EMBO/soda-nel huggingface dataset:
RAB6 and Dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination (2022)
Figure 4

Figure 4. Post-Golgi apical transport of Crumbs is driven by dynein

A. Immuno-staining for Crumbs3 (CRB3) and PALS1 in E15.5 embryonic cortex. Scale bar = 25µm.

B. Schematic representation of the RUSH system. CRB3 is retained in the endoplasmic reticulum until the addition of biotin, which releases it for trafficking. SBP: Streptavidin-binding protein. St: Streptavidin.

C. RUSH assay for CRB3-GFP in control (mcherry) and dynactin-inhibited radial glial cells (CC1-p150-dsRed), electroporated at E.15.5 and imaged at E16.5. Scale bar = 5µm.

D. CRB3 localization at the apical surface upon release.

E. Percentage of cells with 100% of CRB3 signal at the apical surface upon release.

Data information: (D, E) mCherry (control): N= 3 brains per timepoint (361 cells total). CC1-p150: N=3 brains per timepoint (2 brains for T0) (268 cells total). Fisher's exact test and Benjamini-Hochberg procedure, *** p ≤ 0.001. All error bars indicate SD.
RAB6 and Dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination (2022)
Figure 5

Figure 5. LIS1 knock-out leads to ectopically dividing progenitors

A. PAX6 and phospho-Histone 3 (p-H3) staining in WT and LIS1 KO E12.5 brains. Cortices were subdivided into 5 bins of equal size along the radial axis. Scale bar = 25µm.

B. Percentage of PH3+/PAX6+ cells located above the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1192 cells from N=6 brains. LIS1 KO: 589 cells from N=3 brains.

C. Phospho-Vimentin (p-VIM) staining in WT and LIS1 KO E12.5 brains. Scale bar = 25µm.

D. Percentage p-VIM+ cells dividing ectopically, away from the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1056 cells from N=6 brains. LIS1 KO: 879 cells from N=3 brains.

Data information: (B, D) Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.
RAB6 and Dynein drive post-Golgi apical transport to prevent neuronal progenitor delamination (2022)
Figure 6

Figure 6. RAB6A/B and LIS1 are required for Crumbs localization and junctional integrity

A. CRB3 and PALS1 staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.

B. Quantification of CRB3 staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=13 brains), LIS1 KO (E12.5, N=4 brains)) and RAB6A/B dKO (E15.5, N=4 brains).

C. N-Cadherin staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.

D. Quantification of N-Cadh staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=6 brains), LIS1 KO (E12.5, N=3 brains) and RAB6A/B dKO (E15.5, N=3 brains).

E. Model for post-Golgi apical transport of CRB3 to the apical surface of aRG cells.

Data information: (B, D) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.

Expanded View Figure Legends
Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes (2015)
Figure 1

Figure 1: Positions of SNPs in FMR1, FXR1, FXR2, and FMR2, forming the 8SNP model as well as FMR1 and FMR2 repeat polymorphism length distribution in the discovery sample. (A) Schematic overview of FMR1, FXR1, FXR2, FMR2 and position of the 8 selected single nucleotide polymorphisms (SNPs). Line represents introns, grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons. Gene structure plots generated using FancyGene (Rambaldi & Ciccarelli, 2009) (B) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls. (C) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls.
Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes (2015)
Figure 5

Figure 5: Pilot experiments towards first mechanistic insight. (A) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27). (B) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p). (C) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38). (D) Denoted are ∆∆G values for binding of each of the miR-181 family members to different 3’UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ∆∆G values. ∆∆G is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site (Kertesz et al, 2007).
Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling (2020)
Figure 1

Figure 1 - Human HOIP is ubiquitinated at K784 and regulates NF-κB.

A. A scheme of procedures for the UbiCRest-based assays employed to analyze HOIP-modification with ubiquitin chains. Total cell extracts from HEK293T cells transiently expressing Myc-HOIP and Myc-OTULIN C129A, a catalytic inactive mutant, subjected to GST-linear-TUBE pulldown followed by UbiCRest using recombinant deubiquitinases (vOTU, OTULIN and USP21).

B. UbiCRest assays to evaluate ubiquitin chain types on HOIP examined by immunoblotting. Immunoblotting of samples using antibodies as indicated. Ponceau S staining used for monitoring GST-liner-TUBE input. Representative data shown from three independent experiments.

