EMBO/soda-nel huggingface dataset:
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 6.

SNX18 interacts with LC3 family members. (A) GFP, GFP-LC3, or GFP-LC3 G120A were immunoprecipitated from transfected HeLa cells. The immunoprecipitates and 2% of the lysate were analyzed by immunoblotting against SNX18. The GFP proteins were detected by Ponceau staining. (B) Lysates from fed or starved MDBK cells were incubated with the indicated glutathione-Sepharose–bound GST-tagged proteins. SNX18 was detected by immunoblotting. Ponceau staining shows the GST proteins. (C) In vitro translated GFP-p62 or GFP-SNX18 were incubated with the indicated GST-tagged Atg8 family proteins. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 7.

A WDDEW sequence in the SNX18 LC region mediates the interaction with LC3/GABARAP. (A) GFP-p62 or -SNX18 full-length, SH3-LC, or PX-BAR regions were in vitro translated and incubated with GST-LC3B or -GABARAP. The resulting pulldowns were separated by SDS-PAGE. Bound proteins were detected by autoradiography and GST proteins by Coomassie blue staining. (B) 18-mer peptides covering the entire sequence of the SNX18 SH3-LC region were spotted on a membrane that was incubated with GST or GST-GABARAP, which were detected by immunoblotting against GST. The peptide sequences that specifically bound GST-GABARAP are shown with the common WDDEW motif in bold. (C) Peptides with the sequence YGGYQASQGSDDDWDDEWDDSSTVADEPGAL (SNX18 WT) or with the first (SNX18 W154S), the second (SNX18 W158S), or both (SNX18 W154S/W158S) W mutated to S were spotted on membranes that were incubated with GST, GST-LC3B, or GST-GABARAP. Binding was analyzed as in B. (D) GFP-SNX18 WT and the indicated mutants were in vitro translated and incubated with GST-LC3B or -GABARAP, and their binding was analyzed as in A. (E) HeLa GFP-LC3 cells were transfected with myc-SNX18 W154S/W158S mutant, starved for 2 h, immunostained against myc, and analyzed by confocal imaging. Bar, 10 µm. (F) Indicated myc-SNX18 constructs were transfected into HEK GFP-LC3 cells, and the number of GFP-LC3 spots per cell was quantified. The graph shows mean ± SEM (error bars), n = 3. *, P < 0.05.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 8.

SNX18 tubules are positive for TfR. HeLa GFP-LC3 cells were transfected with myc-SNX18 or a control myc plasmid and then immunostained for myc and TfR (A) or Rab11 (B). Bars: (A) 10 µm; (B) 5 µm. Insets show enlarged views of the boxed regions. (C, i) Clusters of tubulo-vesicular structures together with small multivesicular body–like vesicles were observed upon plastic embedding of HeLa GFP-LC3 cells overexpressing myc-SNX18. (ii–vi) Immuno-EM showed that similar clusters labeled strongly for GFP-LC3 (ii, and enlarged in iii from the boxed region), myc-SNX18 (v), and TfR (vi). Strong GFP-LC3 labeling in structures resembling more typical autophagosomal vesicles was also detected (iv). See also Fig. S4.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 9.

Recycling endosomes provide membranes for SNX18-mediated GFP-LC3 membrane trafficking and remodeling. (A) HeLa cells were starved or not starved for 2 h before fixation and immunostaining against endogenous SNX18, Rab11, and TfR. (B) Cells were transfected to express flag-Atg16L1, starved, and immunostained against Rab11 or TfR and flag. (C) Cells were starved and immunostained against TfR, Atg16L1, and SNX18. (D) Cells were transfected with control or SNX18 siRNA, starved, and immunostained against SNX18 and Rab11. Inset panels show enlarged views of the boxed regions. (E) Cells were transfected with control or Rab11 a+b siRNA, starved, and immunostained against SNX18 and Atg16L1. Cells were imaged by confocal microscopy. Bars, 5 µm.
Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation. (2013)
Figure 10.

