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. 2016 Mar 17;35(8):866–880. doi: 10.15252/embj.201593596

Figure 1. USP19 positively regulates autophagy.

Figure 1

  • A
    Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag‐USP19 (top) or endogenous USP19 in HeLa cells (bottom) treated with USP19‐specific siRNA or scrambled (Scr) siRNA.
  • B, C
    Representative images of GFP‐LC3B puncta in HeLa‐GFP‐LC3B cells transfected with USP19‐specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Pictures (B) were taken using Leica DMI3000 B microscopy with a × 100 oil‐immersion objective. Scale bar, 200 μm. Quantification of GFP‐LC3B puncta in HeLa‐GFP‐LC3B cells transfected with USP19‐specific siRNA or scrambled siRNA (C). Bars represent mean ± standard error of the mean (SEM) of triplicate samples (20 cells per sample). ***P < 0.001 (two‐tailed Student's t‐test).
  • D
    Immunoblot detection of the relative accumulation of LC3‐I and LC3‐II in HeLa cells transfected with USP19‐specific siRNA or scrambled siRNA with rapamycin (250 nM) treatment for 12 h.
  • E
    HeLa cells were transfected with USP19‐specific siRNA or scrambled siRNA and treated with the indicated concentration of rapamycin for 12 h. p62 levels were detected by immunoblot.
  • F
    Human peripheral blood mononuclear cells (PBMCs) transfected with control or USP19‐specific siRNA were treated with rapamycin (250 nM) for 18 h, and the lysates were analyzed with the indicated antibodies.
  • G
    293T cells stably expressing Flag‐USP19 were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3‐I and LC3‐II by immunoblot analysis using anti‐LC3 antibody.
  • H
    Flag‐USP19‐inducible HeLa cells were treated with 200 ng/ml doxycycline (Doxy) overnight to induce the expression of Flag‐USP19. The protein levels of p62 were analyzed after rapamycin treatment for 12 h at the indicated concentrations.
  • I, J
    HeLa cells stably expressing GFP‐LC3B were transfected with pcDNA3.1 empty vector or plasmid expressing the wild‐type and the mutant forms of USP19 (CS or CS/HA). Pictures were taken using Leica DMI3000 B microscopy with a × 100 oil‐immersion objective. Scale bar, 200 μm. Average GFP‐LC3B puncta per cell were calculated (J). Bars represent mean ± SEM of triplicate samples (20 cells per sample). **P < 0.01 and ***P < 0.001 (two‐tailed Student's t‐test).