EMBO/SourceData huggingface dataset:
Figure 6 Cultured hippocampal neurons were co-transfected with mCherry and EGFP-tagged PSD-95 wild-type (WT), E17R, or T19K at 10-11 DIV and treated with BIC (50 μM) or left untreated at 17 DIV for 24-48 h before fixation. (A) Representative confocal microscopic images of PSD-95-EGFP (green) and mCherry (red) used as "fill" to visualize the dendrite with spine heads (scale bar: 2 µm) after control (top) and BIC treatment (bottom). Individual channels are shown in grayscale. (B) BIC treatment reduced spine enrichment of WT but not E17R or T19K PSD-95 as quantified as the ratio of EGFP intensity in spine heads relative to that in the adjacent dendritic shaft. Values were normalized to control treatments, which were set to equal 100% for each PSD-95 construct.(C) mCherry showed no enrichment in spines and BIC had no effect on its localization.(D-F) Spine enrichment and puncta size and intensity of PSD-95 were comparable under basal conditions for WT, E17R, and T19K PSD-95 (A.U., arbitrary unit).(G-I) Spine density, head diameter, and length were comparable under basal conditions for neurons expressing WT, E17R, and T19K PSD-95.
Figure 8Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom).(B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right).(C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).
Figure 1(B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm.(C) Flow cytometry analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown.(D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.(E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C).(F) Opti-prep purified supernatants from (E) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. Mean + SD of 5-10 experiments (each performed with virus produced from an independent transfection) is shown. (G) Virus used in (E) were used to infect SupT1 cells and productive infection was scored at 48-60 hours by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV-MxA served as a negative control. The mean + SD of 5-10 experiments is shown.(H) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP-FLAG-IFITM3 variants and immunostained with anti-FLAG M2 antibody(I) SDS-PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.(J) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 constructs, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag. Scale bar, 5 μm.(K) Opti-prep purified supernatants from (J) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. The mean + SD of 3 experiments is shown.
Figure 2(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm.(B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry.(C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown.(D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies.(E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.
Figure 5(A) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP-FLAG-Human IFITM3 mutated at indicated residues and immunostained with anti-FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22-24, P18L/L23P and P18H/Δ19-23 mutations were introduced into the human IFITM3 background.(B) SDS-PAGE of FLAG-immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP-FLAG-IFITM3 variants and 0.5 μg of pCI-HA-NEDD4 or pCI-HA-NEDD4 C867A. Immunoblotting was performed with anti-FLAG M2, anti-IFITM3 (Abcam, EPR5242), and an anti-ubiquitin antibody (Enzo Life Sciences, recognizing K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins). 'L' indicates the presence of light chain antibody derived from the FLAG antibody-coupled beads used for immunoprecipitation. *, **, and *** indicate mono-, di-, and tri-ubiquitinated forms of IFITM3.(C) SDS-PAGE of whole cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti-FLAG M2, anti-NEDD4, and anti-actin.(D) 293T cells were transfected with 1.0 μg of pNL4-3 and 0.5 μg of pQCXIP-FLAG-IFITM3 variants. Virus-containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post-infection. The means + SD of 3 experiments are shown.(E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The 'Colobus native' sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus H1N1 PR/8/34, (Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.
Figure 1(E) Quantitative RT-PCR (qRT-PCR) of μGLT, αGLT, and AID from siControl- and siPhf5a-treated samples. The values are presented as mean ± sd (n=3).
Figure 2(B) Southern blots of the IgH/c-Myc translocation assay using genomic DNA extracted from CIT-stimulated CH12F3-2A cells treated with the siRNAs shown at the bottom of each panel. Numbers on the top indicate gel lanes. (C) Frequency of IgH/c-Myc chromosomal translocations derived from the number of translocations detected in the total number of PCR reactions per sample (Boboila et al., 2012b). The values are presented as mean ± sd (n=3). Statistical significance was assessed by two tailed unpaired Student's t-test (** p ≤ 0.01, *** p ≤ 0.001).
