX 21654 kb)

Tony Bewersdorff,1 Alexandra Gruber,2 Murat Eravci,1 Malti Dumbani,1 Daniel Klinger,2 Andrea Haase1 1German Federal Institute for Risk Assessment (BfR), Department of Chemical and Product Safety, Berlin, Germany; 2Freie Universität Berlin, Institute of Pharmacy (Pharmaceutical Chemistry), Berlin, GermanyCorrespondence: Andrea HaaseGerman Federal Institute for Risk Assessment, Department of Chemical and Product Safety, Max-Dohrn-Strasse 8–10, Berlin 10589, GermanyTel +49 30 184 122 7600Email Andrea.Haase@bfr.bund.deDaniel KlingerFreie Universität Berlin, Institute of Pharmacy (Pharmaceutical Chemistry), Königin-Luise Street 2-4, Berlin 14195, GermanyTel +49 308 386 0001Email Daniel.Klinger@fu-berlin.deBackground and purpose: Nanogels (NGs) are promising drug delivery tools but are typically limited to hydrophilic drugs. Many potential new drugs are hydrophobic. Our study systematically investigates amphiphilic NGs with varying hydrophobicity, but similar colloidal features to ensure comparability. The amphiphilic NGs used in this experiment consist of a hydrophilic polymer network with randomly distributed hydrophobic groups. For the synthesis we used a new synthetic platform approach. Their amphiphilic character allows the encapsulation of hydrophobic drugs. Importantly, the hydrophilic/hydrophobic balance determines drug loading and biological interactions. In particular, protein adsorption to NG surfaces is dependent on hydrophobicity and critically determines circulation time. Our study investigates how network hydrophobicity influences protein binding, biocompatibility and cellular uptake.Methods: Biocompatibility of the NGs was examined by WST-1 assay in monocytic-like THP-1 cells. Serum protein corona formation was investigated using dynamic light scattering and two-dimensional gel electrophoresis. Proteins were identified by liquid chromatography-tandem mass spectrometry. In addition, cellular uptake was analyzed via flow cytometry.Results: All NGs were highly biocompatible. The protein binding patterns for the two most hydrophobic NGs were very similar to each other but clearly different from the hydrophilic ones. Overall, protein binding was increased with increasing hydrophobicity, resulting in increased cellular uptake.Conclusion: Our study supports the establishment of structure–property relationships and contributes to the accurate balance between maximum loading capacity with low protein binding, optimal biological half-life and good biocompatibility. This is an important step to derive design principles of amphiphilic NGs to be applied as drug delivery vehicles.Keywords: adjustable amphiphilic nanogels, tuneable hydrophilic/hydrophobic balance, biocompatibility, cellular uptake, protein corona, THP-1 cells