EMBO/soda-nel huggingface dataset:
USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling (2021)
Figure 1

Figure 1 - USP42 deubiquitinates ZNRF3 and inhibits Wnt/β-catenin signalling

A, Scheme of the R-spondin complex with ZNRF3 and LGR, which promotes auto-ubiquitination and clearance of ZNRF3. The deubiquitinase (DUB) that stabilises ZNRF3 remains unknown.

B, Example of a TOPflash reporter assay used to screen for DUBs regulating Wnt signalling. Cells were transfected in triplicates with the single siRNAs against the indicated DUBs or the positive control ZNRF3, and stimulated with control or Wnt3a conditioned media.

C, Scheme showing the predicted protein organization of USP42, including the catalytic cysteine of the ubiquitin-specific protease (USP) domain, and the predicted monopartite nuclear localisation signals (NLS) as displayed by NLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi).

D, Representative immunofluorescence microscopy images showing endogenous USP42 (green) in HEK293T cells transfected with the indicated siRNAs from n = 3 independent experiments. Scale bar = 10 μm.

E-H, TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. Where indicated, cells were co-transfected with the indicated siRNAs. In (E), 3 independent siRNAs or a siRNA pool against USP42 were used. In (F), Parental wt HEK293T and ZNRF3-/-/RNF43-/- HEK293T cells were used.

I,J, Ubiquitination of ZNRF3 in HEK293T cells transfected with ubiquitin-HA (Ub-HA). Cells were co-transfected with siUSP42 (I) or GFP-USP42 (J). Cells were treated for 6h with 10 μM MG132 and 5 nM Bafilomycin A1 to prevent ZNRF3-Ub degradation. Ubiquitinated ZNRF3 was isolated by immunoprecipitation (IP) under denaturing conditions and analysed in Western blots with anti-HA antibodies.

K, Representative co-immunoprecipitation experiments in HEK293T cells that were transfected with GFP-USP42 or the indicated GFP-tagged control, together with the stated Wnt signalling component (N = 3 independent experiments). IgG heavy chain is indicated with an asterisk. Dvl1 functions as a positive control that binds both ZNRF3 and Axin1.

L,M, TOPflash reporter assays in HEK293T cells stimulated with either control, Wnt3a, Wnt3a and RSPO1-4 conditioned media, or by transfection with the indicated constructs. HEK293T, Parental HEK293T and USP42-/- HEK293T cells were transfected with wt USP42, the catalytically inactive mutant USP42C120A, ZNRF3 or ZNRF3/RNF43.

Data information: Data is displayed as mean ± SD, and show one representative of n ≥ 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as *P < 0.05, **P < 0.01, ***P < 0.001, or n.s.: not significant.
USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling (2021)
Figure 2

Figure 2 - Cytoplasmic USP42 functionally interacts with the Dishevelled interacting region of ZNRF3

A, Scheme showing the GFP-USP42 and ZNRF3-HA constructs generated for this study. Note that ZNRF3∆C did not express properly, possibly due to misfolding at the ER.

B, Immunofluorescence microscopy showing HEK293T cells transfected with USP42 constructs from (A). Note that depletion of the putative NLS or the whole C-terminal domain containing the NLS leads to increased cytoplasmic expression of USP42. Representative images are shown. Scale bar = 10 μm.

C, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). Representative blots of n ≥ 3 independent experiments are shown.

D, TOPflash reporter assay in HEK293T cells upon overexpression of the USP42 constructs shown in (A) or an empty vector. Cells were stimulated with control or Wnt3a and RSPO3 conditioned medium. Data are displayed as mean ± SD and show one representative of n = 3 independent experiments with three biological replicates. Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as **P < 0.01, ***P < 0.001, or n.s.: not significant.

E,F, Co-immunoprecipitation experiments in HEK293T cells transfected with the constructs shown in (A). In (F) cells were co-transfected with DVL2 as indicated. Unspecific bands resulting from antibody cross-reaction are marked with asterisks. Representative blots of n ≥ 3 independent experiments are shown.