C. Mass spectrometry spectra corresponding to a peptide containing HOIP-K784 with double Gly (114+K).

D. Domains of human HOIP and identified ubiquitination sites at K454, K458, K735 and K784.

E. Multiple sequence alignment of different HOIP orthologues illustrating the position K735 and K784 according to the ClustalX colour scheme. Sequences were retrieved from the NCBI protein database with the following accessions: Homo sapiens (NP_060469.4), Canis lupus (XP_005623312.1), Mus musculus (NP_919327.2), Monodelphis domestica (XP_007479924.1), Xenopus laevis (NP_001090429.1), Alligator mississippiensis (XP_006259801.1), Takifugu rubripes (XP_003968217.2) and Drosophila melanogaster (NP_723214.2).

F. Co-immunoprecipitation analysis of the HOIP K784R mutant with SHARPIN and HOIL-1L using total cell extracts of HEK293T cells transiently expressing Myc-HOIP wildtype (WT), or Myc-HOIP-K784R with HOIL-1L-HA and Flag-SHARPIN. Anti-Vinculin antibody used to monitor protein loading. Representative data shown from three independent experiments.

G. Luciferase-based NF-κB gene reporter assays using Myc -HOIP wildtype (WT), a catalytic inactive mutant C885A, ubiquitination site mutants of K454R, K458R, K735R, K784R co-transfected with HOIL-1L-HA and Flag-SHARPIN. Luciferase signal was normalized to an internal Renilla control signal.

Data information: In (G), data are presented as mean ± SD. **p≤0.01, ****p≤0.0001 (ANOVA). n=4.
Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling (2020)
Figure 2

Figure 2 - The human HOIP K784R mutant as a part of the LUBAC generates linear ubiquitin chains and ubiquitinates its substrate NEMO in vitro and in cells.

A. In vitro ubiquitination assays for the indicated times using the recombinant proteins of ubiquitin (Ub), E1, E2 (UbcH7), HOIP (WT, K784R or C885A mutant), HOIL-1L, SHARPIN and NEMO. Immunoblotting of Linear ubiquitin chains, NEMO, HOIP, HOIL-1L and SHARPIN detected by using antibodies as indicated. Representative data from three independent experiments.

B. Immunoblotting to detect ubiquitination of NEMO in HEK293T cells transiently expressing Flag-NEMO, GFP-SHARPIN, and HOIL-1L-HA with Myc-HOIP wildtype (WT), Myc-HOIP K784R or Myc-HOIP C885A. Total cell lysates in denaturing conditions subjected to SDS-PAGE followed up by immunoblotting using antibodies as indicated. Anti-Vinculin antibody to monitor loading. Representative data from three independent experiments.
Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling (2020)
Figure 3

Figure 3 -No obvious developmental defect in HoipK778R/K778R knockin mice is observed while TNF-responses are suppressed in HoipK778R/K778R cells.

A. Numbers of weaned mice of the indicated genotypes from Hoip+/K778R crosses.

B. A gross appearance image of Hoip+/+ and HoipK778R/K778R male mice at 6-week old.

C,D. Immunoblotting to detect TNF-induced degradation and phosphorylation of IκB-α, or phosphorylation of IKK in immortalized Hoip+/+ and HoipK778R/K778R MEFs treated with human TNF (20ng/ml) for the indicated times. Immunoblots of anti-Vinculin antibody and anti-α-Tubulin antibody shown for monitoring loading amount. Representative data from three independent experiments.

E. Induction of TNF-dependent NF-κB target genes, ICAM, VCAM and IκB-α in Hoip+/+ or HoipK778R/K778R immortalized MEFs determined by qRT-PCR. RNA extraction and cDNA synthesis from MEFs treated with hTNF (20ng/ml) for the indicated time subjected to examine transcripts of ICAM, VCAM and IκB-α. Normalization to β-actin. Representative data from three independent experiments, n=3.

F. TNF-dependent induction of cleavage of PARP and Caspase 3 in primary Hoip+/+ or HoipK778R/K778R MEFs determined by immunoblotting. Total cell extracts of MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for the indicated times subjected to SDS-PAGE followed by immunoblotting using antibodies as indicated. Anti-α-Tubulin antibody used to monitor loading amount. Representative data of two independent experiments.

G. TNF-dependent induction of Caspase 3 activation in immortalized Hoip+/+ or HoipK778R/K778R MEFs measured by using DEVD-AFC. MEFs treated with hTNF (100ng/ml) and CHX (1µg/ml) for 4 hours subjected to the Caspase 3 activity assays.

