EMBO/soda-nel huggingface dataset:
Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling (2020)
Figure 2

Figure 2. Rabl2 deficiency attenuates Hh signaling.

A. Normal SAG-induced ciliary accumulation of Smo. MEFs serum-starved for 24 h were treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Arl13b served as ciliary marker. We also examined Gpr161 but failed to observe specific immunofluorescent signals, though the antibody, prepared by using human GPR161, recognized ciliary GPR161 well in human RPE1 cells (see Fig 3E). Quantification results were pooled from four MEF strains derived from two Rabl2+/+ and two Rabl2-/- embryos.

B. Reduced Gli2 accumulation at the ciliary tip. MEFs were treated as in (A). Ac-tub served as ciliary marker. Arrowheads indicate ciliary tips. Quantification results were pooled from three independent experiments, each using MEFs from different embryos. At least 100 cells were scored in each experiment and condition.

C. Reduced Gli1 expression. MEFs treated as in (A) were lysed for immunoblotting. Gapdh served as the loading control. For quantification, three independent experiments were performed, each using MEFs from different embryos.

D, E. Increased Gli3 FL/R ratios in E11.5 hind limb buds and E10.5 mouse embryos. Relative band intensities of the full-length (FL) and the repressor (R) forms of Gli3 were quantified (red). FL/R ratios were from four different sets of limb bud preparations (D) and three different sets of embryo preparations (E).

Data information: Quantification results are presented as mean ± s.d. Student's t-test: ns, no significance; *P < 0.05; ***P < 0.001.
Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling (2020)
Figure 4

Figure 4. Rabl2-GTP enters cilia through IFT.

A, B. Wildtype Rabl2 was observed in the shaft of both primary cilium (A) and multicilia (B). Ciliated RPE1 cells (A) or mEPCs (B) expressing Rabl2 tagged with 3×mNeonGreen (3NG) were fixed at 48 h or day 7 post serum starvation, respectively.

C. Ciliary 3NG-Rabl2 in living RPE1 cells underwent bidirectional IFT. The 1st frame and kymographs of a representative cilium (also see Movie EV1) are shown. Quantification results, presented as mean ± s.d, were measured from 34 cilia.

D, E. Detection of endogenous Rabl2 in purified multicilia. Multicilia purified from cultured mEPCs (D) were analyzed by immunoblotting (E). Ac-tub, Ift25, and Ift81 served as positive controls for cilia, whereas Lamin B1, GM130, Cep162, and Gapdh served as quality controls for the ciliary preparation to exclude major contaminations from the nucleus, Golgi, basal body, and cytosolic proteins, respectively. 1/400 of the mEPC lysate and 1/20 of the cilia lysate were loaded per lane.

F. Diagram of IFT-B. The core 1, core 2, and peripheral subcomplexes (Nakayama & Katoh, 2018) are depicted in different colors.

G. Rabl2Q80L strongly associated with IFT-B. GFP-Rabl2 or mutants stably expressed in NIH3T3 cells were immunoprecipitated with anti-GFP beads.

H. Rabl2Q80L interacted with the IFT74/81 heterodimer. FLAG-tagged Ift74 and Ift81 and GST-tagged Rabl2Q80L and Rabl2S35N expressed in E. coli were mixed as indicated and pulled down with glutathione-conjugated beads. Antibodies to GST and FLAG were used for immunoblotting.

I. Association of endogenous Rabl2-GTP with IFT-B. Mouse testis lysates with or without 1 mM GTPγS were subjected to sucrose gradient ultracentrifugation. Fractions were collected from top (low MW) to bottom (high MW). Note the obvious enrichment of Rabl2 in fraction #16 in the presence of GTPγS.

J. A model delineating relationships between Rabl2 and IFT machinery. Rabl2-GTP enters cilia by binding to the Ift74-Ift81 heterodimer of IFT-B. After reaching the ciliary tip through anterograde IFT, it undergoes retrograde IFT towards the ciliary base. When its GTP is hydrolyzed into GDP during its IFT, the resultant Rabl2-GDP dissociates from IFT-B. The trafficking of IFT machinery continues in the absence of Rabl2.
Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling (2020)
Figure 5

Figure 5. GTP-locked Rabl2 represses outward passage of the BBSome and SMO through TZ.

A. Rabl2Q80L localized in multicilia. Cultured mEPCs were infected with adenovirus to express GFP-Rabl2 or GFP-Rabl2Q80L at 24 h prior to and fixed at day 7 post serum starvation.