G, Scheme showing the proposed interacting regions of USP42 and ZNRF3.
USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling (2021)
Figure 4

Figure 4 - USP42 inhibits paracrine Wnt signalling in colorectal cancer cells

A, USP42 alterations in colorectal adenocarcinoma (Cancer Genome Atlas, 2012) (n = 524). Information was retrieved from the cBioPortal in 2017, and updated as displayed in 03/2020.

B, FACS analyses of cell surface LRP6 protein levels in HCT116 cells upon knockdown of USP42. Cells were untreated (left panel) or treated with RSPO3 for 12 h (right panel). The grey dotted line in both panels shows background signal upon staining with control IgGs. Representative panels from one out of n = 4 independent experiments are shown.

C, Western blots of lysates from HCT116 cells transfected with the indicated siRNAs. Where indicated, cells were treated for 6 h with RSPO3 conditioned medium. Representative blots from n = 3 independent experiments are shown.

D, TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data is displayed as mean ± SD, and show one representative of n = 3 independent experiments with three biological replicates.

E,F, qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments.

Data information: Statistical significance was calculated by one-way ANOVA analyses with Tukey correction and defined as **P < 0.01, ***P < 0.001, or n.s.: not significant.
USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling (2021)
Figure 5

Figure 5 - Loss of USP42 leads to Wnt-dependent proliferation and EMT

A, Scheme showing the proposed functions of ZNRF3/RNF43 and USP42 in colorectal cancer cells. Auto- and paracrine Wnt secretion is promoted by Evi (WLS) and PORCN.

B,C, Volcano plots representing the genes that conform the Intestinal Stem Cell (ISC) signature (Merlos-Suarez et al., 2011) (B, n = 45 genes, Dataset EV1F), and the EMT signature in cancer cells (Rokavec et al., 2017) (C, n = 171 genes, Dataset EV1G) upon USP42 knockdown in HCT116 cells. Significance was capped at -Log10P = 24.

D, Bright field images of HCT116 cells upon USP42 knockdown. Scale bar = 500 μm.

E-H, Immunofluorescence microscopy showing endogenous E-cadherin levels in wt and WLS (Evi) KO HCT116 cells upon transfection with siUSP42 (E), or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs(G). Scale bar = 20 μm. In (F,G), quantification of the fluorescent signal (Alexa-488) in n ≥ 4 biological replicates with n ≥ 15 cells per replicate are shown.

I-L, Spheroid formation experiments with HCT116 cells upon transfection with siUSP42 (I-K) or electroporation with eSpCas9 empty plasmid (non-edited) or eSpCas9-USP42 gRNAs (L). Scale bar = 500 μm. Where indicated, cells were treated with 5 μM IWP-2 or 20 μM LGK-974 (PORCN inhibitors). The median size was quantified for n ≥ 13 spheroids from a representative experiment repeated independently n ≥ 3 is shown.

Data information: Data is displayed as mean ± SD (F,H) or median (J-L) of the indicated biological replicates, and show one representative of n ≥ 3 independent experiments.
USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling (2021)
Figure 6

Figure 6 - Genetic ablation of Usp42 renders mouse intestinal organoids Wnt hypersensitive.

A, Representative images of mouse small intestinal organoids generated with empty (Non-edited) or USP42gRNA containing CRISPR-concatemers, and cultured for 3 days in matrigel with the indicated growth factors, or the Wnt secretion inhibitor IWP-2. EN, EGF and noggin. Note that knockout of Usp42 (Usp42 gRNAs) rendered the organoids insensitive to RSPO1 withdrawal. Scale bar = 1 mm.

B, Quantification of the number and subtype of organoids from (A).

C, qPCR analysis of Lgr5 and Axin2 expression levels in first passage mouse intestinal organoids cultured in EN + 5% RSPO1.

