Fig. 1
From: USP32 regulates late endosomal transport and recycling through deubiquitylation of Rab7
Deubiquitylating enzyme (DUB) screen reveals USP32 as a regulator of endosome biology. a Small interfering RNA (siRNA)-based screen for DUBs affecting major histocompatability class II receptor (MHC-II) surface levels. MelJuSo cells transfected with siRNAs targeting all human DUBs were analyzed for surface expression of peptide-loaded MHC-II by flow cytometry using monoclonal antibodies (L243-Cy3). Z-scores of DUBs whose depletion resulted in elevated (Z ≥ 3) or diminished (Z ≤ −3) levels of MHC-II on the cell surface are plotted, n = 3 biologically independent samples. Effect of USP32 depletion is highlighted by a red box. b Effect of USP32 loss on the size and distribution of endosomes. Representative confocal overlays of fixed MelJuSo cells transfected with either control (siCtrl) siRNA or oligo #2 targeting USP32 (siUSP32_2) and immunostained against MHC-II (green) and vesicular markers or cargoes (magenta) are shown. EEA1: early endosome (EE) marker, CD63 late endosome (LE)/multi-vesicular body (MVB) marker, mannose-6-phosphate receptor (M6PR): trans-Golgi network (TGN) cargo. Transferrin receptor (TrfR): recycling endosome (RE) marker; PM: plasma membrane. Cell and nuclear boundaries are demarcated with dashed white lines, scale bars = 10 µm. Zooms Z1–Z3 are placed within a schematic of cargo flow. c Percentage cells harboring enlarged MHC-II-positive vesicles, n = 3 independent experiments. Immunoblot of USP32 protein expression in response to depletion using two independent siRNA oligos (siUSP32_1 and siUSP32_2) is provided with actin as loading control. d Vesicle dispersion expressed as fractional distance of MHC-II pixels (black open circles) along a straight line from the center of nucleus (0) to the PM (1.0). Red lines: mean, n = 2 independent experiments. Total number of cells analyzed per condition appears above each bar/scatter. Bar graphs report mean of independent sample values (black circles), error bars reflect ±s.d. All significant values were calculated using Student’s t test: **p < 0.01, ***p < 0.001, NS = not significant. See also Supplementary Fig. 1
