EMBO/soda-nel huggingface dataset:
XIAP inhibits autophagy via XIAP‐Mdm2‐p53 signalling (2013)
Figure 3

XIAP is a novel E3 ligase for Mdm2. (A) Lysates from HCT116 cells were immunoprecipitated separately with anti‐XIAP antibody and an isotype‐matched control IgG. Immunoprecipitates and input were analysed by western blotting. The data are representative of two biological replicates. (B) Recombinant His‐XIAP protein was incubated separately with purified GST and GST‐Mdm2 fusion proteins on glutathione beads for 4 h followed by western blot analysis using anti‐His antibody. GST and GST‐Mdm2 were analysed by Coomassie blue staining. The data are representative of two biological replicates. (C) HCT116 cells were treated with XIAP‐specific or control siRNAs. Twenty‐four hours later, cells were cultured in the presence of 50 μg ml−1 cycloheximide for the indicated periods of time, and subsequently analysed by western blotting. We should mention that amounts of cell lysates were adjusted to achieve similar expression levels of Mdm2 at time 0, while the same amounts of cell lysates were used to examine levels of XIAP and actin. The data are representative of three biological replicates. The ratio of Mdm2 to actin is presented in Supplementary Figure S3B. (D) p53−/−Mdm2−/− MEF cells were co‐transfected with the indicated Mdm2, XIAP, and p53 constructs. Cell lysates were analysed by western blotting with the indicated antibodies. We should mention that XIAP H467A expressing plasmid was always used less than XIAP expressing plasmid to ensure expression levels of XIAP and XIAP H467A were similar. The data are representative of three biological replicates. (E) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins and total cell lysates (TCLs) were analysed by western blot with anti‐Mdm2 antibody. The data are representative of three biological replicates. (F) In all, 2 μM Mdm2 or its C464A mutant and 1 μM XIAP or its H467A mutant proteins were incubated with 100 nM E1, 2 μM E2 and 200 μM Ub in a total 20 μl in vitro ubiquitination reaction buffer at 37°C for 1 h. The reaction mixtures were analysed by western blotting with anti‐Mdm2 antibody. The data are representative of three biological replicates. (G) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. (H) p53−/−Mdm2−/− MEF cells were transfected with the indicated plasmids. Twenty‐four hours after transfection, cells were treated with 20 μM MG‐132 for additional 4 h. Cell lysates were denatured before proteins conjugated to His‐ubiquitin were pulled down by Ni2+‐NTA beads. The bead‐bound proteins were analysed by western blotting with anti‐p53 antibody. The data are representative of three biological replicates.
XIAP inhibits autophagy via XIAP‐Mdm2‐p53 signalling (2013)
Figure 4

XIAP regulates serum starvation‐induced autophagy. (A) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for the indicated periods of time. GFP‐LC3 puncta formation was observed by a microscope. Quantification of LC3 punctate cells was shown on the right. The data are represented as means±s.d. of three independent experiments. (B) HCT116 XIAP WT and XIAP KO cells were treated with EBSS for 4 h followed by western blot analysis with the indicated antibodies. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6B. (C) HCT116 XIAP WT and XIAP KO cells were treated with 1 μM API‐2 for 6 h. Cell lysates were analysed by western blotting. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6C. (D) HCT116 cells were treated with or without EBSS for 4 h. Cell lysates were subjected to immunoprecipitation with anti‐XIAP antibody followed by western blot analysis. The data are representative of two biological replicates. (E) Lysates from HCT116 cells were immunoprecipitated with anti‐Mdm2 antibody. Immunoprecipitates and cell lysates were analysed by western blotting with anti‐p‐XIAP antibody. The data are representative of two biological replicates. (F) HCT116 XIAP KO cells were transfected with Flag‐XIAP, Flag‐XIAP S87A, Flag‐XIAP S87D or control vector. Twenty‐four hours after transfection, cells were harvested. LC3 conversion was evaluated by western blot analysis. The data are representative of three biological replicates. The ratio of LCII/LC3I to actin is presented in Supplementary Figure S6D. (G) p53−/−Mdm2−/− MEF cells were transfected with HA‐Mdm2 plus Flag‐XIAP, Flag‐XIAP S87A or Flag‐XIAP S87D. Twenty‐four hours later, cells were treated with 20 μM MG‐132 for another 4 h. Cell lysates were subjected to immunoprecipitation with anti‐HA antibody. Immunoprecipitates were analysed by western blotting. The data are representative of two biological replicates.
XIAP inhibits autophagy via XIAP‐Mdm2‐p53 signalling (2013)
Figure 5

