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. 2016 Jun 23;35(17):1853–1867. doi: 10.15252/embj.201593148

Figure 2. INPP5E functions in autophagosome–lysosome fusion.

Figure 2

  1. N1E‐115 cells treated with siControl, siINPP5E, or siAtg2a/2b were cultured in growth medium. The postnuclear fraction (PNS) was separated into LSP, HSP, and HSS fractions and then analyzed by immunoblots using anti‐p62, anti‐LC3, and anti‐GAPDH antibodies. The subfractions were treated with proteinase K (Pro. K) with or without Triton X‐100. Quantitation of protein signal intensities from immunoblots showing LC3‐II (left) or p62 (right) levels, following normalization to the control protein GAPDH. Results represent the mean ± s.d. of three independent experiments. **P < 0.01.
  2. NIE‐115 cells stably expressing LAMP1‐mCherry treated with siControl or siINPP5Es were cultured for 2 h in growth medium containing 200 nM Torin1 with protease inhibitors (10 μg/ml pepstatin A and 10 μg/ml E‐64‐d). Because the treatment of protease inhibitors inhibits lysosomal degradation, LC3 puncta persist even if autophagosomes fuse with lysosomes. Cells were fixed and stained with anti‐LC3 antibodies and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Scale bar, 10 μm.
  3. Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **P < 0.01.

Source data are available online for this figure.