Ppackh1 rev

The shRNA expression cassettes were generated from DNA oligonucleotides (Sigma-Aldrich, see sequences in Table S3). shRNAs, under an H1 polymerase III promoter, comprised a 22-bp stem and 10-nt miR-23 loop. We used shScr (scrambled) or shLuc, which targets the luciferase gene, as negative controls of silencing. Pairs of oligonucleotides were annealed and ligated into the pGreenPuro (System Biosciences, Palo Alto, CA, USA) expression plasmid and verified through sequencing. For lentivirus production, the plasmids were cotransfected with the packaging plasmids pPACKH1-GAG, pPACKH1-REV, and pVSVG (System Biosciences) in HEK 293TN cells. The medium was collected at days 2 and 3, and the viral supernatants were passed through 0.45-μm filters and concentrated using PEGit Virus Precipitation Solution (System Biosciences). The lentiviral vectors were resuspended in Opti-MEM (GIBCO), and the virus titers (TU/mL) were determined through flow cytometry (Accuri C6, BD Biosciences) based on copGFP expression. The transduction of fibroblasts was performed at a MOI of 1 and/or 10 in the presence of polybrene (4 μg/mL). Total RNA and protein were harvested at 7 days posttransduction.