Data information: Data is displayed as mean ± SD of n ≥ 4 wells, and show one representative of n = 3 independent experiments.

Expanded View Figure Legends
Histidine dephosphorylation of the Gβ protein GPB-1 promotes axon regeneration in C. elegans (2022)
Figure 3

Figure 3. NDK-1 and PHIP-1 regulate axon regeneration through His-phosphorylation of the Gβ subunit GPB-1.

A PHIP-1 interaction with GPB-1 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-GPB-1, as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.

B His-phosphorylation site in Gβ. Sequence alignments of the His-phosphorylation site and flanking amino acids among human GNB1, GPB-1, and GPB-2 are shown. Identical and similar residues are highlighted with black and gray shading, respectively. The His-phosphorylation site, His-266, is indicated by an arrowhead.

C Relative axon length 24 h after laser surgery at the young adult stage. The number (n) of axons examined from three biological replicates is indicated. The black bar in each violin plot indicates the median. **P < 0.01, ***P < 0.001, as determined by the Kruskal-Wallis test and Dunn's multiple comparison test. NS, not significant.
Histidine dephosphorylation of the Gβ protein GPB-1 promotes axon regeneration in C. elegans (2022)
Figure 4

Figure 4. His-phosphorylation of GPB-1.

A His-phosphorylation of GPB-1 in animals. The phip-1(km96) mutant animals carrying the 3XFLAG::gpb-1 or 3XFLAG::gpb-1(H266F) knock-in allele were lysed. The lysates were treated with or without heating (95°C) and immunoblotted (IB) with anti-3-pHis and anti-FLAG antibodies.

B NDK-1 phosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 or HA-GPB-1(H266F) and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. Immunopurified GPB-1 was subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.

C PHIP-1 dephosphorylates GPB-1 in vitro. COS-7 cells were co-transfected with HA-GPB-1 and T7-GPC-2, and cell lysates were immunoprecipitated (IP) with anti-HA antibodies. The immunopurified HA-GPB-1 was first subjected to the in vitro kinase assay with recombinant GST-NDK-1. Phosphorylated GPB-1 was then equally aliquoted and subjected to the in vitro phosphatase assay with recombinant GST-PHIP-1 or its variants. Phosphorylated GPB-1 was detected by immunoblotting (IB) with anti-3-pHis antibodies.
Histidine dephosphorylation of the Gβ protein GPB-1 promotes axon regeneration in C. elegans (2022)
Figure 6

Figure 6. UNC-51 phosphorylates PHIP-1 at Ser-112.

A PHIP-1 interaction with UNC-51 by yeast two-hybrid assay. The reporter strain PJ69-4A was co-transformed with expression vectors encoding GAL4 DBD-PHIP-1(H45A) and GAL4 AD-UNC-51(274-856), as indicated. Yeast strains carrying the indicated plasmids were cultured on a selective plate lacking histidine and containing 5 mM 5-aminotriazole for 4 days.

B UNC-51 structure. Schematic diagrams of UNC-51 and human ULK2 are shown. The kinase domain is shown in red. The catalytic lysine and four flanking amino acids are shown. Identical and similar residues are highlighted with black and gray shading, respectively. The unc-51(ks49) mutation is a splice site mutation, which significantly reduces the unc-51 mRNA level.

C UNC-51 phosphorylates PHIP-1 at Ser-112. COS-7 cells were co-transfected with Flag-PHIP-1 (WT or mutants) and GFP-UNC-51 [WT or ∆AIKAI (KD)], and cell lysates were analyzed using Phos-tag SDS-PAGE. Total lysates were immunoblotted (IB) with antibodies, as indicated. Filled and open arrowheads indicate unmodified and phosphorylated PHIP-1, respectively. Asterisk indicates non-specific band.

D Schematic representation of the ten Ser/Thr residues and domain structure in PHIP-1.
Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures (2014)
Figure 1

The C-terminal region of ALFY interacts with GABARAP

Transfected GFP-Atg8 homologs were immunoprecipitated with µMACS from total U2OS cell extracts followed by immunoblot analysis with anti-GFP and anti-ALFY antibodies. Data are representative of two independent experiments.

