Cell number, also monitored over 14 days of treatment, was not affected by any of the compounds (data not shown). On day 1, none VX-809 of the selected compounds did induce significant changes in any of the investigated endpoints and thus were not included in the following illustration of results.

CsA was daily administered at concentrations of 0.3, 1 and 3 μM for 14 days. On day 1, 3, 7, 10 and 14, samples were investigated for the selected endpoints. Morphological investigations revealed that the 3 μM CsA exposure resulted into accumulation of vacuoles within the cytoplasm associated with minimal loss of hepatic morphology on day 3 (Fig. 4D). An increased number of vacuoles and disruption of canalicular network was observed after 14 days (Fig. 4P). The presence of vacuoles was visible already at the lower concentrations from day 7 (Fig. 4G, J, N, O). Further biochemical investigations are shown in Fig. 5A. The intracellular ATP levels were not affected within the Alpelisib manufacturer first days of culture, but decreased only after 14 days of treatment with CsA at the concentrations of 1 μM (*p < 0.05) and 3 μM (**p < 0.01) Accordingly, LDH levels increased

at day 14 at both 1 μM (*p < 0.05) and 3 μM CsA concentrations (**p < 0.01). In contrast, CsA affected the Mrp2-mediated canalicular transport already after 3 days of exposure at 3 μM (****p < 0.0001). The inhibition occurred in a time- and concentration-fashion and resulted in a 54% spot average intensity decrease at 0.3 μM (****p < 0.0001) and 76% decrease at 1 μM CsA on day 14. Images click here confirmed that partial inhibition of Mrp2-mediated canalicular transport occurred already after 3 days at 3 μM dose ( Suppl. Fig. 3D). This resulted into a reduced quantification of fluorescent signal, as displayed by the cyan spots overlaying with the DCF ( Suppl. Fig. 3H). As a consequence

of the reduced export of DCF, a clear retention of the dye within the cytoplasm was observed; such effect was exacerbated after 14 days of treatment ( Suppl. Fig. 3N–P). The effect of CsA on lipid metabolism was evaluated by reagents staining respectively neutral lipids and phospholipids. As shown in Fig. 5A, the accumulation of neutral lipids was detected already after 3 days of treatment (3 μM, **p < 0.01). The size of intracellular vacuoles increased over the time of treatment ( Suppl. Fig. 4). On contrast, CsA exposure did not affect the amount of phospholipids. Exposure to AMD did not affect intracellular ATP levels, but affected LDH levels at late stages of treatment (day 10 and 14) at the highest drug concentration only (5 μM, *p < 0.05) ( Fig. 5B). AMD treatment was associated with increase of phospholipids content already after 3 days at a concentration of 5 μM (*p < 0.05), resulting in significant changes after 14 days at concentrations of 2.5 μM (*p < 0.05) as well as a at 5 μM (***p < 0.001) (Images taken at day 3 and 14 ( Suppl. Fig.