SNX18 is a positive regulator of autophagy and its function is conserved in D. melanogaster fat body cells. (A) During starvation, Rab11-positive recycling endosomes are recruited to the SNX18-positive perinuclear area dependent on SNX18 itself. SNX18 and Rab11 are required for the perinuclear localization of Atg16L1 to omegasomes marked by DFCP1. LC3 lipidation and autophagosome formation proceeds. In the absence of SNX18, recruitment of recycling endosomes to the perinuclear site is inhibited, Atg16L1 fails to localize to DFCP1-positive omegasomes, and WIPI2 accumulates. LC3 lipidation and autophagosome formation is inhibited. Upon SNX18 overexpression, Rab11-, TfR-, and LC3-positive membranes accumulate at a perinuclear site, and extensive membrane tubules with SNX18, Atg16L1, LC3, and TfR form from perinuclear recycling endosomes. LC3 lipidation and autophagosome formation is increased. Based on the experimental observations summarized in this model, we propose that SNX18 facilitates LC3 lipidation via Atg16L1 and provides membrane from recycling endosomes to forming autophagosomes. (B and C) RNAi against SH3PX1 (GFP-positive clones) decreases formation of LTR punctae in response to 4 h of starvation in 20% sucrose (B) and inhibits formation of mCherry-Atg8a-positive autophagosomes in response to 2 h starvation (C). (D and E) RNAi against SH3PX1 inhibits formation of LTR (D) and mCherry-Atg8a–positive structures (E) at early stages of developmental autophagy in fat bodies of 110-h-old larvae. (F) There was no change in mCherry-Atg8a in SH3PX1 mutant clones (GFP negative, outlined) in well-fed larvae. (G) Decreased formation of mCherry-Atg8a–positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (B and D) hs-flp; UAS-dicer/+; Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (C and E) hs-flp; UAS-dicer/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/UAS-SH3PX1 RNAi; (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B UAS-2XeGFP/FRT80B SH3PX1HK62b. See also Fig. S5.
Intestinal Apc-inactivation induces HSP25 dependency (2022)
Figure 1

Fig 1. Identification of Hspb1 as Apc-specific heat shock protein.

a, Schematic illustration of the in vitro recombination experiment. TAM, 4OH-tamoxifen

b, Representative phase images of the morphology of WT and Apc-/- organoids before mRNA isolation. Scalebar, 250 μm

c, Heat map illustrating differentially expressed genes (DEGs) encoding heat shock proteins between WT and Apc-/- organoids.

d, Volcano plot of DEGs between normal mouse intestinal tissue and adenomas (GSE65461).

e, f, Relative gene expression of Hspb1 (***P=0.0004, n=6)(e) and protein expression of HSP25 (f) in unrecombined versus recombined murine organoids.

g, Gene expression of Hspb1 in normal (N) versus adenoma (A) tissue (***P=0.0002, n=4).

H, RNA-ISH for Hspb1 in murine intestinal adenoma. Scalebar, 100 μm. Zoom panel, 25 μm

i, Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue, (****P<0.0001, paired t-test, n=59 pairs).

Data information: All data are mean±s.e.m., biological replicates, analysed using unpaired two-sided t-test unless otherwise specified.
Intestinal Apc-inactivation induces HSP25 dependency (2022)
Figure 2

Fig 2. Hspb1 expression is Wnt-regulated and sensitizes Apc-mutant cells to HSP25 inhibition in vitro.

a, Schematic illustration of the doxycycline-inducible dnTCF construct.

b, c, Relative gene expression of AXIN2 (*P=0.0129), P21(*P=0.0103), and HSPB1(*P=0.0236) (b), and protein expression of HSP27 (c) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.

d, e, Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR-99021 (**P=0.0016, one way ANOVA, n=4) (d), and protein expression of HSP25 in WT and Apc-/- organoids (e).

f, Schematic illustration of in vitro recombination experiment using Hspb1 KO clones.

g, h, Representative images of single cell Hspb1 KO clones passaged after loss of Apc (g), and quantification of their clonogenic potential (*P=0.0147(#1), **P=0.0035(#2), **P=0.0032(#3)) (h). Scale bar, 500 μm.

i, j, Representative images of Apc-/- organoids cultured in the absence or presence of 60 μM BVDU (i), and quantification of their clonogenic potential (**P=0.0039) (j). Scale bar, 500 μm.

k, l, Relative gene expression of Wnt target genes Axin2 (**P=0.0080) and Lgr5 (*P=0.0142) in Apc-/- organoids cultured in the absence or presence of 60 μM BVDU.

m, RNA-ISH for Lgr5 in control or BVDU-treated Apc-/- organoids. Scale bar, 50 μm, zoom panel 20 μm.