An overview of the deletion mutant constructs of ALFY used for GST pull-down experiments in (C).

The indicated 35S-labelled in vitro-translated GFP-tagged constructs were incubated with GST, GST-LC3B or GST-GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro-translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments.

Recombinant ALFY (aa 29813526) was incubated with GST-Atg8 proteins conjugated to glutathione Sepharose. The pulled-down complexes were subjected to SDSPAGE and anti-ALFY and anti-GST immunoblotting. 5% of the recombinant ALFY protein used was loaded as input. Data are representative of three independent experiments.

Source data are available online for this figure.
Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures (2014)
Figure 2

Identification of the LC3-interacting region (LIR) in ALFY

Alignment of the potential LIR in human ALFY to the corresponding homolog sequences of representatives of mammals, birds, fish and insects. O indicates an aromatic residue, while ? indicates an aliphatic residue.

35S-labelled in vitro-translated GFP-ALFY (aa 29813526) wild-type and different LIR mutants were incubated with GST-GABARAP or -LC3C and binding evaluated by ARG. 10 and 2% of the in vitro-translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.

MBP, MBP-ALFY (aa 32553526) or the ALFY-LIR mutant (F3346A) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP. The pulled-down complexes were subjected to SDSPAGE and visualized by CBB staining. Data are representative of three independent experiments.

Thermodynamic parameters of ALFY-LIR peptide (aa 33413354) to Atg8 homologs. All titrations were performed at 25°C as described in Supplementary Methods. ITC data were fitted to a one-site binding model. ND, not detected.

35S-labelled in vitro-translated GFP or GFP-Bchs (aa 33263341) were incubated with GST or GST-dAtg8a and binding evaluated by ARG. 5% of the in vitro-translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.

Source data are available online for this figure.
Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures (2014)
Figure 3

Structural analysis of GABARAP-ALFY peptide complex

Overall structure of the complex formed by GABARAP in brown and the ALFY-LIR peptide in yellow with the core LC3-interacting region (LIR) motif highlighted in dark blue.

Alignment of the GABARAP-ALFY-LIR peptide complex structure with the published structure of LC3B (1UGM), using UCSF Chimera. See Supplementary Fig S2C and D for calculation of the distances between the residues highlighted as sticks in the ALFY-LIR peptide and the corresponding annotated residues in GABARAP or LC3B.

Sequence alignment of human Atg8 protein homologs. The alignment was obtained by Clustal W. Black and grey backgrounds represent degree of similarity. Closed circles indicate specific residues of GABARAP involved in the ALFY-LIR peptide interaction.

GFP-LC3B and GFP-LC3B (Q26K/H27Y/H57D) were immunoprecipitated with GFP-TRAP® from total cell lysate of stably transfected HeLa FlpIn cells, followed by SDSPAGE and immunoblotting with the indicated antibodies. Data are representative of three independent experiments.

MBP-ALFY32553526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants. The pulled-down complexes were subjected to SDSPAGE and visualized by immunoblotting with anti-MBP and anti-GABARAP antibodies. Data are representative of three independent experiments.

MBP-tagged ALFY32553526 and p62168391 or their corresponding LIR mutants (F3346A and W340A, respectively) were conjugated to amylose resin and incubated with purified GST, GABARAP, GABARAP mutant (K24Q/Y25H/D54H), LC3B or LC3B mutant (Q26K/H27Y/H57D). The bound proteins were visualized by immunoblotting with anti-GABARAP, anti-LC3 and anti-MBP antibodies. Data are representative of three independent experiments.

35S-labelled in vitro-translated GFP-ALFY (aa 29813526) wild-type and K3343A/D3344A/Y3351A mutant were incubated with GST-LC3B and GABARAP and binding evaluated by ARG. 10% of the in vitro-translated proteins used was loaded. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.