H. TNF-induced Caspase 8 activity in Hoip+/+ or HoipK778R/K778R immortalized MEFs treated with hTNF (100ng/ml) with or without Cycloheximide (CHX) (1µg/ml) or z-VAD (20µM). Representative data from three independent experiments, n=4.

I. TNF-receptor complex II formation in Hoip+/+ or HoipK778R/K778R immortalized MEFs. Total cell extracts of MEFs treated with hTNF (100ng/ml), CHX (1µg/ml) and zVAD (25µM) for the indicated time immunoprecipitated using an anti-FADD antibody. Recruitment of RIPK1, HOIP and SHARPIN monitored by immunoblotting. The anti-Vinculin antibody blot shown for monitoring loading amount. The black arrows indicating HOIP and SHARPIN in the IP samples and unmodified RIPK in the input samples, respectively. Representative data of two independent experiments.

Data information: In (E,G and H), data are presented as mean ± SD. **p≤0.01, ***p≤0.001, ****p≤0.0001 (ANOVA, n=4 in G).

CHX (Cycloheximide), hTNF (human Tumor Necrosis Factor), z-VAD (Z-Val-Ala-Asp fluoromethyl ketone)
Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling (2020)
Figure 4

Figure 4 - SHARPIN-deficiency leads HoipK778R/K778R mice to embryonic lethality, which is rescued by TNFR1 knockout.

A. Numbers of weaned mice of the indicated genotypes from crosses of Hoip+/K778R; Sharpin+/cpdm mice.

B. Number of the embryos of the indicated genotype at E12.5 and E13.5 from crosses of HoipK778R/K778R; Sharpin+/cpdm mice.

C. Gross appearance images of HoipK778R/K778R; Sharpin+/+, HoipK778R/K778R; Sharpin+/cpdm and HoipK778R/K778R; Sharpincpdm/cpdm embryos at E13.5. Representative pictures from 7 embryos each. Scale bars: 1 mm.

D. Gross appearance images of Hoip+/K778R;Sharpincpdm/cpdm;Tnfr1+/-and HoipK778R/+; Sharpincpdm/cpdm ; Tnfr1-/- female mice at 6 weeks of age, and a female HoipK778R/K778R; Sharpincpdm/cpdm ; Tnfr1-/- mouse at 8 weeks of age.

E. H&E staining, Keratin 14 (KRT14) and Cleaved Caspase 3 immunostaining of dorsal skin sections from mice of the indicated genotypes at 4 or 8 weeks of age. Scale bars: 50 µm.
Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling (2020)
Figure 5

Figure 5 - SHARPIN-deficiency in Hoip+/K778R heterozygous mice leads to early onset of skin inflammation accompanied with apoptosis induction.

A. Gross appearance of mice of the indicated genotypes (male mice at 4-week old). Scale bars: 10 mm.

B. Immunostaining (H&E, Keratin 14 (KRT14), Ly6G, F4/80 and Cleaved Caspase 3) of mouse dorsal skin sections of the indicated genotypes. Scale bars: 50 µm.

C-E. Measurements (Total epidermis skin thickness, Keratin layer thickness and Squamous cell layer thickness) of dorsal skin sections obtained from male mice of the indicated genotypes. Each dot on the scatter dot plot represents one focus point of the measurement. (N=20, 20, 30, 40, 40 for Hoip+/+; Sharpin+/+, HoipK778R/K778R; Sharpin+/+ , Hoip+/K778R; Sharpin+/cpdm, Hoip+/+; Sharpincpdm/cpdm, Hoip+/K778R; Sharpincpdm/cpdm, respectively.)

F. Immunoblotting to examine TNF-induced degradation and phosphorylation of IκB-α in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (20ng/ml) for the indicated times. Representative of three independent experiments.

G. Immunoblotting of TNF-induced cleavage of PARP and caspase 3 in immortalized MEFs of the indicated genotypes using total cell extracts of MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) for indicated times. Representative of three independent experiments.

H,I. TNF-dependent induction of Caspase 3 activation in immortalized MEFs of the indicated genotype measured by DEVD-AFC. MEFs treated with hTNF (100ng/ml) alone, or with CHX (1µg/ml) for 4 hours (H) or 2 hours (I) subjected to the Caspase 3 activity assays. A representative data of two independent experiments, n=4.

