Dounce tissue grinder set

Muscle tissue was washed in 1× PBS, cleaned of any visible fat depositions and minced to obtain fragments of approximately 1 mm³. Per sample, approximately 0.3 g of minced tissues was homogenized in 3 ml of buffer A (250 mM sucrose, 10 mg ml⁻¹ BSA, 5 mM MgCl₂, 0.12 U μl⁻¹ RNaseIn, 0.06 U μl⁻¹ SUPERasIn, 1× protease inhibitor) using Dounce tissue grinder set (Merck) with 50 strokes of the loose pestle (A). The homogenate was filtered through a 100-μm cell strainer (Corning) and the strainer was washed with 1 ml and then 750 μl of buffer A. After the addition of Triton X-100 (final concentration 0.5%), the mixture was further homogenized with 50 strokes of the tight pestle (B). After filtering through a 40-μm strainer, nuclei were centrifuged (3,000g, 5 min, 4 °C), resuspended in 1 ml of buffer B (320 mM sucrose, 10 mg ml⁻¹ BSA, 3 mM CaCl₂, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, 1 mM DTT, 1× protease inhibitor, 0.12 U μl⁻¹ RNaseIn, 0.06 U μl⁻¹ SUPERasin) and purified using a 27% Percoll gradient solution. The Percoll mixture was centrifuged at 20,000g (15 min, 4 °C) and the pellet was resuspended in 200 μl of buffer B, followed by centrifugation (20,000g, 3 min, 4 °C). After Trypan Blue staining, the intact nuclei were counted using a haemocytometer. Nuclei were profiled using a Chromium Controller (10X Genomics) according to the manufacturer’s protocol.

TAMRA-Tat, TAMRA-Tat-NR2B9c, and TAMRA-Tat-N-dimer were dissolved in 0.9% NaCl and administered as a 100 µL bolus injection (3 nmol/g body weight) into the tail vein of 12-week-old male NMRI mice (27–33 g). Following 1 h of circulation, the mice were deeply anesthetized by i.p. injection of 0.05 mL/10 g body weight Ketaminol/Dexdormitor (2.25 mg ketamine/0.03 mg meteoidin per 10 g body weight in 0.9% NaCl). The chest cage was opened and the mice transcardially perfused with 0.01 M PBS (pH 7.4) prior to isolation of the brain, heart, lungs, spleen, kidney, liver, and 2 cm of the upper part of the intestine. All tissues were immediately put on ice and kept at −80 °C until analysis.
The tissues were homogenized in 1 mL of ice-cold lysis buffer using a 7-mL Dounce tissue grinder set (Merck, St. Louis, MO, USA). Homogenates were left on ice for 10 min prior to centrifugation at 14,000× g and 4 °C for 10 min. Supernatants (100 µL) were transferred to a black clear-bottom 96-well plate for quantification of the TAMRA-peptides using a NovoStar fluorescence plate reader (BMG Labtech, Offenburg, Germany) with excitation and emission set to 542 and 568 nm, respectively. The molar peptide amounts were calculated using standard curves that were freshly prepared in lysates from relevant blank tissue at concentrations ranging from 30 nM to 2 µM.

RNA was extracted from hepatoma cells using the NucleoSpin RNA plus kit (Macherey-Nagel) according to the manufacturer’s instructions.
GLT1 infected human liver chimeric mice were sacrificed at 8 weeks post infection. After collection of the livers, 100 mg of tissue samples were preserved frozen in 1.5 ml RNAlater solution (ThermoFisher Scientific). To extract RNA, 20 mg of tissue were grinded to powder using a Dounce tissue grinder set (Merck) on dry ice. RNA was then isolated using the Bio&SELL RNA-Mini Kit (Bio&SELL) according to the manufacturer’s protocol.
RNA was extracted from the human GLT1 patient serum as well as from the serum of GLT1-mut4B, -20M and GLT1cc infected human liver chimeric mice using the NucleoSpin Virus kit (Macherey-Nagel) according to the manufacturer’s instructions.