XIAP‐modulated autophagy is associated with the tumorigenecity. (A–C) 1 × 106 HCT116 XIAP WT and 1 × 106 XIAP KO cells (group 1), 1 × 106 HCT116 XIAP KO cells (KO‐Ctrl) and 1 × 106 HCT116 XIAP KO cells stably expressing wild‐type XIAP (KO‐XIAP) (group 2), 1 × 106 HCT116 XIAP KO cells (KO‐Ctrl) and 1 × 106 HCT116 XIAP KO cells stably expressing XIAP S87A mutant (KO‐XIAP SA) (group 3), or 1 × 106 HCT116 XIAP KO cells (KO‐Ctrl) and 1 × 106 HCT116 XIAP KO cells stably expressing XIAP S87D mutant (KO‐XIAP SD) (group 4) were individually injected to the left flank (Left) and right flank (Right) of nude mice as indicated. Five weeks after injection, the mice were sacrificed and photographed (A). The volumes of all flank tumours excised from six mice in each group were compared (n=6) (B). Tumour weights were represented as means±s.d. from six mice in each group (C). (D–F) 1 × 106 HCT116 XIAP KO cells or 1 × 106 HCT116 XIAP KO cells stably expressing XIAP D148A/310A were injected to the right flank of nude mice as indicated (n=3). Five weeks after injection, the mice were sacrificed and photographed (D). The volumes of excised tumours were shown (E). Tumour weights were represented as means±s.d. from three mice in each group (F). (G, H) The human tumour tissues (T) from Oesophagus (G) and Colon (H) and their adjacent normal tissues (N) were homogenized for protein extraction. Protein extracts were analysed by western blotting with the indicated antibodies. The data are representative of three biological replicates. The blots in (G, H) were qualified and the ratio of LC3II/LC3I to actin was then calculated and shown in Supplementary Figure 9A and B. (I) The schematic model of autophagic regulation by XIAP is shown. Under non‐stressed condition, phosphorylated XIAP interacts with Mdm2 and mediates its rapid degradation, thus maintaining the relative high levels of p53 and inhibiting basal levels of autophagy. In response to serum starvation, XIAP undergoes dephosphorylation due to AKT inhibition, thereby promoting the dissociation of XIAP from Mdm2. This in turn accelerates the degradation of p53 and facilitates serum starvation‐induced autophagy.
CCDC50 suppresses NLRP3 inflammasome activity by mediating autophagic degradation of NLRP3 (2022)
Figure 2

Figure 2. Deficiency of CCDC50 increases the activity of NLRP3 inflammasome in THP-1 cells.

A-C qRT-PCR detection of mRNA levels of IL-1β, TNFα and IL-6 mRNA levels in CCDC50-WT and CCDC50-KO THP-1 cells primed with LPS and then treated with ATP and Nigericin (A), or infected with Listeria monocytogenes at a multiplicity of infection (MOI) of 10 and Salmonella typhimurium (MOI of 10) for 6 h (B) or MSU (40 μg/ml ) and SiO2 (40 μg/ml) for 6 h (C) (n = 3 biological replicates).

D Enzyme-linked immunosorbent assays (ELISA) of IL-1β production in supernatants of THP-1 cells treated as in (A-C) (n = 2 biological replicates).

E-G CCDC50-WT and CCDC50-KO THP-1 cells were stimulated as in (A-C). The cells were collected for immunoblot analysis of pro-caspase-1, and the supernatants were subjected to cleaved caspase-1 and IL-1β analysis. The expression levels of cleaved-caspase-1 and cleaved-IL-1β were quantitated using ImageJ software.

Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; Casp1, caspase-1; mRNA results are normalized to GAPDH and relative to untreated wild-type cells; actin was used as a loading control. Data are representative of three biological replicates and shown as mean with SEM (A-C); data are representative of two biological replicates (D). **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.
CCDC50 suppresses NLRP3 inflammasome activity by mediating autophagic degradation of NLRP3 (2022)
Figure 3

Figure 3. CCDC50 negatively regulates NLRP3 inflammasome activity in mouse primary immune cells.