B, C. Rabl2Q80L selectively induced ciliary accumulation of BBS proteins. Multicilia were purified from adenovirus-infected mEPCs harvested at day 10 and subjected to label-free quantitative (LFQ) mass spectrometric analyses (B) or immunoblotting (C). The abundance (B) is relative to LFQ intensities in the GFP-Rabl2 sample. Relationships among the BBS proteins, IFT-B, and IFT-A are depicted according to literature (Eguether et al, 2014; Nakayama & Katoh, 2018). Lztfl1 has been proposed to link BBSome to IFT-B through interaction with Ift27 (Eguether et al, 2014). Lamin B1, GM130, Cep162, and Gapdh served as quality controls for the ciliary preparations (C). The values in red are relative band intensities. 1/400 of the mEPC lysates and 1/20 of the cilia lysates were loaded per lane.

D, E. Ciliary accumulation of BBS proteins in Rabl2Q80L-expressing RPE1 cells. The white arrows (D) point to positions of cilia. Quantification results (E) were from three independent experiments. At least 100 GFP-positive cells were scored in each experiment and condition.

F. FLIP assays revealed that ciliary Rabl2Q80L impaired BBSome export. RPE1 cells co-expressing 3NG-Bbs5 with RFP-Rabl2Q80L or RFP-Rabl2 were serum-starved to induce ciliogenesis. Photobleaching of 3NG in cells around the ciliary base was performed at t = 0 s as illustrated. Ciliary 3NG autofluorescence in the living cells was imaged at 30-s intervals after a snapshot for RFP. Images of representative cilia and half-life (t1/2) of ten cilia from one typical set of experiments are shown.

G. Ciliary SMO was accumulated above the transition zone (TZ) (yellow line) in RPE1 cells expressing GFP-Rabl2Q80L. Images were acquired through 3D-SIM. CEP290 served as TZ marker. DA, distal appendage. Also see Fig EV3.

H, I. BBSomes and IFT-B were able to reach the ciliary base through retrograde IFT in the presence of Rabl2Q80L. Ciliary 3NG-Bbs5 (H) or Ift27-GFP (I) in IMCD3 cells co-expressing RFP-Rabl2 or RFP-Rabl2Q80L was live imaged at 4 fps. Snapshots for the ciliary RFP proteins, the first frames from Movies EV2-EV5, corresponding kymographs, and quantification results from the indicated numbers of cilia are presented. For processivity analysis (I), only traceable puncta were scored.

Data information: Data are presented as mean ± s.d. Student's t-test: ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.
Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling (2020)
Figure 6

Figure 6. TZ passage of ciliary BBSome-GPCR requires dissociation of Rabl2 from IFT-B.

A. Rabl2D73GQ80L localized in the ciliary shaft and did not affect ciliogenesis efficiency. RPE1 cells infected with adenovirus to express GFP-Rabl2D73GQ80L were serum-starved for 48 h. The white arrow denotes the cilium shown in insets. Quantification results were from three independent experiments. Uninfected RPE1 cells served as control. At least 107 cells were scored in each experiment and condition.

B. Rabl2D73GQ80L underwent bidirectional IFT. Ciliary GFP-Rabl2D73GQ80L in RPE1 cells was live imaged at 5 fps. The 1st frame and kymograph of a representative cilium (also see Movie EV6) are shown. Velocities were quantified from 10 cilia.

C, D. Rabl2D73GQ80L did not induce ciliary accumulation of BBS proteins. Ciliated RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were immunostained (C). The white arrows (C) point to positions of cilia. In quantification results (D), at least 100 cells were scored in each experiment and condition.

E, F. Rabl2D73GQ80L did not repress ciliary translocation of SMO and GPR161. RPE1 cells expressing GFP-tagged Rabl2Q80L or Rabl2D73GQ80L were serum-starved for 24 h and treated with DMSO (mock) or SAG for an additional 24 h prior to immunostaining (E). The white arrows (E) point to positions of cilia. In quantification results (F), at least 100 cells were scored in each experiment and condition.

G. Ciliary Rabl2D73GQ80L did not repress the BBSome export. FLIP assays were performed in RPE1 cells co-expressing 3NG-Bbs5 with RFP-tagged Rabl2Q80L or Rabl2D73GQ80L as depicted in Fig 5F. Photobleaching was executed at t = 0. Quantification results on half-life (t1/2) were from 10 cilia for each condition.

