BioProject

To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. More...

To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines 18.5 and 19.5, (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Overall design: To generate cell lines in which PP6-C was reduced in expression (PP6-C knock-down; PP6KD), HepG2 cells were seeded into 96-well plates and transduced with lentiviral particles (multiplicity of infection of 5) corresponding to different short hairpin RNA (shRNA) constructs targeted to the PP6-C gene (Mission shRNA; Sigma-Aldrich, St. Louis, MO). Procedures followed the manufacturer’s protocol. Cell lines were selected in 2 µg/ml puromycin and surviving colonies of cells were expanded as mass cultures in puromycin. Five individual shRNA lentiviral particle constructs were tested. Cell lines 18.5 and 19.5 (TRCN0000297274 and TRCN0000379835 Mission lentiviral designation, respectively) were chosen for further experiments. Mission TRC2 non-target shRNA control transduction particles were used for control transduction. Less...