C qRT-PCR detection of Il6, IL-1β and Tnfα mRNA levels in Ccdc50+/+ and Ccdc50-/-

BMDMs primed with LPS for 3 h and then induced with ATP and Nigericin (A), L.m and S.t (B), or MSU and SiO2 (C) for indicated time points (n = 3 biological replicates).

D-F Ccdc50+/+ and Ccdc50-/- BMDCs treated as BMDMs were collected and subjected to qRT-PCR analysis (n = 3 biological replicates).

G ELISA analysis of the protein level of secreted IL-1β in supernatants of Ccdc50+/+ and Ccdc50-/- BMDMs (top panel) and BMDCs (bottom panel) treated as in (A-C) (n = 3 biological replicates).

H,I Ccdc50+/+ and Ccdc50-/- BMDMs (H) and BMDCs (I) were treated as in (A-C). The cells were lysed for western blotting analysis of pro-caspase-1 and the supernatant was collected for immunoblots of cleaved-caspase-1. The expression levels of cleaved-caspase-1 were quantitated using ImageJ software.

Data information: ND, not detectable; mRNA data are normalized to GAPDH and relative to untreated wild-type cells; actin was used as a loading control. Data are representative of three biological replicates and are shown as the mean with SEM (A-G). *P <0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.
CCDC50 suppresses NLRP3 inflammasome activity by mediating autophagic degradation of NLRP3 (2022)
Figure 4

Figure 4. CCDC50 interacts with NLRP3 and mediates its degradation.

A,B Co-Immunoprecipitation (Co-IP) with anti-HA (A) or anti-Flag (B) was performed for the analysis of the association between HA-CCDC50 and Flag-AIM2, -NLRP3, and -ASC in cotransfected HEK293 cells.

C Immunoprecipitation (IP) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP-1 cells activated by LPS plus ATP. IgG was used as a negative control.

D Representative endogenous colocalization analysis of CCDC50 and NLRP3 in PMA-induced THP-1 cells treated with LPS and ATP. Scale bar, 10 μm.

E Representative confocal microscopy image of HCT116 cells overexpressing GFP-CCDC50 and stimulated with LPS as well as ATP. Scale bar, 10 μm.

F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag- AIM2, -NLRP3, -ASC and HA-CCDC50 or empty control vector. The expression levels of Flag-tagged proteins and Actin were quantitated using ImageJ software.

G Immunoblot analysis of HEK293 cells transfected with FLAG-NLRP3 and an increasing amount of HA-CCDC50 plasmids.

H Immunoblot analysis of lysates from CCDC50-KO and CCDC50-WT THP-1 cells induced with LPS plus ATP and then treated with cycloheximide (CHX) for indicated time points. The expression levels of NLRP3 and Actin were quantitated using ImageJ software.

I HEK293 cells were co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h and then treated with MG132, NH4Cl, 3-MA, CQ or DMSO for another 6 h.

J Immunoblot analysis of NLRP3 in CCDC50-WT and CCDC50-KO THP-1 cells stimulated with LPS in combination with ATP.

K Western blotting analysis of ATG5 wild-type or knockout HEK293T cells co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h.

L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs.

Data information: L.C, light-chain; actin was used as a loading control. All the experiments were repeated at least once.
CCDC50 suppresses NLRP3 inflammasome activity by mediating autophagic degradation of NLRP3 (2022)
Figure 5

Figure 5. CCDC50 suppresses NLRP3 inflammasome assembly.

A Denature-IP (with anti-Flag) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag-NLRP3, HA-Ub (ubiquitin) as well as HA- CCDC50 or empty vector for 24 h.

B Denature-IP analysis (with anti-Flag) of K63-linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h.

C The colocalization analysis of K63-Ub (Red), Flag-NLRP3 (Purple) and GFP- CCDC50 in HeLa cells; scale bars, 5 μM.

D,E Images of HeLa cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3 (Purple) and GFP-LC3B for 24 h and then induced with LPS plus ATP; scale bars, 5 μM.