H. Rabl2D73GQ80L displayed an attenuated association with IFT-B as compared to Rabl2Q80L. mEPCs infected with adenovirus to express GFP-Rabl2Q80L or Rabl2D73GQ80L were lysed and immunoprecipitated using anti-GFP resin. Gapdh served as the negative control.

I. A model delineating the effects of Rabl2 and mutants on the TZ passage of ciliary BBSome and its cargo GPCR: (1) In physiological conditions, the GTP hydrolysis of Rabl2-GTP results in Rabl2-free IFT-B capable of mediating the TZ passage of the BBSome and its cargos together with the retrograde IFT machinery; (2) Rabl2D73GQ80L dissociates from IFT-B to allow similar TZ passage; (3) The BBSome sheds from IFT-B in the presence of Rabl2Q80L or Rabl2-GTP during the passing process and is therefore blocked in the ciliary shaft with its cargos.

Data information: Quantification results are presented as mean ± s.d. Student's t-test: ns, no significance; *P < 0.05; ***P < 0.001.
Rabl2 GTP hydrolysis licenses BBSome-mediated export to fine-tune ciliary signaling (2020)
Figure 7

Figure 7. Rabl2Q80L knock-in mice display neonatal death and duplex kidney that phenocopy Ift27-deficient mice.

A. Experimental scheme for the knock-in (KI) mice production and characterization. The single-stranded DNA donor-mediated repair of the Cas9-induced Rabl2 DNA breaks is expected to introduce the Q80L mutation in fertilized eggs or early embryos. Neonatal F1 mice were obtained through the caesarean section at E18.5. Genotyping was performed by directly sequencing the PCR products that covered the expected Cas9-cleavage region of Rabl2.

B. Representative sequencing results for Rabl2KI/KI mice, shown with corresponding sequences for wildtype Rabl2 and the 3' end of the sgRNA. The TTG codon for the Q80L mutation is underlined.

C. Viability of neonatal mice in the day of birth after the caesarean section at E18.5. +, wildtype allele of Rabl2; KI, knock-in allele with Q80L mutation; mt, allele with Cas9-induced mutation; x, ambiguous allele. Refer to Fig EV5 for additional genotyping results. As these "heterozygous" mice might contain chimeric ones, the putative genotypes are used only for simplicity.

D. Morphologically normal and duplex kidneys of Rabl2KI/KI mice. The whole kidneys were from a single mouse. The histological sections, stained with hematoxylin and eosin, were from different mice. Arrows point to the constriction of the duplex kidneys.

E. Renal ducts of Rabl2KI/KI kidney displayed normal ciliary formation as compared to wildtype kidney. Nuclear DNA was stained with DAPI.

F, G. Endogenous Rabl2Q80L entered ciliary shaft to repress the export of the BBSome (Bbs5) and its cargo GPCR (Smo). mEPCs cultured from Rabl2KI/KI and wildtype neonatal mice were fixed at day 7 post serum starvation. Anti-Rabl2 antibody was used to visualize Rabl2 and Rabl2Q80L. Ac-tub or Arl13b served as ciliary marker. Representative micrographs of multicilia (F) and primary cilia (G) are shown. The white arrows point to positions of cilia.

H. A model delineating the switch role of ciliary Rabl2 in the BBSome-mediated GPCR turnover: (1) GTP hydrolysis induced presumably by a GTPase-activating protein (GAP) dissociates Rabl2 from IFT-B to enable a tight BBSome-IFT-B interaction in the retrograde IFT machinery for concomitant TZ passage; (2) Without the GTP hydrolysis, Rabl2-GTP (or Rabl2Q80L) binds persistently to IFT-B, resulting in shedding of the BBSome and its cargos from the IFT machinery in the TZ passing process and accordingly their ciliary retention. See Discussion for details. For simplicity, kinesin motor is not illustrated.

Expanded View Figure Legends
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 1

mATG9 Is Internalized by Clathrin-Mediated Endocytosis

(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson’s coefficient: 0.452 ± 0.034 (SEM).

(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson’s coefficient: 0.413 ± 0.020 (SEM).

(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson’s coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.

(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson’s coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.

(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.

(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.

(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.

(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.

(H) HeLa cells treated with Dynasore were processed for immunogold labeling on cryosections and double labeled with ATG16L1 (10 nm) and mATG9 (15 nm). Scale bar, 100 nm.