J. TNF-induced Caspase 8 activity in immortalized MEFs of the indicated genotype. Luminol-dependent activity of Caspase 8 in immortalized MEFs treated with hTNF (100ng/ml) with or without CHX (1µg/ml) . A representative data of three independent experiments, n=4.

Data information: In (C-E and H-J), data are presented as mean ± SD. **p≤0.01 , ***p≤0.001, ****p≤0.0001, ANOVA.

hTNF (human Tumor Necrosis Factor), CHX (Cycloheximide),
C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1 (2022)
Figure 4

Figure 4: GRH-1 depletion extends molt duration in a dose-dependent manner.

A, Quantification of the percentage of grh-1::aid animals constitutively expressing luciferase (HW2434) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated. Appendix Figure S4 shows biological replication with a different set of concentrations.

B, Boxplot showing the duration of M1, M2 and M3 of animals treated with indicated concentrations of auxin. Animals that failed to develop beyond M3 in (A) were excluded. The horizontal line represents the median, hinges extend to first and third quartiles, and the whiskers extend up to the most extreme value within 1.5*IQR (interquartile range) of the median. Significant differences relative to 0 nM auxin are indicated. P-values were determined by a non-parametric Wilcoxon rank sum test that does not assume normal distribution. ns: not significant,* p<0.05, ** p<0.01, *** p<0.001. Appendix Figure S4 shows biological replication with a different set of concentrations.

C Quantification of intermolt durations for the same animals as in (B).

Data information: See also Appendix Figures S3, S4.
C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1 (2022)
Figure 5

Figure 5: GRH-1 is required for successful completion of each molt.

A-D, Heatmaps showing trend-corrected luminescence (Lum, arbitrary units) of grh-1::aid animals constitutively expressing luciferase (HW2434). T = 0h corresponds to time of plating embryos, which subsequently hatch at different times. Arrow indicates time point when 250 μM auxin was added, i.e. prior to the first molt (A; M1), M2 (B), M3 (C) or M4 (D) larval stage. Note that for technical convenience in (A), auxin was provided at time of plating. Animals are sorted by entry into M1 (A), M2 (B), M3 (C), M4 (D), respectively.
C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1 (2022)
Figure 6

Figure 6: GRH-1 protein accumulates rhythmically before each molt

Time-lapse imaging of grh-1::gfp::3xflag animals producing endogenously GFP-tagged GRH-1 protein. Average +/- 95% confidence interval (cyan shading) are shown; gray boxes indicate average time of molts. See also Figure EV4, Appendix Figures S5.
C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1 (2022)
Figure 7

Figure 7: GRH-1 functions prior to the molt

A,B Heatmap showing trend-correct luminescence (Lum) of grh-1::aid animals (HW2434) treated with vehicle (0.25% ethanol) (A), or 250 μM auxin (B) at 29 hours after plating (white dashed line). Black intensities reflect low luminescence during lethargus (molt). Embryos of various stages were plated to obtain an asynchronously hatching population. Luminescence traces are sorted by entry into molt 2 (M2) such that traces of early hatched animals are at the bottom and those of late hatched animals are at the top.

C, Plot of phenotype onset over time of auxin application relative to entry into molt 2 (M2 entry). Dots represent individual animals from (B) sorted by time of entry into M2 and colored according to whether the last observed molt in the luminescence trace was M2 (M2 exit phenotype; green) or M3 (M3 exit phenotype; blue). A white, dashed line indicates time of auxin application; dots to the left represent animals that entered M2 before auxin application, dots to the right represent animals that entered M2 after auxin application.

D, Western blot revealing rapid GRH-1 depletion in the grh-1::aid strain (HW2434) upon addition of 250 µM auxin. A synchronized culture of animals was grown in liquid at 20°C. After 21 h (denoted t=0 h in the figure), the culture was split in two and either auxin or vehicle were added as indicated. Cultures were sampled hourly and protein lysates probed by Western blotting using anti-FLAG and anti-actin antibodies as indicated.

Data information: See also Figure S6
C. elegans molting requires rhythmic accumulation of the Grainyhead/LSF transcription factor GRH-1 (2022)
Figure 8

Figure 8: GRH-1 binds to promoters of oscillating genes

A, Scatter plot of GRH-1::GFP::3xFLAG ChIP-seq peak enrichments across two biological replicates, using an anti-GFP antibody to immunoprecipitate the endogenously tagged protein from L2 stage larval lysates. Peaks assigned to genes encoding rhythmically expressed transcription factors are plotted in red, all others in blue. Peaks assigned to candidate core clock genes identified in this study are labeled.

