EMBO/soda-nel huggingface dataset:
A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis (2020)
Figure 2

Figure 2: Impact of decreasing procaspase-8 on DISC assembly and caspase activation. (A) Western blot analysis of FADD, FLIP and Caspase 8 at the TRAIL-R2 DISC in HCT116 cells treated with escalating doses (0-30nM) of caspase-8 siRNA for 48 hours. Samples were incubated with anti-TRAIL-R2 (AMG655) beads (4x) for 90 minutes prior to collection. A scrambled control siRNA (SC) was transfected (30nM) for comparison. (B) Quantification of Caspase 8 (p55, p43/41 and p26/24) and FLIP (FLIP(L), FLIP(S) and p43-FLIP) at the TRAIL-R2 DISC by densitometry and normalised to known protein standards (see Fig EV1). (C) Ratio of Caspase-8:FLIP calculated from values presented in (B). (D) Caspase-8 (IETDase) and -3/7 (DEVDase) activity assay in the unbound soluble fraction from panel (A). (E) Western blot analysis of FLIP, Caspase 8, and FADD recruitment to the TRAIL-R2 DISC in PC3 cells stably expressing an empty vector (EV) or flag tagged FLIP L (FL) treated with 2.5µM MS-275 for 48 hours, followed by a 90 minute DISC IP. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L). (F) Table of the relative ratios of Caspase-8 (p55, p41/43 and p24/26) and FLIP (endogenous and Flag-tagged) at the TRAIL-R2 DISC in MS-275 treated and untreated cells after a 90 minute DISC IP. (G) Caspase-8 (IETDase) activity assays of the unbound soluble fraction from (E).
A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis (2020)
Figure 3

Figure 3: Although FLIP can interact directly with FADD in a caspase-8-independent manner, its DISC recruitment is highly caspase-8-dependent. (A) Western Blot analysis of Caspase 8, Caspase 10, FLIP and FADD recruitment to the TRAIL-R2 DISC in procaspase 8 (WT) or procaspase 8 deficient (Null) A549 cells incubated with AMG655 beads (4x) for either 30, 60 or 180 minutes. Unbound fractions shown in Fig EV6A. (B) Western blot analysis of the recruitment of FLIP, Caspase 8, Caspase 10 and FADD to the TRAIL-R2 DISC in HCT116 Parental, caspase 8 null (CASP8), caspase 10 null (CASP10) or CASP 8/10 cells treated with 4x AMG655 conjugated beads for 90 minutes. Unbound fractions shown in Fig EV6B. * modified form of FLIP, potentially mono-ubiquitinated p43-FLIP(L). (C) AlphaScreen® assessment of the interaction of recombinant FLIP DED1/2 and recombinant FADD DED. (D) AlphaScreen® assessment of the impact on the FLIP-FADD interaction of unstapled and stapled peptides corresponding to the FADD α1 helix. (E) NanoBiT® assay to quantify FLIP/FADD interactions in a U2OS cell line stably expressing the NanoBiT® constructs, FLIP (LgBiT) and FADD (smBiT) following 48 hour silencing of FLIP, FADD, or caspase 8. Western blot analysis was used to confirm successful knockdown of target proteins by siRNA. (F) NanoBiT® assay of HCT116 and U20S wild type (WT) and procaspase 8 null (Null) cells transiently co-transfected with FLIP (LgBiT) and FADD (SmBiT) NanoBiT® constructs for 24 hours. Construct expression was analysed by Western blotting.
A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis (2020)
Figure 4

Figure 4: FADD recruitment to the TRAIL-R2 DISC is impaired in the absence of procaspase-8. (A) Western blot analysis of FLIP, Caspase 8 and FADD recruitment to the TRAIL-R2 DISC in U20S parental cells (EV) and 3 independent caspase 8 null clones (#1, #2, #3) after incubation with AMG655 conjugated beads for 30, 60 or 180 minutes. (B) Western blot analysis of FLIP, Caspase 8 and FADD in the soluble unbound fraction from panel (A). (C) Quantification of FADD recruitment to the TRAIL-R2 DISCs from (A); FADD levels were normalised to TRAIL-R2 levels in the pull-downs. Densitometry was performed using ImageJ®. (D) The structure of FADD was previously published [20]. Here, we present the Connolly (solvent-excluded) surface, with the positions of the H9 and F25 residues highlighted. The accompanying chevron is a short-hand representation of the 6 α-helices of FADD's DED. (E) Western blot analysis of FADD recruitment to the TRAIL-R2 DISC in HCT116 cells transiently transfected with an empty vector (EV), Flag-tagged FADD (WT), or Flag tagged FADD with point mutations at either Phe25 (F25A) or His9 (H9G). Western blotting analysis of the soluble unbound fraction was used to monitor transfection efficiency. (F) NanoBiT® assay of U20S cells transiently co-transfected with caspase 8 (LgBiT) and either WT FADD smBiT or FADD (smBiT) constructs with a point mutation at either phenylalanine 25 (F25A) or histidine 9 (H9G) for 48 hours. Western blot analysis was used to assess the expression level of each construct.
A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis (2020)
Figure 5

