Differential effects of 5-fluorouracil and oxaliplatin on autophagy in chemoresistant colorectal cancer cells.

Macroautophagy (hereafter autophagy) is one of the adaptive pathways that contribute to cancer cell chemoresistance. Despite the fact that autophagy can both promote and inhibit cell death, there is mounting evidence that in the context of anticancer treatment, it predominantly functions as a cell survival mechanism. Therefore, silencing of key autophagy genes emerges as a potent strategy to reduce chemoresistance. Though the importance of autophagy in chemoresistance is established, the changes in autophagy in the case of acquired chemoresistance are poorly understood. In this study, we aimed to determine the changes of autophagy in the cellular model of acquired chemoresistance of colorectal cancer cell lines HCT116 and SW620, induced by 5-fluorouracil (5-FU) or oxaliplatin (OxaPt) treatment, and determine the susceptible factors for autophagy inhibition. Our results demonstrate that in the context of autophagy, 5-FU and OxaPt have different effects on HCT116 and SW620 cell lines and their chemoresistant sublines. 5-FU inhibits autophagic flux, while changes in the flux after OxaPt treatment are cell type- and dose-dependent, inducing autophagy reduction or increase. The chemoresistant subline of HCT116 cells derived by OxaPt differs from the subline derived by 5-FU treatment - it responds to OxaPt by upregulating ATG7 protein level and autophagic flux, in contrast to downregulation in cells derived by 5-FU. Moreover, 5-FU and OxaPt treatments significantly modulate protein levels of core-autophagy proteins ATG7 and ATG12. The potential effects of 5-FU and OxaPt on ATG protein levels should be taken into account to reduce chemoresistance by applying small interferingRNAs, targeting ATG proteins.

Solid-phase reverse transfection for intracellular delivery of functionally active proteins.

Delivery of large and functionally active biomolecules across cell membranes presents a challenge in cell biological experimentation. For this purpose, we developed a novel solid-phase reverse transfection method that is suitable for the intracellular delivery of proteins into mammalian cells with preservation of their function. We show results for diverse application areas of the method, ranging from antibody-mediated inhibition of protein function to CRISPR/Cas9-based gene editing in living cells. Our method enables prefabrication of "ready to transfect" substrates carrying diverse proteins. This allows their easy distribution and standardization of biological assays across different laboratories.

miRNA target identification and prediction as a function of time in gene expression data.

The understanding of miRNA target interactions is still limited due to conflicting data and the fact that high-quality validation of targets is a time-consuming process. Faster methods like high-throughput screens and bioinformatics predictions are employed but suffer from several problems. One of these, namely the potential occurrence of downstream (i.e. secondary) effects in high-throughput screens has been only little discussed so far. However, such effects limit usage for both the identification of interactions and for the training of bioinformatics tools. In order to analyse this problem more closely, we performed time-dependent microarray screening experiments overexpressing human miR-517a-3p, and, together with published time-dependent datasets of human miR-17-5p, miR-135b and miR-124 overexpression, we analysed the dynamics of deregulated genes. We show that the number of deregulated targets increases over time, whereas seed sequence content and performance of several miRNA target prediction algorithms actually decrease over time. Bioinformatics recognition success of validated miR-17 targets was comparable to that of data gained only 12 h post-transfection. We therefore argue that the timing of microarray experiments is of critical importance for detecting direct targets with high confidence and for the usability of these data for the training of bioinformatics prediction tools.

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet-Microarray Platform.

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo "microenvironment" compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96- and 384-well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic-superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single-spheroid-microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24-48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high-throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.

An RNAi screen identifies KIF15 as a novel regulator of the endocytic trafficking of integrin.

α2ß1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. α2ß1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of α2ß1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on α2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of α2 integrin. Of these, 83 were found to be putative regulators of α2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of α2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate α2 integrin internalization.

Hypothalamic miR-103 protects from hyperphagic obesity in mice.

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines.

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

Live-cell assays to identify regulators of ER-to-Golgi trafficking.

