Piercetm bca assay

The oxygen consumption rates (OCRs) in homogenates from frozen livers were measured with a Seahorse Extracellular Flux (XF) ‐96 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Frozen liver tissues were thawed and washed two times with ice-cold PBS. The ‘Respirometry in Frozen Samples (RIFS)’ protocol was adapted from Acin-Perez R et al. 2020.^{44 (link)} Approximately, 50 mg of tissue was cut into small pieces and homogenized in MAS buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH₂PO₄, 5 mM MgCl₂, 1 mM EGTA, 2 mM HEPES, pH 7.4) with 20 strokes in a glass Dounce homogenizer. The homogenate was centrifuged at 1,000 g for 10 min at 4°C, the supernatant was collected and protein concentration was determined by Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). In Seahorse XF96 microplate, 10 µg (proteins) of homogenate was loaded into in 20 µl of MAS. The loaded plate was centrifuged at 2,000 g for 5 min at 4°C and an additional 130 µl of MAS containing cytochrome c (10 µg/ml) was added to each well. Substrates were delivered by port A (pyruvate + malate (5 mM each)) or NADH (1 mM), or 5 mM succinate + rotenone (5 mM + 2 µM). The inhibitors, Rotenone and antimycin A (2 µM + 4 µM) were delivered by port B. TMPD + ascorbic acid (0.5 mM + 1 mM) at port C; and azide (50 mM) at port D.

Lactate release in media was performed by colorimetric determination with Cobas® C311 (Hitachi, Tokio, Japan). Therefore growth media from cell culture was centrifuged at 1000 rpm for 10 minutes to remove floating cells and debris and 1.3 ml were removed to a sodium fluoride vessel (Sarstedt, Nümbrecht, Germany). To eliminate influences of phenol red and fetal calf serum, medium was used as negative control and deducted from measurements. Further the lactate content was normalized to intracellular protein concentration to prevent influences of cell numbers and volume. Protein concentration was assessed with Pierce^TM BCA Assay (Thermofisher Scientific) as described in manufacturer´s instructions.

ELISA (MyBioSource #MBS2885065) was performed to assess the levels of HNF1α comparing slices cultured in transwells and PFC. The results were normalized to total tissue protein content using a Pierce^TM BCA assay (Thermo Scientific #23227).

Livers were homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 50 mM Tris-HCl pH 7.4) containing protease and phosphatase inhibitors (Roche, Rotkreuz, Switzerland). Protein concentration was measured with the Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, blocked for 1 h with 5% nonfat milk or BSA, then incubated overnight at 4 °C with primary antibodies (Table S2). After incubation with peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Rockford, IL, USA), signals were revealed with enhanced chemiluminescence (Amersham ECL Prime, GE Healthcare, Glattburg, Switzerland) and a Fusion CCD camera coupled to a computer equipped with Fusion Capt Fx Software (Vilber-Lourmat, Marne-la-Vallée, France). Signals were quantified with the Bio-1D Advanced software (Vilber-Lourmat).

Livers were homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 50 mM Tris-HCl pH 7.4) containing protease and phosphatase inhibitors (Roche, Rotkreuz, Switzerland). Protein concentration was measured with the Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, blocked for 1 h with 5% nonfat milk or BSA, then incubated overnight at 4 °C with primary antibodies. After incubation with peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Rockford, IL, USA), signals were revealed with enhanced chemiluminescence (Amersham ECL Prime, GE Healthcare, Glattburg, Switzerland) and a Fusion CCD camera coupled to a computer equipped with Fusion Capt Fx Software (Vilber-Lourmat, Marne-la-Vallée, France). Signals were quantified with the Bio-1D Advanced software (Vilber-Lourmat). Uncropped and unprocessed scans of the blots are supplied as Supplementary Figs. in the Supplementary Information.

Frozen liver tissues were thawed and washed twice with ice-cold PBS. Livers were homogenized with 0.5 mm zirconium oxide beads (Next Advance, Troy, NY, USA) in a bullet blender (Next Advance, Troy, NY, USA) in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 7.4) containing Halt™ protease and phosphatase inhibitor (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentration was measured with the Pierce^TM BCA assay (Thermo Fisher Scientific, Rockford, IL, USA). 15 µg of total lysate was loaded into 10–12% sodium dodecyl sulfate polyacrylamide gel and proteins were resolved by electrophoresis. The proteins were transferred to nitrocellulose membranes, blocked for 1 h with 5% nonfat milk, and then incubated overnight at 4°C with primary antibodies (Supplementary table 1S). Next, the membranes were incubated with peroxidase-conjugated secondary antibody (Thermo Fisher Scientific, Rockford, IL, USA) for 1 h and signals were revealed with enhanced chemiluminescence (Amersham ECL Prime, GE Healthcare, Glattburg, Switzerland) and a Fusion CCD camera coupled to a computer equipped with Fusion Capt Fx Software (Vilber-Lourmat, Marne-la-Vallée, France). Band densitometry was measured with the Bio-1D Advanced software (Vilber-Lourmat).