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. 2015 Jul 2;34(17):2272–2290. doi: 10.15252/embj.201590992

Figure 7.

Figure 7

UVRAG phosphorylation regulates lysosomal tubulation

  1. U2OS cells stably expressing LAMP1-mCherry and wild-type (WT) or S550A+S571A (dblA) GFP-UVRAG were transfected with 100 nM control or UVRAG siRNA ∼40 h prior to live cell imaging in complete medium. Representative image are shown from Supplementary Movie S2A–C. Scale bar, 10 μm.
  2. Quantitation from (A) of mean tubules per cell and tubule length as indicated ± SEM for n = 3 independent experiments, significance was determined by one-way ANOVA and Dunnett’s multiple comparison test to the siCTRL, **P < 0.01.
  3. U2OS cells stably expressing LAMP1-mCherry and WT or dblA GFP-UVRAG were transfected with 100 nM UVRAG siRNA 40 h prior to live cell imaging in complete medium and treatment with 50 nM lysotracker-648 (LysoTR). Magnified images of tubules from dblA-expressing cells are shown below. Scale bar, 5 μm.
  4. HeLa cells or those stably expressing WT or dblA GFP-UVRAG were transfected with 100 nM control or UVRAG siRNA ∼40 h prior to treatment. Cells were serum-starved for 2 h before the addition of complete medium and 50 ng/ml EGF before lysis at 0 or 30 min in the presence or absence of 1 μM KU0063794 (KU).
  5. MEFs stably expressing WT or dblA GFP-UVRAG were transfected with 100 nM control or UVRAG siRNA ∼40 h prior to treatment. Cells were incubated in complete medium (ctrl) or EBSS for 1 h in the presence or absence of 50 nM bafilomycin A1 (Baf).
  6. Quantitation of normalised LC3-II from (E), mean ± SEM for n = 3 independent experiments.

Source data are available online for this figure.