Data information: All data are mean±s.e.m. n=3 biological replicates, analysed using unpaired two-sided t-test unless otherwise specified.
Intestinal Apc-inactivation induces HSP25 dependency (2022)
Figure 3

Fig 3. Inhibition of HSP25 prevents oncogenic transformation and adenoma development in vivo.

a, Experimental set-up of short-term assay using Villin-CreERT2;Apcfl/fl mice. p.o., per os, orally

b, c, Visualization of proliferating cells in tissues incubated with EdU for 2-hours (b), and quantification of the hyperproliferative zone (**P=0.0057, n=3 mice) (c). Scalebar, 50 μm.

d, Experimental set-up of short-term assay using Lgr5-CreERT2;Apcfl/fl mice.

e, f, RNA-ISH for Notum reveals Apc-mutant clones in crypt bottoms of control or BVDU-treated mice (e), and quantification of the abundance of mutant crypts (n=2 mice per condition, n=5 technical replicates per mouse) (f). Scalebar, 50 μm.

g, h, Clone size distributions of Notum+ crypts in control (n=265 crypts) or BVDU-treated (n=260 crypts) mice, the average clone size and number of fixed clones are included in the figure.

i, Experimental set-up of tumour development assay using Lgr5-CreERT2;Apcfl/fl mice, mice were sacrificed once they develop signs of adenoma development.

j-l, Survival curves for control and BVDU-treated mice (P=0.0351, Mantel-Cox test) (j), macroscopic images of adenoma development in the distal small intestine (k), and boxplots for the number of adenomas per intestinal region(**P=0.0013) (l), n=8 control, n=11 treated.

Data information: All data are mean±s.e.m., boxplots, the box represents the 25th-75th percentile and the median (line), whiskers represent min to max values, biological replicates, analysed using unpaired two-sided t-test unless otherwise specified.

Extended View Figure legends
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 1.

Mutations in epg-7 cause defective degradation of SQST-1 aggregates. (A and B) In wild-type embryos, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. (C) Elevated expression level and accumulation of many SQST-1::GFP aggregates in epg-7 mutant embryos. (D) Compared with wild type and epg-2 mutants, levels of SQST-1 are dramatically elevated in atg-3 and epg-7 mutant embryos in an immunoblotting assay. (E–G) Compared with wild-type embryos (E and F), endogenous SQST-1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg-7 mutant embryos (G). ∼200-cell stage embryos are shown in E–G. (E) DAPI image of the embryo shown in F. (H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants. (I–L) At the ∼100-cell stage, far fewer SQST-1 aggregates are formed in epg-7 mutants than in atg-3 mutants. (I and K) DAPI images of the embryos shown in J and L, respectively. (M) SQST-1 directly interacts with ZK1053.4 in a GST pull-down assay. (N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants. (O–R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O–P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P. (S) Survival curve of L1 larvae under food depletion conditions in wild type, epg-1 mutants, and epg-7 mutants. This experiment was completed once (wild type: n = 2,964; epg-7: n = 602; epg-1: n = 215). Statistical analysis of L1 survival shows that epg-7 mutants exhibit significant difference to wild type and also to epg-1 mutants (log-rank test, P = 0.000). (T) Loss of function of epg-7 does not ameliorate the degeneration of mec-4::gfp–labeled touch neurons caused by mec-4(u231). (U) Survival curves of wild type and various mutants. sqst-1;epg-7 mutants have a shortened mean and maximum life span. (P = 0.000 for epg-7 or wild type vs. sqst-1;epg-7 in all three repeats; log-rank test). (V) Survival of hydrogen peroxide–treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test). (W) Survival of animals at the indicated time after heat shock treatment. (P < 0.05 for wild type vs. epg-7 or epg-7 vs. sqst-1;epg-7 at 5, 6, and 7 h). (X) Dauer formation of daf-2(e1370) mutants in different backgrounds. Bars: (A–C, E–G, I–L, and O–R) 10 µm. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, bars for embryos are only shown once in each figure.
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 2.

