Isolation of Primary Mouse Hepatocytes, RNA Isolation, and Quantitative Real-Time PCR (qPCR)

The primary hepatocytes were isolated from male C57BL/6N mice, treated like explained above. Isolation was performed by a collagenase perfusion technique as described before (Gebhardt et al., 2003). The cell suspension was cleared of non-parenchymal cells by differential centrifugation steps (Matz-Soja et al., 2014). Pure hepatocyte fraction was used for further procedures.

Total RNA from hepatocytes was extracted using RNeasy^® Mini Kit (Qiagen, Hilden) and the quality was controlled by agarose gel electrophoresis. The reverse transcription was performed with the Proto Script M-MuLV First Strand cDNA Synthesis Kit (New England Biolabs). Gene expression quantification by qPCR was performed in duplicates using the Rotor-Gene SYBR^® Green PCR Kit and a Rotor-Gene Q (Qiagen). Gene specific intron-spanning primers were designed with Primer 3 software and are listed in Supplementary Table 1. The specific qPCR products were quantified using internal amplification standards; 18S was used as reference gene. Values are plotted as average of biological replicates (n = 3) ± standard deviation. The statistical evaluation was performed with the unpaired Student’s t-test (GraphPad Prism 7). The null hypothesis was rejected at ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 levels.