Figure 5: Impact of FLIP deletion on DISC assembly and caspase activation. (A) Western blot analysis of FLIP, caspase 8 and FADD recruitment to the TRAIL-R2 DISC in parental (WT) and CFLAR deficient (KO) HAP-1 cells treated with AMG655 conjugated beads for either 45 minutes (45'), 90 minutes (90'), 180 minutes (180'), 3 hours (3h), 6 hours (6h) or 9 hours (9h). (B) Caspase activity assays of the unbound soluble fraction from panel (A). (C) Caspase-8 (i) and -3/7 (ii) activity assays and high content microscopy assessment of cell death (iii) in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 6 hours. (D) Caspase-8 (i) and -3/7 (ii) activity assays in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with escalating doses of isoleucine zipper TRAIL (IZ-TRAIL) for 24 hours. (iii) Flow cytometry assessment of cell death induction in Parental (PAR) and FLIP knockout (CFLAR CRISPR) cells treated with IZ-TRAIL for 24h. (E) Western Blot analysis of Parental (WT) and FLIP knockout (KO) HAP-1 cells treated with 10ng/mL IZ-TRAIL for 0, 3, 6 or 24 hours. (F) Cell viability assay of Parental (PAR) and FLIP knockout (CFLAR CRISPR cells) treated with an escalating dose (0-1000ng/mL) of IZ-TRAIL for 72 hours.
A revised model of TRAIL-R2 DISC assembly explains how FLIP(L) can inhibit or promote apoptosis (2020)
Figure 6

Figure 6. Proposal of a novel DISC assembly model. (A) Schematic representation of a model for DISC structure consistent with the experimental observations in this paper. Key: green chevrons: FADD DED; orange chevrons: FLIP DED1/2; purple chevrons: caspase-8 DED1/2; black triangle: trimeric TRAIL-R2. (B) NanoBiT® assay performed on TRAIL-R2 DISC IPs (90') carried out in U20S Caspase-8 null cells co-transfected with wild-type or F114A FLIP-LgBiT fusion proteins and wild-type or F122A caspase-8-SmBiT fusion proteins. (C) Docking Connolly surface models of interactions between the DEDs of FADD (green), FLIP (orange) and caspase-8 (purple). The structures of the FADD, FLIP and caspase-8 DEDs was previously published [20]. (D) Potential DISC structure linking two receptor trimers via DED chains. Putative caspase domain interactions are depicted. (E) Western blot analysis of size exclusion chromatography experiments in which HCT116 cells transfected with Flag-tagged wild-type (WT) FLIP(S) were treated with 100ng/mL IZ-TRAIL for 3h prior to cell lysis and separation of low and high MW complexes using a size exclusion column. The fractions eluted at 2MDa and >2MDa are presented.

Expanded View Fig Legends
High-resolution analysis of the conformational transition of pro-apoptotic Bak at the lipid membrane (2021)
Figure 1

Figure 1 - Functional and biophysical characterization of BakΔTM bound to liposomes. All liposome permeabilization assays and CD measurements were performed with C-terminally His-tagged BakΔTM and BclxLΔTM attached to Ni2+-lipid doped MOM-like liposomes.

(a-d) Liposome permeabilization assays detecting the release of a fluorescence dye from liposomes. (a) Pore formation of 50 nM BakΔTM (red) is activated by 20 nM cBid (blue). 20 nM cBid alone (orange) shows no liposome permeabilization. Addition of 25 nM BclxLΔTM (green) inhibits pore-formation. (b) Increasing amounts of cBid are titrated to 50 nM BakΔTM in the absence (blue) and presence (green) of 25 nM BclxLΔTM. (c,d) BakΔTM becomes autoactive at increasing concentrations. The concentrations are color coded as indicated in (d). (c) The averaged kinetics for increasing BakΔTM concentrations. Bar diagrams in (b) and (d) represent data from the kinetic experiments averaged between 9000 -10000 s. The standard deviation was calculated from three measurements.

(e) Far-UV-CD spectra of BakΔTM +/- liposomes and the activators cBid or Puma-BH3 peptide as indicated.

(f) CD-detected thermal melting experiments of BakΔTM (black) with liposomes (red) and Puma-BH3 (blue).
High-resolution analysis of the conformational transition of pro-apoptotic Bak at the lipid membrane (2021)
Figure 6

Figure 6 - The structural state of Bak helix 1 triggers pore formation.

Design of Bak variants with reduced pore-forming activity. Mutated amino acids are highlighted in red.