(I) HeLa cells treated as in (H) were processed for triple labeling on cryosections (clathrin, 5 nm; ATG16L1, 10 nm; and mATG9, 15 nm). Scale bar, 150 nm.

(J) Table shows quantification of numbers of clathrin-coated structures carrying mATG9 and ATG16L1 alone or together or neither protein. A minimum of 50 clathrin-coated structures were examined from three different experiments. We constructed a 2 × 2 table with 0 or 1–3 gold particles for mATG9 or ATG16L1 for a X2 test to avoid statistical cells with 0 counts. X2 = 10.4, df = 1, and p = 0.001.

See also Figure S1.
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 2

ATG16L1 Is Found in Recycling Endosomes

(A) HeLa cells transfected with GFP-ATG16L1 for 24 hr were incubated for 15 min with 2.5 μg/ml with HRP-cholera toxin subunit B at 4°C and then for 10 min at 37°C. Cells were then fixed and treated for immunogold labeling on cryosections. ATG16L1 was detected with anti-ATG16L1 antibody (15 nm), and cholera toxin was detected using anti-HRP antibody. Scale bar, 150 nm.

(B) HeLa cells were transiently transfected with GFP-ATG16L1 for 20 hr. Cells in basal conditions were fixed and labeled with anti-RAB11 antibody.

(C) HeLa cells transfected with mStrawberry-ATG16L1 (20 hr) and starved (4 hr) were processed for immunogold labeling on cryosections. Double labeling was performed with rabbit anti-ATG16L1 (10 nm gold) and rabbit anti-RAB11 (15 nm gold). Scale bar, 150 nm. Schematic diagram shows membrane outlines from EM. Green lines indicate structures positive for both ATG16L1 and RAB11, whereas red lines are only positive for RAB11. Histogram shows the percentage by EM of ATG16L1 tubulovesicular structures that carry RAB11. 281 tubulovesiclular structures from different experiments were analyzed. Error bars, SD.

(D) HeLa cells were transfected with EGFP-ATG16L1 and mCherry-RAB11, and 5 min movies were recorded. Representative images from movies are shown.

(E) HeLa cells were loaded with mouse anti-transferrin receptor antibody (5E9C11) for 30 min and fixed for immunogold labeling for cryosections. The sample was labeled with rabbit anti-mouse antibody to recognize transferrin receptor (5 nm), anti-ATGL116 (10 nm), and anti-mATG9 (15 nm). Scale bar, 150 nm.

(F) HeLa cells were transfected with pmStrawberry-ATG16L1 and ATG9L1-pEGFP, and 5 min movies were recorded. Representative images from the movies are shown.

(G) HeLa cells were transfected with pmStrawberry-ATG16L1 and pEGFP-LC3, starved for 2 hr, fixed, and labeled for mATG9.

See also Figure S2 and Movies S1 and S2.
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 3

mATG9 and ATG16L1 Meet in Recycling Endosomes

(A and B) (A) HeLa cells were incubated for 4 hr at 37°C or 18°C and processed for LC3II western blot. Some cells were incubated overnight with 20 mM NH4Cl in full medium (to block lysosomal degradation) at 37°C or 18°C (B).

(C) HeLa cells were transfected with pEGFP-LC3 for 20 hr and incubated for 4 hr at 37°C or 18°C.

(D and E) (D) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr and incubated for 4 hr at 37°C or 18°C. The size and the number (E) of vesicles were scored (a minimum of 20 cells were examined for each condition). Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.

(F and G) (F) HeLa cells were transfected with pEGFP-ATG16L1 for 20 hr, incubated for 4 hr at 37°C or 18°C, and labeled with EEA1 or RAB11 to visualize early endosomes or recycling endosomes. Histogram in (G) shows the amount of colocalization of ATG16L1 in EEA1 or in RAB11 compartment (Manders’ coefficient). Error bar, SEM. NS, not significant; ∗∗p < 0.01.

(H) HeLa cells were transfected with pmStrawberry-ATG16L1 for 20 hr, fixed, and labeled for mATG9. The histograms show quantification of colocalization and the size of the vesicles labeled with ATG16L1 and mATG9 at 37°C or 18°C. Error bar, SEM. ∗∗∗p < 0.001 and ∗∗p < 0.01.

(I) HeLa cells were transfected with pEGFP-RAB11 or pEGFP-Myosin Vb tail (MVb) and incubated during the last 16 hr with Bafilomycin A1 (Baf) or DMSO and processed for LC3-II western blot. Control cells were transfected with pEGFP empty vector.

