Fig. 1
From: The IAP family member BRUCE regulates autophagosome–lysosome fusion
shRNA-based screen identifies BRUCE as an autophagy regulator. a Schematic representation of the FACS-based screen using a library of short hairpin RNAs with the miR-E structure (shRNAmir). shRNAs (4,184) targeting 710 ubiquitin- and autophagy-associated genes were cloned into a retroviral gene expression vector (pSBEN). A single copy of shRNA was delivered into the mCherry-EGFP-LC3B-expressing MEF line (clone 1) by transduction. A cell population selected with neomycin was starved and sorted based on the GFP fluorescent signal. Subsequently, genomic DNA was isolated from sorted cells and analyzed by next-generation sequencing. miR-E, microRNA-element 3’ backbone; NeoR, Neo reporter gene cassette; PGK, phosphoglycerate kinase; SFFV, spleen focus-forming virus; TagBFP, monomeric blue fluorescent protein. b Gating strategy of “GFP low/high” cell population of shRenilla and shAtg5 MEFs, compared with ubiquitin shRNAmir library transduced cells (replicate #1). After 6 h starvation, cells were enriched in “GFP low” gate (blue) and depleted in “GFP high” gate (green). Percentage of cells in corresponding gates is indicated. c Relative mCherry and GFP signals compared with basal condition (fully supplemented medium (FM)) analyzed by flow cytometry. Control (shRenilla (Renilla.713)), two BRUCE knockdown (KD) (shBruce#1 (Birc6.766.), and #2 (Birc6.14744)) and ATG5 KD (shAtg5 (Atg5.1063)) MEF lines were analyzed. Data are presented as mean±SD from three biological replicates (****p < 0.0001). Representative data are shown from four independent experiments. d, e Knockdown efficiency of BRUCE and ATG5 in shBruce#1 and #2, and shAtg5 MEFs, respectively, analyzed by immunoblotting using anti-BRUCE and anti-ATG5 antibodies. Anti-Vinculin antibody was used for loading control
