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.