Figure 3(D-E) Detection of Sμ DNA double-strand breaks by LM-PCR in CH12F3-2A cells transfected with the indicated siRNA combinations. The DNA samples were either left untreated (none) or treated with indicated end processing enzymes prior to linker ligation.
Figure 4(B) Effect of Phf5a knockdown on I-SceI-induced NHEJ. Plot represents the GFP-positive cells obtained by FACS analysis after co-transfection of the I-SceI expression plasmid and the indicated siRNAs into a cell line harboring the NHEJ reporter construct. The values are presented as mean ± sd (n=3). Statistical significance was evaluated against corresponding siControl by two-tailed unpaired Student's t-test (*** p ≤0.001).
Figure 5(C) Top: schematic diagram of the position of the ChIP assay PCR products specific to the S region. Bottom: ChIP assay of the indicated DNA repair proteins using CIT-stimulated CH12F3-2A cells transfected with siControl or siPhf5a. The values are presented as mean ± sd (n=3). Statistical significance was evaluated in reference to siControl by two-tailed unpaired Student's t-test (*p ≤ 0.05, ** p ≤0.01, *** p ≤0.001).
Figure 6(C) Western blot analysis of the whole cell extract from CIT-stimulated CH12F3-2A cells transfected with the indicated siRNAs. Expression of various proteins including AID, variant H2As and Ku80 were not decresed by Phf5a KD. (ns, non-specific)
Figure 7(C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot.
Figure 8(B) Western blot showing expression of endogenous and exogenously introduced Phf5a.(C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.
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.
Figure 2.(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.
Figure 3.(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.
Figure 4.(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.
Figure 5.(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)(C and D) Coimmunoprecipitation reveals that EPG-7 associates with 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.
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.
Figure 7.(A2) Levels of W07G4.5::GFP in wild type, epg-7 mutants, and atg-3 L1 larvae before and after 6 h starvation treatment. (B2) Model for the role of epg-7 in degradation of SQST-1 aggregates. SQST-1 directly interacts with EPG-7 and is recruited into EPG-7 aggregates. EPG-7 also associates with multiple Atg proteins, which may be recruited to the SQST-1-EPG-7 complex in a hierarchical order. IM, isolation membrane. (C2) Hierarchical relationship of the scaffold proteins EPG-2 and EPG-7 and other essential ATG and EPG proteins in the aggrephagy pathway(A and B) C17E4.2::GFP is weakly expressed and diffusely localized in a wild-type embryo. (A) DIC image of the embryo shown in B. (C and D) C17E4.2::GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg-1 (C) and epg-7 (D) mutant embryos.(E and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants.(I) Levels of C33D9.6::GFP and W07G4.5::GFP are dramatically elevated in atg-3 and epg-7 mutant embryos.(J and K) C17E4.2 aggregates in atg-3 mutants overlap with EPG-7 aggregates (J), but are separable from SQST-1 aggregates (K). Insets show a magnified view. (L) Colocalization frequency of C17E4.2, C33D9.6, and W07G4.5 aggregates with EPG-7 and SQST-1 aggregates in atg-3 mutants.(M) SQST-1::GFP accumulates in hypodermal cells in epg-7 mutants at the larval stage. (N) SQST-1::GFP aggregates disappear in rheb-1(RNAi);epg-7 mutant larvae. (O) Dramatic accumulation of SQST-1::GFP aggregates in hypodermal cells in atg-3 mutant larvae. (P) SQST-1::GFP aggregates persist in rheb-1(RNAi);atg-3 mutant larvae.(Q-T) At larval stages, W07G4.5::GFP forms aggregates in intestinal cells (Q and R). These aggregates disappear upon RNAi knockdown of let-363 (S and T). (U-X) W07G4.5::GFP aggregates accumulate in intestinal cells in epg-7 mutant larvae (U and V). These aggregates disappear when let-363 is simultaneously depleted (W and X). (Y and Z) In atg-3 mutants, W07G4.5::GFP aggregates accumulate in intestinal cells (Y) and persist after RNAi knockdown of let-363 (Z).