Graduate School of Frontier Biosciences, Osaka University

Japanese

Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells

Journal FEBS LETT 590, 2671-2680 (2016)
Authors Tsuchiya M (1), Ogawa H (1, 2), Koujin T (2), Kobayashi S (2), Mori C (2), Hiraoka Y (1, 2), Haraguchi T (1, 2).
  1. Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
  2. Advanced ICT Research Institute Kobe, National Institute of Information and Communications Technology, Kobe, Japan.
Title Depletion of autophagy receptor p62/SQSTM1 enhances the efficiency of gene delivery in mammalian cells
PubMed 27317902
Laboratory Nuclear Dynamics Group 〈Prof. Hiraoka〉
Abstract Novel methods that increase the efficiency of gene delivery to cells will have many useful applications. Here we report a simple approach involving depletion of p62/SQSTM1 to enhance the efficiency of gene delivery. The efficiency of reporter gene delivery was remarkably higher in p62-knockout murine embryonic fibroblast (MEF) cells compared with normal MEF cells. This higher efficiency was partially attenuated by ectopic expression of p62. Furthermore, siRNA-mediated knockdown of p62 clearly increased the efficiency of transfection of murine embryonic stem (mES) cells and human HeLa cells. These data indicate that p62 acts as a key regulator of gene delivery.
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Fig. Knockdown of p62 increases the efficiency of mES cell transfection.
(A) Fluorescence micrographs (upper panels, GFP; lower panels, DAPI) of untreated mES cells (untreat; a, d), negative control siRNA-treated cells (control-siRNA; b, e), and p62-targeting siRNA-treated cells (p62-siRNA; c, f) 24 h following transfection of a GFP reporter construct. (B) Western blot analysis of cell lysates was performed 24 h after transfection of a GFP-expressing construct to measure levels of GFP, p62, and GAPDH. (C) Immunofluorescence analysis of p62 expression in mES cells treated with p62-siRNA 24 h following transient transfection with a GFP reporter construct. DeltaVision microscope images of GFP (a), p62 (b), DAPI (c), and a merged image (d) are shown. The enclosed area within the white dashed line indicates GFP positive cells.