(J) HeLa cells were transfected with ATG16L1-mStrawberry and RAB11-EGFP or MVb tail-EGFP. The size of ATG16L1 vesicles was scored in RAB11 and MVb tail overexpression conditions. The inserts show just the ATG16L1 vesicles as quantified in histogram. Error bar, SEM. ∗∗∗p<0.001.

(K) HeLa cells were transfected as in (C) and loaded with transferrin Alexa-647 for 30 min. Histogram and the inserts show the correlation between ATG16L1 and the loaded transferrin (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001.

(L) HeLa cells were transfected as in (C) and labeled for mATG9. Histogram and the inserts show the correlation between ATG16L1 and mATG9 in RAB11 and MVb tail overexpression conditions (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001.

(M) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium, loaded with transferrin Alexa-488 (Tf). Pictures and histogram show the correlation between ATG16L1 and transferrin in basal and starving condition (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001.

(N) HeLa cells were transfected with mStrawberry-ATG16L1 for 20 hr, starved in HBSS for 4 hr, or maintained in full medium and then fixed and labeled with anti-mATG9 antibody. Pictures and histogram show the correlation between ATG16L1 and mATG9 in basal and starved cells (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001.

See also Figure S3 and Movies S3 and S4.
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 4

mATG9/ATG16L1 Vesicle Fusion Is SNARE Dependent

(A) HeLa cells were transfected with mStrawberry-ATG16 for 20 hr and treated for 10 min with N-ethylmaleimide (NEM; 100 μM) in full medium, fixed, and stained for mATG9. Histogram shows correlation between mATG9 and ATG16L1 (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001.

(B) Schematic diagram of the in vitro fusion assay of ATG16L1 and mATG9 vesicles. Postnuclear supernatants (PNS) of cells expressing mStrawberry-ATG16L1 were mixed with PNS from cells expressing ATG9L1-GFP for 1 hr in the presence of ATP, ATP and NEM at 37°C, or with ATP on ice. The samples were fixed and mounted on a microscope slide with Mowiol 4-88 for confocal analysis.

(C and D) (C) Representative fields of in vitro fusion assay of ATG16L1 and mATG9 vesicles. The quantification in (D) was performed on ten fields (similar to that shown in the figure) per experiment. The quantification is expressed as Pearson’s coefficient. Error bar, SEM. ∗∗p < 0.001.
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 5

VAMP3 Regulates Autophagy

(A) HeLa cells were transfected with siRNAs for different R-SNAREs, and we assessed the levels of the autophagy substrate p62 versus actin.

(B) HeLa cells transfected with control or VAMP3 siRNA (oligo A) for 4 days were treated during the last 16 hr with Bafilomycin A1 (Baf A1) or DMSO (basal) and processed for LC3-II western blots.

(C) Endogenous LC3 immunocytochemistry in HeLa cells transfected with control or VAMP3 siRNA for 4 days. The histogram shows the number of vesicles (LC3 positive) in a minimum of 30 cells. Error bar, SEM. ∗∗∗p < 0.001.

(D) HeLa cells were transfected with VAMP3-HA for 20 hr and stained for EEA1 or RAB11. Histogram shows colocalization between VAMP3-HA and EEA1 or VAMP3-HA and RAB11 (Manders’ coefficient). Error bar, SEM.

(E) HeLa cells were transfected for 20 hr with VAMP3-HA, incubated for 1 hr at 37°C or 18°C, fixed, and labeled for HA and mATG9. Histogram shows the percentage of colocalization between VAMP3 with mATG9 (Manders’ coefficient). Error bar, SEM. NS, not significant.

See also Figure S4.
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 6

VAMP3 Regulates mATG9 and ATG16L1 Trafficking

(A–C) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with ATG9L1-pEGFP and labeled with anti-EEA1 (for early endosomes) or anti-RAB11 (for recycling endosomes). The histogram in (C) shows the quantification of the colocalization of mATG9 in EEA1 or RAB11 (Manders’ coefficient). Error bars, SEM. ∗∗∗p < 0.001.

(D–F) HeLa cells were treated as in (A)–(C) and transfected during the last 20 hr with pEGFP-ATG16L1 and labeled with anti-EEA1 (early endosomes) or anti-RAB11 (recycling endosomes). Histogram in (D) shows quantification of the colocalization of ATG16L1 in EEA1 or RAB11 (Manders’ coefficient). Error bars, SEM. ∗∗∗p < 0.001; NS, not significant.