Animal experiments were performed according to the guidelines of Hannover Medical School, Germany. BALB/c mice were purchased from Charles River Laboratories (Germany). Hepa 1-6 mouse hepatoma cells (American Tissue Culture Collection,
ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (PAA Laboratories) supplemented with 10% fetal bovine GSK2118436 cell line serum (FBS) (PAA), L-glutamine (PAA), and penicillin/streptomycin (PAA). Cells were transduced with retrovirus expressing short hairpin RNA (shRNA) against DGCR8 and DROSHA (Addgene plasmid) as described.13 After transduction, shRNA-expressing cells were selected in medium supplemented with puromycin (Invitrogen) at a concentration of 1 μg/mL. Loss of DGCR8 and DROSHA
was confirmed by western blots. Primary mouse hepatocytes were isolated by two-step collagenase (Roche) perfusion followed by Percoll (Sigma) density gradient centrifugation as described.14 Purified mouse hepatocytes were cultured in Primaria dishes (BD Labware) in the presence of hepatocyte basal medium supplemented with hepatocyte single quotes (Lonza). Targefect hepatocyte reagent Palbociclib ic50 (Targetingsystems) for plasmid transfection and Targefect F2 reagent (Targetingsystems) were used for small interfering RNA (siRNA) transfection into primary hepatocytes. For in vitro apoptosis induction, a final concentration of 0.5 μg/mL Jo2 antibody was added to the hepatocyte culture medium. In vitro apoptosis ID-8 by TNF-α was induced as described.15In vivo apoptosis was induced by intraperitoneal injection of 0.4 μg/g body weight Jo2 antibody (BD Pharmingen) in 8 to 10-week-old BALB/c mice. Mice were sacrificed at indicated timepoints. Liver tissues were harvested
and immediately snap-frozen in liquid nitrogen and fixed in 4% paraformaldehyde (Sigma). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured as described.16 Western blots were performed as described.17 DGCR8 antibody (Abcam, dilution 1:250), p53 up-regulated modulator of apoptosis (PUMA) (Abcam, 1:1000), p27 (BD Pharmingen, 1:500), phosphatase and tensin homolog (PTEN) (Cell Signaling 1:1000), FAS (Santa Cruz, 1:250), and Tubulin (Sigma, 1:1,000) were used. Liver tissues were fixed in 4% paraformaldehyde for 4 hours at 4°C, washed in phosphate-buffered saline (PBS), and embedded in OCT to prepare frozen blocks. The 7-μm sections were cut and air-dried for 20 minutes before staining. A terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Millipore) was performed according to the manufacturer’s guidelines. miRNA profiling was performed and analyzed by FeBIT (Heidelberg, Germany). Briefly, total RNA was isolated from liver tissue using the miRNeasy kit (Qiagen). Following on-column DNase treatment, total RNA quality was determined by Nanodrop (NanoDrop Technologies) and Bioanalyzer (Agilent).