F The colocalization analysis of NLRP3 and LC3B in CCDC50-WT and CCDC50- KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM.

G The oligomerization analysis of NLRP3 in CCDC50 wild-type and knockout THP-1 cells stimulated with LPS as well as ATP. LPS-primed (1 μg/ml, 3 h) THP-1 cells were stimulated with ATP (5 mM) for another 30 min and then were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.

H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag-NLRP3, HA-CCDC50 or empty vector for 24 h, and then treated with DMSO or CQ for 6 h. After treatment, the cells were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.

I Co-IP (with anti-HA) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h, and treated with or without CQ for 6 h.

J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM.

K Confocal microscopy analysis of ASC speck formation in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM.

Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.
Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy (2014)
figf1

(a–f) Atg8 was co-incubated with Atg19 or Atg34 (wild type and mutants) at concentrations of 40 μM and 800 μM, respectively, and run on a size exclusion column. Aliquots of individual fractions were run on SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels and Coomassie stained. The white box indicates the N-terminal domain, the light grey box the coiled-coil domain, the dark grey box the Ams1-binding domain and the purple box the C-terminal domain. (g–i) Atg8 was co-incubated with the indicated Atg19 domains at 40 μM and run on a size exclusion column. Individual elution profiles are shown above; Coomassie stained SDS–PAGE gels below. Columns: (a–f,i) S200 3.2/30; (g,h) S75 3.2/30. M: size marker. All experiments shown have been carried out twice.
Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy (2014)
figf2

(a) Anti-Atg8 western blot of the input and pellet fractions of a pulldown experiment using GST–Atg19 as bait and Atg8 as prey. The black vertical line indicates that unrelated samples were run on the same gel but in between the two samples on the left and the one on the right (Supplementary Fig. 7). (b) Coomassie stained gels of pulldown experiments using the GST–prApe1 propeptide as bait to pull down Atg19, the Atg19–Atg34 chimaera and Atg34, respectively. The Atg19–Atg34 chimaera contained residues 1–361 of Atg19 and residues 348–412 of Atg34. (c) Yeast two-hybrid (Y2H) assay testing for the interaction of Atg11 with Atg19, the Atg19–Atg34 chimaera and Atg34, respectively. (d) Anti-Atg8 western blot using the indicated GST fusion proteins as bait to pull down Atg8. The pulldowns were conducted in the presence or absence of the prApe1 propeptide. See Supplementary Fig. 1 for input gel. All experiments shown have been carried out twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.
Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy (2014)
figf3

(a,b) Anti-Atg8 western blots using the indicated GST fusion proteins shown on the left as bait to pull down Atg8. See Supplementary Fig. 1 for input gels. (c) Alignment of the Atg19 C-terminus lacking the LIR motif with the region around the LIR motif of human p62 (NP_003891.1). The amino acids shown in magenta and green indicate the residues mutated in the experiments shown in d–f. (d) Anti-Atg8 western blots with the indicated GST fusion proteins as bait to pull down Atg8. Quantification of the amount of Atg8 pulled down by the indicated GST fusion protein. The graph is based on three independent experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. WT, wild type. (e) Green fluorescent protein (GFP)-trap pulldown experiment using yeast cells with integrated GFP–Atg8 transformed with the indicated Myc–Atg19 constructs. (f) Pulldown experiment using the GST–prApe1 propeptide (propep.) to pull down Atg8 through the indicated Atg19 proteins. The graph is based on three experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. See Supplementary Fig. 2 for input gel. (g) Representative profile of a size exclusion chromatography run using a 10/300 S200 column. Atg8 was run at 400 μM and the Atg19 C-terminus at 40 μM. For the complex run both proteins were preincubated at the concentrations mentioned. The average number of Atg8 molecules shifted into the complex peak per Atg19 C-terminus was 4.14 (average of three experiments). The experiments shown in a,b,d,f,g have been conducted three times; the experiment shown in e has been conducted twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.
Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy (2014)
figf4

