The absorbance was measured by an automatic microplate reader (GENios Tecan reader, Tecan, Männedorf, Switzerland) at 570 nm. The results were expressed as percent living cells compared to untreated control cells. TNF-α ELISA. In the supernatant of Huh7 cells, the levels of TNF-α were measured according to the manufacturer‘s instructions (Bioscience, San Diego, USA). NFκB activation assay: The activation of NFκB was Ribociclib cell line investigated using the TransAM-NFκB p65 assay according to the manufacturer‘s instructions (Active Motif. LaHulpe, Belgium) The employed SiO2-NPs previously analyzed by [12] were characterized

by heterogeneous size distribution of the SiO2-NPs with a mean size of 273 nm, a BET of 115 m2/g and a Zeta potential of -12.7 mV. For confirmation,

SiO2-NPs were measured again. The heterogeneous size distribution with particles with a size smaller than 100 nm and particles bigger than 500 nm were determined. The majority of particles showed a size between 100 and 300 nm with an average of 225 + - 32 nm (Fig. S1). In our previous study, we demonstrated the up-take of the SiO2-NPs into Huh7 cells by transmission electron microscopy [12]. Based on our previous data demonstrating an induction of ER stress in Huh7 cells after exposure to SiO2-NP, here we made a more detailed analysis of ER stress and induction of the UPR. We investigated three well known Selleck NVP-BKM120 ER stress markers associated with three distinct branches of the UPR, namely ATF-4, BiP and XBP-1s. Huh7 cells were

exposed to 0.005, 0.05 and 0.5 mg/ml SiO2-NPs for 24 h followed by quantification of ATF-4, BiP and XBP-1s mRNA. SiO2-NPs lead to a strong induction of BiP and XBP-1s at all concentrations and a moderate these but significant induction of ATF-4 at 0.05 and 0.5 mg/ml ( Fig. 1A). In addition to the transcript BiP protein was induced at 0.05 mg/ml SiO2-NPs ( Fig. 1B). These data clearly demonstrate that exposure to SiO2-NP lead to ER stress and associated induction of UPR. In addition we analyzed the expression of Noxa, a gene up-regulated in response to ER stress. We found a strong up-regulation of Noxa after exposure to 0.05 and 0.5 mg/ml SiO2-NPs ( Fig. 1 C). One consequence of ER stress is the induction of TNF-α. Therefore we analyzed the expression of TNF-α on the mRNA and protein level in Huh7 cells after 24 h exposure to SiO2-NPs. Figure 2A shows a significant and dose-dependent induction of TNF-α mRNA. In addition, we analyzed the TNF-α protein level in the supernatant of Huh7 cells. An induction of TNF-α protein occurred after a 24 h exposure to SiO2-NPs at 0.005 mg/ml, which was significant at 0.05 mg/ml ( Fig. 2B). Another known consequence of ER stress is the induction of PP2Ac. A significant induction of PP2Ac mRNA was detected after exposure of Huh7 cells to 0.05 and 0.5 mg/ml SiO2-NPs ( Fig. 2 C). PP2Ac was also induced at the protein level ( Fig. 2D). ER stress and TNF-α can both lead to an activation of NFκB.

The same approach as used for FD contributed

decisively to identify and name Gnathodiaphyseal Dysplasia as a separate disease, distinct from both FD and Osteogenesis Imperfecta, and to predict from the cell-autonomous properties of stromal progenitors [32], its genetic nature, which was to be identified shortly thereafter [33]. Specific dysfunction in skeletal and dental progenitors was recognized in Cleidocranial Selleckchem Torin 1 Dysplasia [34], while heterotopic transplants of stromal progenitors from patients with Hurler’s disease, conversely, dispel an inherent disruption of stromal cell differentiation [35]. However, the use of novel types of heterotopic transplantation assays [6] reveals specific changes in cartilage metabolism in Hurler’s disease (Serafini et al., manuscript in preparation). Heterotopic transplantation of stromal progenitor cells serves also to demonstrate in vivo the functional impact of gene knockout or of transgenes [36] and [37]. The adoption of stem cells as a model of disease has been remarkably productive in the specific area in which it was Selleck MK2206 most intensively pursued,

Fibrous Dysplasia. Use of cultures of FD-derived bone marrow stromal cells resulted in the development of simple diagnostic tests for the identification of the causative GNAS mutations [28] and [38], and for the quantification of the mutational load in a somatic mosaic disease [39]. Correlation of quantitative estimates of mutational load with patient age and clinical and pathological assessment of organ lesions led to the recognition that GNAS-mutated and wild-type stromal progenitors have different

lifespans and self-renewal kinetics, explaining the natural occurrence of a spontaneous sterilization over time of the bone marrow progenitor compartment from the disease gene in some patients [40]. Using clonal populations of GNAS-mutated stromal progenitors, it was also selleck chemicals possible to determine the imprinting profile of GNAS transcripts in skeletal progenitors. This revealed that while alternative transcripts of GNAS are expressed in osteoprogenitors and imprinted, Gsα is asymmetrically expressed in different clones in a random fashion, independent on imprinting, but potentially contributing to disease heterogeneity [41]. Finally, recognition of FGF23 as a product of the osteogenic lineage, and consequently of the role of bone as an endocrine organ regulating phosphate metabolism in the kidney, came from the use of stromal osteoprogenitors as an in vitro and in vivo model of FD. Overproduction of FGF23 in FD can account for the occurrence of hypophosphatemic rickets/osteomalacia in patients with severe panostotic forms of the disease [42].