(a) Bak∆TM_1 (green frame): R156Q stabilizes the interaction between the latch domain (α6-8) and helix 1 (gold).

(b) Bak∆TM_2 (blue frame): Combines R156Q and I80E, N86E, which target the interaction between the BH3 region and helix 1.

(c, d) Liposome permeabilization assay comparing the variants in (a, green) and (b, blue) to wild-type BakΔTM (black). (c) Comparison of 50 nM of wild-type BakΔTM (black) to the two variants after activation by 25 nM cBid.

(d) Bar diagram comparing the behavior of the BakΔTM variants alone and in the presence of 25 nM cBid and 25 nM BclxLΔTM. The data derived from the liposome permeabilization kinetics shown in panel (c) is averaged for the time interval of 9000-10000 s and the standard deviations were derived from three technical replicates.

(e) Model of the structural changes of Bak during pore formation. Stage 1 depicts the resting state on the OMM surface. The linker between the TMH and soluble domain is most likely flexible. Stage 2 shows the effect induced by the binding of a BH3-only protein to the Bak BH3 binding groove, where α3 and α4 are pushed apart, most likely weakening the interaction of α2 and α3 with α1, facilitating its detachment. Thereby, α1 unfolds and most likely binds to the membrane (stage 3). This enables the detachment of the core and latch domains, homodimerization (stage 4) and ultimately pore formation. Possibly, the unfolding of α1 and the detachment of the latch domain (α6-α8) might happen simultaneously. Since no structural information is available for the final Bak pore, only the dimeric form is depicted here. A membrane surface-bound position of the latch domain is suggested by a recent crystal structure (Birkinshaw et al, 2021).

Expanded View Figure Legends
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 1

Figure 1. Omomyc inhibits GSC proliferation, self-renewal, and migration while promoting differentiation.

A Scheme of the lentiviral vector with inducible FlagOmomyc (top) and immunoblotting (bottom) of FlagOmomyc and actin loading control upon Dox treatment of BT168FO cells for 0-48 hours.

B Proliferation curves of BT168FO and BT168GFP cells upon Dox treatment for 0-96 h (n = 3; mean ± SD). Viable cells were counted using a haemocytometer.

C-D Self-renewal assay upon Dox treatment. C: histograms showing the percentage of cells capable of re-forming a neurosphere seven days after dissociation (n = 3; mean ± SD). D: representative micrographs of BT168FO cell neurospheres.

E qRT-PCR of proliferation, stem cell and differentiation markers (CCND1, PTEN, SOX2, NOTCH1, NESTIN, MYC) in BT168FO cells after 48 h Dox treatment, compared to uninduced cells (n = 3; mean ± SD). Expression levels in non-induced cells were set as 1.

F. Transwell migration assay of BT168FO cells after 3 days with or without Dox (n = 3; mean ± SD). 10 fields were counted per assay.

G. Immunofluorescence analyses of GSC differentiation. To stimulate differentiation BT168FO were grown as monolayer in the presence of serum and treated with doxycycline for up to 7 days. The upper panel displays immunofluorescence images of NESTIN, GFAP, ß-tubulin III, SOX2, and FlagOmomyc expression. FlagOmomyc blunted SOX2 expression and decreased GFAP and NESTIN protein levels, while inducing the onset of ß-tubulin III. The lower panel shows the percentage of positive cells for each cell marker evaluated (n = 4; mean ± SD). 16 fields for each assay were examined; scale bar = 100 μm.
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 2

Figure 2. Reduced migration and vascularization, and increased differentiation in xenografts of Omomyc expressing GSCs.

A-H Immunostaining of representative xenografts derived from naïve (control) and Omomyc expressing BT168FO cells. A-D) Flag, Ki67, CD31, and DCX (doublecortin) immunostaining of adjacent sections in control and Omomyc xenografts. E-G) Differentiation phenotype investigated by GFAP, ß-tubulin III, and OLIG2 staining. H) Immunostaining for anti-human nuclei. Representative images are shown. Scale bar = 100μm.
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 3

Figure 3. Loss of MYC and appearance of Omomyc ChIP-seq signals around TSSs of MYC promoter-target genes.

A-B Seqminer heatmaps of MYC and Omomyc levels around TSSs of all MYC promoter-target genes in BT168FO (A) and U87FO (B) cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in units of tags per 50bp. The plots adjacent to each heatmap depict MYC and Omomyc binding patterns at genes in the two gene clusters denoted by arrows, in cells treated or not with Dox.
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 4

Figure 4. Transcription at some MYC target promoters is modulated by Omomyc.

A Heatmap of RNA Pol II levels around TSSs of MYC promoter-target genes in BT168FO cells, treated or not with Dox for 24 h. TSS regions are ranked by decreasing MYC occupancy in untreated cells. Each row shows the ± 5 kb region centred on TSSs. Colour scaled intensities are in tags/50bp.

