Knock down of LARP6 decreases expression of type I collagen.

<p>A. Knock down of LARP6. Expression of LARP6 was analyzed by western blot in HLFs after transfection of LARP6 siRNA (lane 1) and control siRNA (lane 2). Loading control: actin (ACT). B. Cellular level of collagen polypeptides after LARP6 knock down. Collagen α1(I) (COL1A1) and α2(I) (COL1A2) polypeptides were analyzed by western blot in LARP6 knock down HLFs (lane 1) and control HLFs (lane 2). C. Secretion of collagen polypeptides into the cellular medium. Cellular medium of cells in B was analyzed by western blot. Loading control: fibronectin (FIB). D. Partitioning of collagen mRNAs in control cells. Upper panel: HLFs transfected with control siRNA were fractionated into cytosolic (SOL) or membrane (MEM) fraction and analyzed for presence of collagen mRNAs and actin (ACT) and fibronectin (FIB) mRNAs by RT-PCR. Bottom panel: distribution of the marker proteins. E. Partitioning of collagen mRNAs in LARP6 knock down cells. Experiment as in D, except LARP6 siRNA was transfected. F. Partitioning of collagen mRNAs in LARP6 knock down cells after puromycin treatment. Experiment as in E, except the cells were treated with puromycin for 3 h. G. Rescue of partitioning of collagen mRNAs by supplementing LARP6. Upper panel: expression of LARP6 in LARP6 knock down cells (lane 1), in control cells (lane 2) and in LARP6 knocked down cells transduced with LARP6 adenovirus (lane 3). Loading control: actin (ACT). Bottom panel: Distribution of collagen mRNAs in the soluble (S) and membrane (M) fractions of control cells (lanes 1 and 2), LARP6 knock down cells (lanes 3 and 4) and LARP6 knock down cells supplemented with exogenous LARP6 (lanes 5 and 6).