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. 2017 Aug 23;18(10):1727–1739. doi: 10.15252/embr.201744069

Figure EV3. Rab7b depletion causes LC3‐II accumulation but does not affect the fusion of autophagosomes with lysosomes.

Figure EV3

  • A
    Cell lysates from cells transfected with control siRNA or siRNAs against Rab7b were subjected to Western blot analysis with the indicated antibodies.
  • B, C
    Quantification of LC3‐II levels normalized against tubulin and plotted relative to the intensities obtained in cells transfected with control siRNA in CM (B) and of LC3‐II/LC3‐I ratio (C). Data represent the mean ± s.e.m. of four independent experiments.
  • D
    U2OS cells transfected with control siRNA (siCtrl) or siRNA against Rab7b #1 (siRab7b) were incubated for 2 h in either CM, CM with BafA1, EBSS, or EBSS with BafA1, fixed, and stained with antibodies against LC3 and Lamp‐1. The percentage of LC3 vesicles also positive for Lamp‐1 per cell is represented in the graph. Data represent the mean ± s.e.m. of four independent experiments (n > 60 cells).
  • E
    Representative confocal fluorescence images showing LC3 (green), Lamp‐1 (red), and Hoechst (blue) under normal or starvation conditions (with or without BafA1) in control cells transfected with a non‐targeting siRNA or in cells depleted of Rab7b. The insets show magnifications of the boxed areas. Scale bar: 20 μm.
Data information: Statistical significance was evaluated in (B–D) using paired Student's t‐test. *P < 0.05; **P < 0.01.