We applied fluorescence microscopy-based quantitative assays to living cells to identify regulators of endoplasmic reticulum (ER)-to-Golgi trafficking and/or Golgi complex maintenance. We first validated an automated procedure to identify factors which influence Golgi-to-ER relocalization of GalT-CFP (ß1,4-galactosyltransferase I-cyan fluorescent protein) after brefeldin A (BFA) addition and/or wash-out. We then tested 14 proteins that localize to the ER and/or Golgi complex when overexpressed for a role in ER-to-Golgi trafficking. Nine of them interfered with the rate of BFA-induced redistribution of GalT-CFP from the Golgi complex to the ER, six of them interfered with GalT-CFP redistribution from the ER to a juxtanuclear region (i.e. the Golgi complex) after BFA wash-out and six of them were positive effectors in both assays. Notably, our live-cell approach captures regulator function in ER-to-Golgi trafficking, which was missed in previous fixed cell assays, as well as assigns putative roles for other less characterized proteins. Moreover, we show that our assays can be extended to RNAi and chemical screens.

MicroRNAs Influence the Migratory Ability of Human Umbilical Vein Endothelial Cells.

To identify miRNAs that are involved in cell migration in human umbilical vein endothelial cells (HUVECs), we employed RNA sequencing under high glucose incubation and text mining within the databases miRWalk and TargetScanHuman using 83 genes that regulate HUVECs migration. From both databases, 307 predicted miRNAs were retrieved. Differentially expressed miRNAs were determined by exposing HUVECs to high glucose stimulation, which significantly inhibited the migratory ability of HUVECs as compared to cells cultured in normal glucose. A total of 35 miRNAs were found as differently expressed miRNAs in miRNA sequencing, and 4 miRNAs, namely miR-21-3p, miR-107, miR-143-3p, and miR-106b-5p, were identified as overlapping hits. These were subjected to hub gene analysis and pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG), identifing 71 pathways which were influenced by all four miRNAs. The influence of all four miRNAs on HUVEC migration was phenomorphologically confirmed. miR21 and miR107 promoted migration in HUVECs while miR106b and miR143 inhibited migration. Pathway analysis also revealed eight shared pathways between the four miRNAs. Protein-protein interaction (PPI) network analysis was then performed to predict the functionality of interacting genes or proteins. This revealed six hub genes which could firstly be predicted to be related to HUVEC migration.

High-content analysis of microRNAs involved in the phenotype regulation of vascular smooth muscle cells.

In response to vascular injury vascular smooth muscle cells (VSMCs) alternate between a differentiated (contractile) and a dedifferentiated (synthetic) state or phenotype. Although parts of the signaling cascade regulating the phenotypic switch have been described, the role of miRNAs is still incompletely understood. To systematically address this issue, we have established a microscopy-based quantitative assay and identified 23 miRNAs that induced contractile phenotypes when over-expressed. These were then correlated to miRNAs identified from RNA-sequencing when comparing cells in the contractile and synthetic states. Using both approaches, six miRNAs (miR-132-3p, miR-138-5p, miR-141-3p, miR-145-5p, miR-150-5p, and miR-22-3p) were filtered as candidates that induce the phenotypic switch from synthetic to contractile. To identify potentially common regulatory mechanisms of these six miRNAs, their predicted targets were compared with five miRNAs sharing ZBTB20, ZNF704, and EIF4EBP2 as common potential targets and four miRNAs sharing 16 common potential targets. The interaction network consisting of these 19 targets and additional 18 hub targets were created to facilitate validation of miRNA-mRNA interactions by suggesting the most plausible pairs. Furthermore, the information on drug candidates was integrated into the network to predict novel combinatorial therapies that encompass the complexity of miRNAs-mediated regulation. This is the first study that combines a phenotypic screening approach with RNA sequencing and bioinformatics to systematically identify miRNA-mediated pathways and to detect potential drug candidates to positively influence the phenotypic switch of VSMCs.

Involvement of a Golgi-resident GPI-anchored protein in maintenance of the Golgi structure.

The Golgi apparatus consists of a series of flattened cisternal membranes that are aligned in parallel to form stacks. Cytosolic-oriented Golgi-associated proteins have been identified that may coordinate or maintain the Golgi architecture. Here, we describe a novel GPI-anchored protein, Golgi-resident GPI-anchored protein (GREG) that has a brefeldin A-sensitive Golgi localization. GREG resides in the Golgi lumen as a cis-oriented homodimer, due to strong interactions between coiled-coil regions in the C termini. Dimerization of GREG as well as its Golgi localization depends on a unique tandem repeat sequence within the coiled-coil region. RNA-mediated interference of GREG expression or expression of GREG mutants reveals an essential role for GREG in maintenance of the Golgi integrity. Under these conditions, secretion of the vesicular stomatitis virus glycoprotein protein as a marker for protein transport along the secretory pathway is inhibited, suggesting a loss of Golgi function as well. These results imply the involvement of a luminal protein in Golgi structure and function.