B, Consensus DNA binding motifs of Grainyhead family proteins. Cel_GRH-1 is the top scoring motif of length 12 observed in the GRH-1 ChIP-seq data. Cel_GRH-1 in vitro was re-built using binding site sequences provided in (Venkatesan et al., 2003); hsa_GRHL1 & 2 were extracted from JASPAR 2018 (https://jaspar2018.genereg.net), and Dme_Grainyhead was extracted from FlyFactorSurvey (https://mccb.umassmed.edu/ffs/). [RC] denotes motifs that have been automatically reverse-complemented for better alignment with other motifs.

C, Smoothed rose plot showing peak phase distributions of putative direct GRH-1 targets among oscillating genes. Genes are defined as GRH-1 targets if they contain a GRH-1 ChIP-seq peak with a fold enrichment over input at or exceeding 80 % of all peaks that additionally overlaps a putative binding site scoring above the HOMER-derived threshold (Heinz et al., 2010) (Appendix Figure S7). Figure EV5 shows phase distributions for enrichment cut-offs of various stringencies. Approximate time of molt and the peak phase of grh-1 mRNA accumulation are indicated by an arc and a dot, respectively.

Extended View Figure Legends
High affinity interactions and signal transduction between Aβ oligomers and TREM2 (2018)
Figure 1

Figure 1. Soluble TREM2 and soluble TREML1-Fc bind Aβ42 fibrils.

A- Media from soluble TREM2-Fc transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original media samples. Top right panel shows the presence of soluble TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10.

B- RIPA lysate from TREM2 transfected HEK293T cells incubated with Aβ42 fibrils. Top left panel shows original RIPA lysate samples and top right panel shows the presence of TREM2 associated with Aβ42 pellet. The bottom two panels represent the same samples as the top, but probed with 6E10.

C- Media from soluble TREM2-Fc and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Left panels show original media samples. Right panels show the purified soluble TREM2-Fc (bottom) and the presence of APOE3 with the purified soluble TREM2-Fc (top).

D- Media from soluble TREM-Fc family member transfected HEK293T cells incubated with Aβ42 fibrils. Top panel shows original media sample and the presence of TREM family members associated with Aβ42 pellet. Bottom panel shows original media and Aβ42 pellet probed with 6E10.

E- Media from soluble TREM-Fc family members and APOE3 co transfected HEK293T cells incubated with agarose beads anti-Human IgG Fc. Top panel shows original media sample and the presence of soluble TREM family members associated with APOE3. Bottom panel shows original media and purified soluble TREM-Fc family members probed with anti-V5.

Western blot in schematic A, B and C had been cropped to facilitate the reading and originals with the supernatant fractions are available in Fig EV1. All experiments were replicated 3 independent times.
High affinity interactions and signal transduction between Aβ oligomers and TREM2 (2018)
Figure 2

Figure 2. Binding affinity of soluble TREM-Fc family members and AD variants with Aβ42 oligomers, Aβ42 monomers, Aβ40 monomers, APOE3 and APOE4.

BioLayer Interferometry experiments were performed to measure ligand binding affinity with soluble TREM-Fc family members and mouse soluble TREM-Fc family members.

A- Schematic representation of a full-length experiment.

B- Schematic representation of Aβ42 oligomers (right panel) and APOE4 (left panel) binding with soluble TREM2-Fc at various concentrations. Curves were fit by setting buffer control (no ligand) to y=0.

C- Calculated Kon, Kdis and KD values for Aβ42 oligomers or Aβ42 monomers, Aβ40 monomers, APOE3 or APOE4 association/dissociation with soluble TREM2-Fc and AD variants. Kinetic constants were calculated using five different concentrations of ligand and the entire experiment (including new ligand preparations and purifications of soluble TREM-Fc) was repeated twice (# p < 0.05, ANOVA, Tukey's Multiple Comparison Test, see Appendix table S1 for p value).

D- Calculated kon, Kdis and KD value for Aβ42 oligomers association/dissociation with soluble human vs. mouse TREM-Fc family members. Kinetic constants were calculated using five different concentrations of ligand and the entire experiment (including new ligand preparations and purifications of soluble TREM-Fc) was repeated twice

Abbreviation: Kon; constant of association, kdis; constant of dissociation, KD; equilibrium constant of dissociation.