Source data are available online for this figure.
Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B‐positive structures (2014)
Figure 4

Physiological role of the interaction between ALFY and GABARAP

HeLa FlpIn GFP-GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti-ALFY antibodies. Scale bar, 10 µm.

GFP-tagged full-length ALFY wild-type or LC3-interacting region (LIR)-mutant was expressed into Alfy-deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti-LC3B or anti-GABARAP antibodies. Scale bars 10 µm.

HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti-LC3B, anti-ALFY and anti-p62 antibodies. Scale bars, 10 µm.

HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX-100-soluble and insoluble fractions. The TX-100-soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.

ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X-100 (TX-100)-soluble and insoluble fractions. The TX-100-soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.

Schematic model of ALFY-mediated selective autophagy.

Source data are available online for this figure.
Involvement of circulating factors in the transmission of paternal experiences through the germline (2020)
Figure 2

Fig. 2. Analyses of PPAR expression and PPAR activity in sperm and germ-like GC-1 cells

(a) PPARγ mRNA expression in sperm from control and MSUS mice. Control n = 15, MSUS n = 13, two-tailed Student's t-test, P = 0.029, t = 2.30, df = 26.

(b) Schematic of serum treatment of GC-1 cells. On day 1, cells are transfected with PPRE and luciferase normalization plasmids. Serum is mixed in culture medium at 10% concentration and exposed to cells on day 2. Cells are harvested 24h later (Day 3) and luminescence is measured.

(c) Relative luciferase luminescence in transfected GC-1 cells exposed to serum from control or MSUS mice. Numbers correspond to serum from individual animals applied to different cell culture wells. Control n = 17, MSUS n = 22, two-tailed Student's t-test, P = 0.042, t = 2.1, df = 37.

Data information: Data reported as mean ± s.e.m.
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 1

Figure 1. Expression of PAX6 isoforms in forebrain and retinal development

Schematic diagram of the PAX6 isoforms in human.

Western blot detection of PAX6 isoforms in human fetal forebrain and retina. The antibody used targets the C terminus of PAX6 and can detect all the 3 isoforms at different molecular weight. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform. #1, #2 are #3 are from ~8-week-old human embryos. #4 is from ~12-week-old human fetus.

qPCR quantification of overall PAX6 gene expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).

qPCR quantification of PAX6A&B isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).

qPCR of PAX6D isoform expression during human forebrain and retinal differentiation (n=3 biological replicates for each group).

Western blot detection of PAX6 isoforms in cells during forebrain and retina differentiation from hESCs. D10 indicates day 10 of differentiation. The green arrow indicates the PAX6A&B isoforms. The red arrow indicates the PAX6D isoform.

Data information: In (C-E), data are presented as mean ± SD.
PAX6D Instructs Neural Retinal Specification from Human Embryonic Stem Cell-Derived Neuroectoderm (2020)
Figure 2

Figure 2. Effect of PAX6 knockout on NR differentiation

Schematic strategy for generating PAX6 knockout in hESCs. The exon 8 is shared by all the isoforms and was replaced by the PGK-Puromycin element. The primers indicated in the panel are used to detect the gene knockout.

qPCR quantification of PAX6 expression in Wildtype and PAX6 KO during retinal differentiation (n=3 biological replicates for each group).

Western blot detection of PAX6 in Wildtype and PAX6 KO cells at day 20 of retinal differentiation.

Immunostaining of PAX6 in Wildtype and PAX6 KO cells. C'-PAX6 antibody detects PAX6A, B and D. N'-PAX6 antibody detects PAX6 A and B but not D. Scale bars: 50 µm.

qPCR quantification of retinal related genes in Wildtype and PAX6 KO cells. Samples are collected at day 6, 10, 14 and 19 after the start of differentiation.

Data information: In (B, E), data are presented as mean ± SD. Significance was assessed by unpaired Student's t‐test; **P < 0.01, ***P < 0.001.