Transcriptome profiling reveals Silibinin dose-dependent response network in non-small lung cancer cells.

Silibinin (SIL), a natural flavonolignan from the milk thistle (Silybum marianum), is known to exhibit remarkable hepatoprotective, antineoplastic and EMT inhibiting effects in different cancer cells by targeting multiple molecular targets and pathways. However, the predominant majority of previous studies investigated effects of this phytocompound in a one particular cell line. Here, we carry out a systematic analysis of dose-dependent viability response to SIL in five non-small cell lung cancer (NSCLC) lines that gradually differ with respect to their intrinsic EMT stage. By correlating gene expression profiles of NSCLC cell lines with the pattern of their SIL IC50 response, a group of cell cycle, survival and stress responsive genes, including some prominent targets of STAT3 (BIRC5, FOXM1, BRCA1), was identified. The relevancy of these computationally selected genes to SIL viability response of NSCLC cells was confirmed by the transient knockdown test. In contrast to other EMT-inhibiting compounds, no correlation between the SIL IC50 and the intrinsic EMT stage of NSCLC cells was observed. Our experimental results show that SIL viability response of differently constituted NSCLC cells is linked to a subnetwork of tightly interconnected genes whose transcriptomic pattern can be used as a benchmark for assessment of individual SIL sensitivity instead of the conventional EMT signature. Insights gained in this study pave the way for optimization of customized adjuvant therapy of malignancies using Silibinin.

Epistatic Analysis of the Contribution of Rabs and Kifs to CATCHR Family Dependent Golgi Organization.

Multisubunit members of the CATCHR family: COG and NRZ complexes, mediate intra-Golgi and Golgi to ER vesicle tethering, respectively. We systematically addressed the genetic and functional interrelationships between Rabs, Kifs, and the retrograde CATCHR family proteins: COG3 and ZW10, which are necessary to maintain the organization of the Golgi complex. We scored the ability of siRNAs targeting 19 Golgi-associated Rab proteins and all 44 human Kifs, microtubule-dependent motor proteins, to suppress CATCHR-dependent Golgi fragmentation in an epistatic fluorescent microscopy-based assay. We found that co-depletion of Rab6A, Rab6A', Rab27A, Rab39A and two minus-end Kifs, namely KIFC3 and KIF25, suppressed both COG3- and ZW10-depletion-induced Golgi fragmentation. ZW10-dependent Golgi fragmentation was suppressed selectively by a separate set of Rabs: Rab11A, Rab33B and the little characterized Rab29. 10 Kifs were identified as hits in ZW10-depletion-induced Golgi fragmentation, and, in contrast to the double suppressive Kifs, these were predominantly plus-end motors. No Rabs or Kifs selectively suppressed COG3-depletion-induced Golgi fragmentation. Protein-protein interaction network analysis indicated putative direct and indirect links between suppressive Rabs and tether function. Validation of the suppressive hits by EM confirmed a restored organization of the Golgi cisternal stack. Based on these outcomes, we propose a three-way competitive model of Golgi organization in which Rabs, Kifs and tethers modulate sequentially the balance between Golgi-derived vesicle formation, consumption, and off-Golgi transport.

High-Density Cell Arrays for Genome-Scale Phenotypic Screening.

Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.

Measuring secretory membrane traffic: a quantitative fluorescence microscopy approach.

In this chapter the authors describe automated imaging methods to quantify the transport rates of transmembrane as well as soluble cargo, and to evaluate the integrity of the Golgi complex. The quantification of cargo transport rates serves as an example of fluorescence intensity-based assays, the quantification of the Golgi complex integrity--as an example of morphology-based assays. These quantitative assays could be applied for single experiments as well as for middle- and high-throughput screening approaches. Each of these assays can be used to appreciate effects caused by gene silencing by RNAi, cDNA overexpression or application of chemical compounds. For each assay the authors discuss protocols for sample preparation, parameters for automated image acquisition, strategies of image analysis, and data quantification.