(a) Giant unilamellar vesicles (GUVs) incubated with mCherry–Atg19. The membrane was marked by incorporation of an Oregon Green labelled lipid. (b) GUV coated with GFP–Atg8 incubated with mCherry–Atg19. (c) Schematic representation showing the proteins and reaction resulting in GFP–Atg8 conjugation to the GUV shown in b. (d) GUVs containing nickel lipids incubated with GFP–Atg8–6×His and wild-type mCherry–Atg19. (e) Set-up of the experiment shown in d. (f) Quantification of membrane binding by the indicated mCherry–Atg19 proteins. The experimental set-up is shown in e. The graph is based on three experiments. Shown are the averages and s.d. of these three experiments (N = 3, on the basis of 318 GUVs for Atg19, 171 GUVs for Atg19 W412A, 166 GUVs for Atg19 F376A, F379A, W412A, 188 GUVs for the enhanced GFP (eGFP) control). Scale bars, 5 μm. Abbreviations: 3, Atg3; 5, Atg5; 7, Atg7; 8, Atg8; 12, Atg12; 16, Atg16; 19, Atg19 redundant. Experiments shown in a,b have been conducted twice and the experiment in d three times.
Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy (2014)
figf5

(a) prApe1 propeptide recruitment to Atg8-harbouring GUVs in the presence and absence of Atg19. The experimental set-up is shown on the right. (b) Atg19-dependent envelopment of prApe1-coated beads with Atg8-positive membranes. The experimental set-up is shown on the right. The membrane was labelled by incorporation of a rhodamine-labelled lipid. (c) Quantification of the percentage of GUV-associated eGFP–Ape1-coated beads showing detectable membrane bending (N = 3; 40 GUVs per condition). The experimental set-up is shown in b. The density of Atg8–6×His on the membrane was titrated by different amounts of nickel lipids in the GUV membrane. The graph is based on at least three independent experiments. Shown are the averages and s.d. (d) Quantification of the percentage of GUV-associated eGFP–Ape1-coated beads showing detectable membrane bending. The quantification is based on three independent experiments for the mutant Atg19 versions and 16 independent experiments for wild-type Atg19 and the no-Atg19 control (Atg19, N = 16, total GUVs = 382; Atg19 W412A, N = 3, total GUVs = 55; Atg19 F376A, F379A, N = 3, total 66 GUVs; Atg19 F376A, F379A, W412A, N = 3, total GUVs = 84; Atg19 F376A, F379A, P385A, E386A, W412A, N = 3, total GUVs = 60; Atg19–34, N = 4, total GUVs = 110; no Atg19, N = 16, total GUVs = 382). Shown are the averages and s.d. The experimental set-up is as shown in b; the p-values in c and d were calculated using a two-tailed Student t-test. (e) Visualization of Atg19 during the envelopment of prApe1-coated beads with Atg8-containing membranes. Note that Atg19 directly binds the prApe1-coated beads (arrows). The experimental set-up is shown on the right. Scale bars, 5 μM. Abbreviations: 8, Atg8; 19, Atg19; DIC, differential interference contrast. All experiments have been conducted at least three times.
Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane–cargo apposition during selective autophagy (2014)
figf6

(a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Rap., rapamycin; Log., (b) Representative electron micrographs of ypt7Δ, atg19Δ yeast cells grown under cytoplasm-to-vacuole targetting (Cvt) conditions expressing the indicated proteins labelled with an anti-Myc antibody. The white arrowheads indicate the isolation membrane. The dashed line indicates the circumference of the prApe1 oligomer. Gold particles, 10 nm. Scale bars, 200 nm. See Supplementary Fig. 5 for full images. (c) Quantification of electron micrographs of yeast cells expressing either Myc–Atg19 or Myc–Atg19–Atg34 co-labelled with anti-Myc (10 nm gold) and anti-prApe1 (5 nm gold). See also Supplementary Fig. 5. Three independent experiments; Myc–Atg19, N = 48, Myc–Atg19–34, N = 24. (d) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Supplementary Fig. 6 shows the expression of the Myc-tagged proteins and a quantification of the assay. (e) Y2H assay testing for the interaction of Atg11 with Atg19 and the indicated Atg19 mutants. (f) Atg19Δ yeast cells expressing the indicated Atg19 proteins and prApe1–RFP (red fluorescent protein) were labelled with the vacuolar membrane dye MDY-64. Scale bars: 2 μM. The experiments in a–d,f have been conducted three times, the experiment in e twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.