epg-7 encodes a protein with similarity to mammalian FIP200 and yeast Atg11 and is degraded by autophagy. (A) Cloning of epg-7. (B) Protein sequence of EPG-7. The molecular lesions in identified mutant alleles are highlighted in red. (C) Schematic structures of EPG-7, FIP200, and Atg11. The coiled-coil domains are highlighted in green. The Atg11 domain is depicted in pink. (D) A large number of SQST-1::GFP aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del Atg11) transgene. (E and F) No SQST-1 aggregates are detected in epg-7 mutants carrying an epg-7::gfp reporter. (E) DAPI image of the embryo shown in F. (G) EPG-7::GFP is weakly expressed and diffusely localized in the cytoplasm in wild-type embryos. (H–I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages. (J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants. (K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos. (L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos. Bars: (D–G and K–O) 10 µm; (H) 10 µm; (I) 10 µm.
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 3.

EPG-7 colocalizes with SQST-1 aggregates in autophagy mutants and directly interacts with SQST-1. (A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view. (B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants. (C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1. (D) EPG-7 interacts with SQST-1 in a pull-down assay. (E) EPG-7 self-associates in a pull-down assay. (F) SDS-PAGE and blue-native PAGE of purified EPG-7(520–714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively. (G) An EPG-7 fragment containing amino acids 531–631 self-interacts. (H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531–631)::gfp transgene. (H) DAPI image of the embryo shown in I. (J and K) EPG-7(del 531–631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H–K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time. (L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26–181 (M). (N) Levels of SQST-1(del 26–181)::GFP are dramatically elevated in epg-7 and atg-3 mutants. (O and P) SQST-1(del 26–181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P. (Q and R) SQST-1(del 26–181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants. Bars: (A, H–K, and O–R), 10 µm; (A, insets) 5 µm.
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 4.

Loss of epg-7 activity impairs the colocalization of SQST-1 aggregates with LGG-1 puncta in autophagy mutants. (A–D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B. (E) Loss of function of epg-7 does not increase levels of LGG-1 and also does not suppress accumulation of LGG-1 in epg-3 mutant embryos. (F–H) Expression of LGG-1 in epg-7 mutant embryos before the ∼100-cell stage (F), at the ∼200-cell stage (G), and at the fourfold stage (H). Images in B–D and F–H were taken using the same exposure time. (I) SQST-1 aggregates are separable from LGG-1 puncta in epg-7 mutant embryos. Insets show a magnified view. (J) In epg-6 mutant embryos, LGG-1 puncta and SQST-1 aggregates are enlarged and colocalized. (K) LGG-1 and SQST-1 aggregates are smaller in size and largely separable in epg-6;epg-7 double mutants. (I–K) DAPI images of the embryos shown in the same row. (L–O) Enlarged LGG-1 and SQST-1 aggregates are colocalized in epg-4 mutants (L and M). Simultaneously depleting epg-7 reduces the size of LGG-1 puncta and SQST-1 aggregates, which become largely separable (N and O). (L and N) DAPI images of the embryos shown in M and O, respectively. (P) In atg-9 mutant embryos, LGG-1 puncta largely colocalize with SQST-1 aggregates at the comma stage. Notably, there are more SQST-1 aggregates than LGG-1 puncta. (Q) SQST-1 aggregates are separable from LGG-1 puncta in atg-9;epg-7 mutants at the comma stage. (R) Percentage of LGG-1 puncta colocalized with SQST-1 aggregates in indicated autophagy mutants. Comma-stage embryos were examined for atg-9 and atg-9;epg-7 mutants. ∼200-cell stage embryos were examined for other autophagy mutants. Bars: (A–D and F–Q) 10 µm; (insets in I–K, M, O, and P–Q) 5 µm.
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 5.

