Fig 2: Rab7b dynamically interacts with autophagic membranes Live‐cell imaging of U2OS GFP‐LC3 cells transiently transfected with mCherry‐Rab7b. The arrows indicate Rab7b‐ and LC3‐positive vesicles moving together. Scale bars: 15 μm and 5 μm.Frequency histogram representing the quantification of how long Rab7b‐ and LC3‐positive vesicles move together before separating. Total number of objects counted (n) is shown for 60 different cells from three independent experiments.Live‐cell imaging of U2OS cells transiently transfected with GFP‐Rab7b and mCherry‐LC3. 3D volume rendering (top) and the relative Imaris isosurface 3D rendering (bottom) are shown. Scale bar: 15 μm. Magnification of the boxed area is shown in the inset on the right (scale bar: 2 μm).Double immunogold labeling of U2OS cells stably transfected with GFP‐LC3 and transiently transfected with HA‐Rab7b. HA‐Rab7b (10 nm gold) localizes to outer (open arrowheads) and inner (filled arrowheads) membranes of autophagosomes, identified by LC3 labeling (5 nm gold) and characteristic ultrastructure (cytoplasmic mass surrounded with electron lucent area representing double membrane), or to small vesicles close to autophagosomes (arrows). Scale bar: 500 nm.

Fig 3: Atg4B interacts with Rab7b AU2OS cells were transiently co-transfected with myc-Atg4B and either GFP-Rab7b wt, GFP-Rab7b Q67L, or GFP-Rab7b T22N, starved, and lysed, followed by co-IP with GFP-Trap magnetic agarose beads. Whole-cell lysates (WCL) and immunoprecipitates (IP) were subjected to Western blot analysis.BBacterially expressed and purified His-Rab7b Q67L, His-Rab9, and His-Rab9 Q66L were loaded with GTP?S and incubated with cell lysates of human MDDCs. His-tagged proteins pulled down using cobalt-coated Dynabeads were subjected to Western blot analysis.C–FProximity ligation assay (PLA) of U2OS cells incubated in CM (C) or starved for 2 h (D), fixed, and stained with anti-Rab7b, anti-Atg4B, and DAPI. As negative control, PLA was performed on U2OS cells starved for 2 h and stained with anti-Atg4B and DAPI only (E) or on cells transiently transfected with HA-Rab7b wt, starved for 2 h, and stained with anti-HA, anti-EEA1, and DAPI (F). Scale bar: 30 µm.GLive-cell imaging of U2OS cells co-transfected with GFP-Atg4B and mCherry-Rab7b. Red and white arrows indicate vesicles positive for both GFP-Atg4B and mCherry-Rab7b. Scale bar: 10 µm.

Fig 4: Rab7b depletion causes LC3-II accumulation but does not affect the fusion of autophagosomes with lysosomes ACell lysates from cells transfected with control siRNA or siRNAs against Rab7b were subjected to Western blot analysis with the indicated antibodies.B, CQuantification 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.DU2OS 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).ERepresentative 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.

Fig 5: Rab7b depletion increases autophagic flux and sequestration rate AU2OS mRFP-GFP-LC3 cells were transfected with either control siRNA or siRNA against Rab7b or Rab7a and incubated for 2 h in CM, EBSS, or EBSS with BafA1 before fixing. The percentage of autolysosomes (vesicles positive for RFP but not GFP) per cell is represented in the graph. Data represent the mean ± s.e.m. for three independent experiments (n > 60 cells).B, CCell lysates from U2OS cells treated with control siRNA and siRNA against Rab7b (B) or Rab7a (C) were subjected to Western blot analysis with the indicated antibodies.DU2OS cells transfected with control siRNA or siRNA against Rab7b were subjected to a LLPD assay. The quantification of the degradation rate for long-lived proteins is presented as mean ± s.e.m. of at least four independent experiments.EU2OS cells transfected with control siRNA, siRNA against Rab7b, or depleted of Rab7b and then transfected with HA-Rab7b were subjected to a LDH sequestration assay. In the graph, the net sequestered LDH per hour (%/h) is represented as mean ± s.e.m. of at least three independent experiments.FU2OS cells transfected with control siRNA or siRNA against Rab7b were starved or not for 2 h before fixation and then stained with antibodies against LC3 and WIPI2. The insets show magnifications of the boxed areas. Scale bar: 20 µm.G, HQuantification of the number (G) and size (H) of WIPI2-positive spots per cell. The data represent the mean ± s.e.m. of three independent experiments (n > 60 cells).Data information: Statistical significance was evaluated in (A, D, E, G, and H) using paired Student's t-test. *P < 0.05; **P < 0.01; ***P < 0.001.