Fig 2: Autophagic program of colorectum in experiment colitis mouse model after Erbin was deleted(A) LC3 (green) in colorectum of DSS -treated GFP-LC3+/- mice by immunofluorescent staining and confocal microscopy. The nuclei were stained with DAPI (blue). (B) Ultrastructural changes of autophagic/lysosomal vacuoles (labelled as 1 and mitochondrial (labelled as 2) in colorectal mucosa of Erbin-/- mice treated with DSS. (C-D) Expression of Erbin, LC3, ATG16L1, ATG7, ATG2A of colorectum in DSS-induced wild type (WT) and Erbin-/- mice by Western Blots (WB) (C), and immunohistochemical staining (IHC) in IL-10-/- mice (D). Scale bar, 1 μm and 0.5 μm (B). Scale bar, 50 μm (A and D).

Fig 3: HCK regulated macrophage polarization through autophagy.A Endogenous HCK interacted with autophagy proteins ATG2A and CBL in macrophage cell line Raw264.7 by IP/WB. B Overexpression of HCK inhibited autophagy flux as more LC3 HiBiT reporter remained. HCK knockdown promoted autophagy flux as less LC3 HiBiT reporter remained. C HCK KO could promote autophagy by increasing LC3II/LC3I and decrease P62 levels with and without 3-MA treatment in mice BMDM cells. Western blots (D) and quantification (E) showed phospho-PI3K and phospho-AKT decreased in HCK KO BMDMs. However, there were no significant differences for phospho-Erk1/2 and phospho-EGFR with HCK KO. F Effect of HCK KO was abrogated in autophagy inducing by PP242 treatment. Baf1: bafilomycin A1. 3-MA: 3-methyladenine. Source data are provided as a Source Data file.

Fig 4: TfR Is Recruited to LC3 Vesicles in Starvation Conditions(A) HeLa cells transfected with GFP-LC3, were loaded with transferrin Alexa 555 for 1 hr in Hank’s balanced salt solution or full medium and chased for 15 min. The Tf/LC3 and LC3/Tf signal overlaps are shown. Data are means ± SEM, n = 3; two-tailed paired t test, ***p < 0.001.(B) HeLa cells treated as in (A) were processed for immunogold labeling on cryosections. Arrows specify autophagosome double membranes, black outer, white inner.(C) HeLa cells starved for 1 hr and loaded with anti-TfR antibody were processed for pre-embedding electron microscopy.(D) HeLa cells treated with ATG2A/B siRNA (left) were processed as in (C). HeLa cells transfected for Flag-ATG4BC74A were loaded with Tf and processed as in (B). LD, lipid droplet. Arrows specify autophagosome double membranes as in (B).(E) HeLa cells treated with control or ATG7 siRNA and/or transfected with ATG7 were processed for immunoblotting and the TfR levels measured. Data are means ± SD, n = 3; two-tailed paired t test, ***p < 0.001.(F) HeLa cells treated with control or RAB11A or WIPI2 siRNAs were processed as in (E). Data are means ± SD, n = 3; two-tailed paired t test, **p < 0.01, ***p < 0.001.(G) HeLa cells starved for 1 hr, loaded with Ferrofluid-Tf Alexa 488 for 1 hr and chased for 15 min in starvation medium. The cells were then fragmented and the membranes containing Ferrofluid-Tf488 (bound) or not containing Ferrofluid-Tf488 (unbound) were separated and processed for immunoblotting (see Figure S5G).(H) HeLa cells transfected with control or ATG2A/B siRNA were processed as in (G). The amount of LC3II in bound fraction in ATG2 knockdown condition is expressed as percentage of control. Data are means ± SEM, n = 3.(I) HeLa cells treated with control or ULK1 siRNA and processed as in (G). The LDH activity was measured in the bound fraction. Data are means ± SEM, n = 3; two-tailed paired t test, ***p < 0.001.

Fig 5: Mitochondria Are Engulfed by RAB11A-Positive Membranes during Parkin-Dependent and -Independent Mitophagy(A) HeLa cells treated with ATG2A/B siRNA were processed as in Figure 5D.(B) GFP-RAB11A HeLa cells treated with DFP, labeled with MitoTracker Red, and imaged. Line scan analysis of the area under the arrow is shown in the graph on the right.(C) HeLa cells treated as in (B) were stained for LC3 and RAB11A. Z-stacks of the area in the inset were processed by ZEN software and presented as max z-projected confocal images.(D) HA-PARKIN HEK cells transfected with GFP-RAB11A, labeled with MitoTracker Red, and treated with CCCP were imaged. Line scan analysis of the area under the arrow is shown in the graph on the right.(E) Lysates from HA-PARKIN HEK cells treated with control, RAB11A, or WIPI2 siRNA and incubated with DMSO or 5 µM CCCP for 6 hr were probed as indicated. Data are means ± SD, n = 3; two-tailed unpaired t test, *p < 0.05.(F) The cartoon summarizes events that lead to formation of the autophagosome: coincident detection of PI3P and RAB11A by WIPI2 (1) and recruitment of ATG16L1 complex (2–3). Here we show that all the relevant events preceding the completion of autophagosome formation (4) occur on the same membrane district (the platform), the RAB11A-positive membranes. Light green, inactive RAB11A, dark green, active RAB11A.