EPG-7 directly interacts with multiple Atg proteins. (A) EPG-7 coimmunoprecipitates with ATG-9::HA. (B) EPG-7 directly interacts with ATG-9 in a GST pull-down assay. (C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-3 (C) and LGG-1 (D). (E) EPG-7 directly interacts with LGG-3. (F) EPG-7 fragments containing amino acids 852–1003 or 1004–1142, but not 762–851, interact with LGG-1. (G) Fragments of EPG-7 containing amino acids 762–851 or 852–1003 directly interact with ATG-18. (H) Fragments of EPG-7 containing amino acids 1–704 or 1143–1330 directly interact with UNC-51. (I) SQST-1 associates with LGG-1 in vivo. Embryonic extracts from epg-7 mutants show that interaction of SQST-1 with LGG-1 is EPG-7 independent. (J) Fragments of SQST-1 containing amino acids 351–499 and 604–693, but not 500–603, interact with LGG-1. (K) Fragments of SQST-1–containing amino acids 418–499 and 604–630 bind to LGG-1. Mutating the putative LIR in the 604–630 fragment (605YPDL608 to APDA) does not affect the binding. (L and M) SQST-1(del LGG-1-1+2)::GFP, in which the two LGG-1–binding fragments (amino acids 418–499 and 604–630) are deleted, is weakly expressed and diffusely localized in the cytoplasm in sqst-1(ok2892) mutants. (N and O) Loss of function of lgg-1 dramatically elevates the expression level of SQST-1(del LGG-1-1+2)::GFP, which forms a large number of aggregates. (L and N) DIC images of the embryos shown in M and O, respectively. (P) SQST-1(del LGG-1-1+2)::GFP aggregates colocalize with EPG-7 in lgg-1;sqst-1 mutants. Insets show a magnified view. Bars: (L–P) 10 µm; (P, insets) 5 µm.
The scaffold protein EPG-7 links cargo-receptor complexes with the autophagic assembly machinery. (2013)
Figure 6.

Formation of ATG-9 puncta in various autophagy mutants. (A and B) In wild-type embryos, ATG-9::GFP is diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. Insets show a magnified view. (C) ATG-9::GFP forms a large number of small, intense puncta in epg-1 mutants. (D and E) Accumulation of ATG-9::GFP puncta in unc-51(D) and epg-1 (E) mutants is independent of lgg-1. (F) The ATG-9::GFP puncta largely disappear in epg-1;epg-7 mutants. (G) ATG-9::GFP accumulates into large punctate structures in epg-6 mutants. (H) Loss of function of lgg-1 suppresses the accumulation of ATG-9::GFP puncta in epg-6 mutants. (I) Loss of function of epg-7 has no effect on the formation of ATG-9::GFP puncta in epg-6 mutants. The ATG-9::GFP puncta are smaller in epg-6;epg-7 mutants than those in epg-6 single mutants. (J–L) ATG-9::GFP accumulates into large punctate structures in epg-4 mutants (J) and the accumulation is suppressed by simultaneously depleting the activity of lgg-1 (K) or epg-7 (L). (M and N) In epg-8 mutants, ATG-9 accumulates into aggregates (M) that are suppressed by loss of lgg-1 activity (N). (O) epg-7 mutants exhibit the same distribution pattern of ATG-9::GFP as wild-type embryos. (P) Percentage of ATG-9::GFP puncta colocalized with EPG-7 and SQST-1 aggregates in indicated autophagy mutants. (Q–T) The ATG-9::GFP puncta in epg-6 (Q), epg-8 (R), unc-51 (S), and atg-18 (T) mutants colocalize with EPG-7 aggregates. (U and V) ATG-9::GFP puncta in epg-6 (U) and epg-8 (V) mutants colocalize with SQST-1 aggregates. (W and X) In unc-51 (W) and atg-18 (X) mutants, ATG-9::GFP partially colocalizes with SQST-1 aggregates. Bars: (A–O and Q–X) 10 µm; (insets in B–O and Q–X) 5 µm.