Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner.

BackgroundKrüppel-like factor 6 (KLF6) is an evolutionarily conserved and ubiquitously expressed protein that belongs to the mammalian Sp1/KLF family of transcriptional regulators. Though KLF6 is a transcription factor and harbors a nuclear localization signal it is not systematically located in the nucleus but it was detected in the cytoplasm of several tissues and cell lines. Hence, it is still not fully settled whether the tumor suppressor function of KLF6 is directly associated with its ability to regulate target genes.Methodology/Principal FindingsIn this study we analyzed KLF6 expression and sub-cellular distribution by immunohistochemistry in several normal and tumor tissues in a microarray format representing fifteen human organs. Results indicate that while both nuclear and cytoplasmic distribution of KLF6 is detected in normal breast tissues, breast carcinomas express KLF6 mainly detected in the cytoplasm. Expression of KLF6 was further analyzed in breast cancer tissues overexpressing ERBB2 oncoprotein, which is associated with poor disease prognosis and patient's survival. The analysis of 48 ductal carcinomas revealed a significant population expressing KLF6 predominantly in the nuclear compartment (X2 p = 0.005; Fisher p = 0.003). Moreover, this expression pattern correlates directly with early stage and small ductal breast tumors and linked to metastatic events in lymph nodes.Conclusions/SignificanceData are consistent with a preferential localization of KLF6 in the nuclear compartment of early stage and small HER2-ERBB2 overexpressing ductal breast tumor cells, also presenting lymph node metastatic events. Thus, KLF6 tumor suppressor could represent a new molecular marker candidate for tumor prognosis and/or a potential target for therapy strategies.

No abstract

c-Fos is a proto-oncogene involved in diverse cellular functions. Its deregulation has been associated to abnormal development and oncogenic progression. c-fos−/− mice are viable but present a reduction in their body weight and brain size. We examined the importance of c-Fos during neocortex development at 13.5, 14.5 and 16.5 days of gestation. At E14.5, neocortex thickness, apoptosis, mitosis and expression of markers along the different stages of Neural Stem Progenitor Cells (NSPCs) differentiation in c-fos−/− and wild-type mice were analyzed. A ∼15% reduction in the neocortex thickness of c-fos−/− embryos was observed which correlates with a decrease in the number of differentiated cells and an increase in apoptosis at the ventricular zone. No difference in mitosis rate was observed, although the mitotic angle was predominantly vertical in c-fos−/− embryos, suggesting a reduced trend of NSPCs to differentiate. At E13.5, changes in differentiation markers start to be apparent and are still clearly observed at E16.5. A tendency of more AP-1/DNA complexes present in nuclear extracts of cerebral cortex from c-fos−/− embryos with no differences in the lipid synthesis activity was found. These results suggest that c-Fos is involved in the normal development of NSPCs by means of its AP-1 activity.

The lysosomal cell death (LCD) pathway is a caspase 3-independent cell death pathway that has been suggested as a possible target for cancer therapy, making the development of sensitive and specific high-throughput (HT) assays to identify LCD inducers highly desirable. In this study, we report a two-step HT screening platform to reliably identify such molecules. First, using a robust HT primary screen based on propidium iodide uptake, we identified compounds that kill through nonapoptotic pathways. A phenotypic image-based assay using a galectin-3 (Gal-3) reporter was then used to further classify hits based on lysosomal permeabilization, a hallmark of LCD. The identification of permeabilized lysosomes in our image-based assay is not affected by changes in the lysosomal pH, thus resolving an important limitation in currently used methods. We have validated our platform in a screen by identifying 24 LCD inducers, some previously known to induce LCD. Although most LCD inducers were cationic amphiphilic drugs (CADs), we have also identified a non-CAD LCD inducer, which is of great interest in the field. Our data also gave new insights into the biology of LCD, suggesting that lysosomal accumulation and acid sphingomyelinase inhibition are not sufficient or necessary for the induction of LCD. Overall, our results demonstrate a robust HT platform to identify novel LCD inducers that will also be very useful for gaining deeper insights into the molecular mechanism of LCD induction.

No abstract

No abstract

BackgroundDuring its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite.Methodology/Principal FindingsIn this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present.ConclusionTaken together, these results strongly support the hypothesis that SmZF1 acts as a transcription factor in S. mansoni.