Comparison of Cell Arrays and Multi-Well Plates in Microscopy-Based Screening.

Multi-well plates and cell arrays enable microscopy-based screening assays in which many samples can be analysed in parallel. Each of the formats possesses its own strengths and weaknesses, but reference comparisons between these platforms and their application rationale is lacking. We aim to fill this gap by comparing two RNA interference (RNAi)-mediated fluorescence microscopy-based assays, namely epidermal growth factor (EGF) internalization and cell cycle progression, on both platforms. Quantitative analysis revealed that both platforms enabled the generation of data with the appearance of the expected phenotypes significantly distinct from the negative controls. The measurements of cell cycle progression were less variable in multi-well plates. The result can largely be attributed to higher cell numbers resulting in less data variability when dealing with the assay generating phenotypic cell subpopulations. The EGF internalization assay with a uniform phenotype over nearly the whole cell population performed better on cell arrays than in multi-well plates. The result was achieved by scoring five times less cells on cell arrays than in multi-well plates, indicating the efficiency of the cell array format. Our data indicate that the choice of the screening platform primarily depends on the type of the cellular assay to achieve a maximum data quality and screen efficiency.

Transfected cell microarrays: an efficient tool for high-throughput functional analysis.

Transfected cell microarrays are considered to be a breakthrough methodology for high-throughput and high-content functional genomics. Here, recent advances in the cell microarray field are reviewed, along with its potential to increase the speed of determining gene function. These advances, combined with an increasing number and diversity of gene perturbing systems, such as RNAi and ectopic gene expression, provide tools for expanding our understanding of biology at the systems level.

The genome sequence of the extreme thermophile Thermus thermophilus.

Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.

A novel type of uracil-DNA glycosylase mediating repair of hydrolytic DNA damage in the extremely thermophilic eubacterium Thermus thermophilus.

Spontaneous hydrolytic deamination of DNA cytosine and 5-methyl-cytosine residues is an abundant source of C/G (5-meC/G) to T/A transition mutations. As a result of this pressure, at least six different families of enzymes have evolved that initiate repair at U/G (T/G) mispairs, the relevant pre-mutagenic intermediates. The necessarily higher rate of the process at elevated temperatures must pose a correspondingly accentuated problem to contemporary thermophilic organisms and may have been a serious bottleneck in early evolution when life passed through a phase of very high ambient temperatures. Here we show that Thermus thermophilus, an aerobic, Gram-negative eubacterium thriving at up to 85 degrees C, harbors two uracil-DNA glycosylases (UDGs), termed TTUDGA and TTUDGB. According to both amino acid sequence and enzymatic properties, TTUDGA clearly belongs to the family of 'thermostable UDGs'. TTUDGB shares with TTUDGA 23% sequence identity, but differs from it in profound functional aspects. TTUDGB, unlike TTUDGA, does not act upon uracil residues in the context of single-stranded DNA whereas both enzymes process various double-stranded substrates, albeit with different preferences. TTUDGB shows a number of sequence features characteristic of the UDG superfamily, but surprisingly lacks any polar residue within its so-called motif 1 (GLAPG-X(10)-F). This finding is in conflict with a previously assumed crucial catalytic role of motif 1 in water activation and supports a more recently suggested alternative of a dissociative ('S(N)1-type') reaction mechanism. Together, the characteristics of TTUDGB and its homologs in other organisms define a novel family of UDG repair enzymes.

High-Content Analysis of the Golgi Complex by Correlative Screening Microscopy.

The Golgi complex plays a central role in a number of diverse cellular processes, and numerous regulators that control these functions and/or morphology of the Golgi complex are known by now. Many of them were identified by large-scale experiments, such as RNAi-based screening. However, high-throughput experiments frequently provide only initial information that a particular protein might play a role in regulating structure and function of the Golgi complex. Multiple follow-up experiments are necessary to functionally characterize the selected hits. In order to speed up the discovery, we have established a system for correlative screening microscopy that combines rapid data collection and high-resolution imaging in one experiment. We describe here a combination of wide-field microscopy and dual-color direct stochastical optical reconstruction microscopy (dSTORM). We apply the technique to simultaneously capture and differentiate alterations of the cis- and trans-Golgi network when depleting several proteins in a singular and combinatorial manner.