B Tracks of Pol II binding signals (ChIP-seq) at NCL, miR-17-92, OLIG2, HDAC1, and DUSP10 genes in BT168FO cells treated or not with Dox for 24 h. The y-axis shows Pol II binding signals as tags/500bp/million reads. The x-axis displays genomic positions; introns as lines, exons as boxes. Arrowheads denote direction of transcription. Blue and green bars denote the presence of MYC or Omomyc peaks. Grey boxes are TSS regions.

C Transcript levels (FPKM from RNA-seq data) in BT168FO cells of the genes shown in B. Data are means of 3 independent biological replicates.

D Ratio between fold enrichments (specific antibody / IgG signal) derived from qChIPs of Pol II-Ser2p and Pol II-Ser5p (n = 3; mean ± SD).

E RNA Polymerase II traveling ratio distribution from the Pol II ChIP-seqs, in the presence or absence of Dox.
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 5

Figure 5. Omomyc-dependent gene expression changes.

A Promoter occupancy by MYC correlates with transcript levels in cells untreated with doxycycline (black line). In doxycycline treated cells promoter occupancy by MYC is lower, and no longer increases together with transcript levels. The scatter plot displays MYC ChIP-seq reads at promoters (-1000, +100 regions with respect to TSS) in BT168FO cells - untreated (black) or upon 48 h Dox treatment (blue) - versus transcript levels (FPKM from RNA-seq data).

B Distribution of transcript level differences (Δ FPKM) of single genes classified as promoter-targets (green), intragenic-targets (blue), intergenic-targets (red) in Dox treated (24 h) versus untreated cells, plotted against expression in untreated cells (n=3). Δ FPKM = FPKM_24h Dox – FPKM_no Dox.

C The table first row shows the number of genes significantly up or down regulated at 24 h of Dox treatment, subdivided in promoter-, intragenic-, intergenic-targets (± 50 kbp from TSS), and the rest. The following rows depict – for each gene class – mean (average) values of FPKM in cells treated or not with Dox for 24 h and the corresponding Δ FPKM and Fold change (Fc). Data are from 3 independent biological replicates.

D Transcript level distribution of MYC promoter-targets significantly down (red dots) or up (green dots) modulated in cells treated with Dox for 24 h vs. untreated cells. Transcript levels, expressed as FPKM, represent averages of three independent experiments.
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 6

Figure 6. Omomyc resets gene expression programs of glioblastoma stem-like cells.

A RNA-seq expression values of select genes in BT168FO GSCs along a 48 h time course of Omomyc induction (n = 3 independent biological replicates). The block on the left represents DUSP (dual specificity phosphatase) family genes. The middle block contains transcription factors (TFs): the upper thirteen (ASCL1-SALL1) are GSC-specific and the remaining ones are oncogenic. The right block contains genes, including well-known MYC targets, with functions in proliferation, neurodifferentiation, and gliomagenesis. The first column of each block represents the average expression (log2 FPKM) in untreated cells (0 h) in the colour scale illustrated by the lower bar: violet indicates low and blue high expression. The other columns depict relative expression versus untreated cells (average log2 FC) at different times (8-48h) of Dox treatment, according to the scale shown by the upper bar: red indicating low and green high expression. The table in the upper left summarizes the GSEA score of the set 19 GSC-specific transcription factors [40], showing a significant downregulation by Omomyc.

B Enrichment plots obtained by GSEA (Gene Set Enrichment Analysis) of RNA-seq data from BT168FO cells. Left: genes downregulated by Omomyc in BT168FO identify the set of genes targeted by the GSC core TFs [40]. Middle: Omomyc downregulated genes identify a gene set upregulated by MYC in cancer cells [66]. Right: genes upregulated by Omomyc identify genes downregulated in small cell lung cancers carrying MYC amplification [2]. .

C The tables show Enrichment Analyses of various examples of MYC-regulated gene sets present in MSigDB (http://www.broadinstitute.org/gsea/msigdb) and of the gene set targeted by the GSC core TFs [40], versus genes repressed (left table) or induced (right table) by Omomyc in the RNA-seq dataset. NES: Normalized Enrichment Score; FDR: False Discovery Rate.
Resetting cancer stem cell regulatory nodes upon MYC inhibition (2016)
Figure 7

Figure 7. Omomyc-modulated microRNAs in glioblastoma stem-like cells.

A List of KEGG pathways significantly enriched in the genes targeted by miRs up- or down-regulated upon Omomyc induction (Table 2). The number of affected genes and the number of miRs targeting them are indicated for each pathway together with merged p-values computed as in [48], denoting the probability that the examined pathway is not enriched with gene targets of at least one selected microRNA.

B Immunoblots of EGFR, ZEB1 and GAPDH loading control expression in BT168FO and BT275FO after 2 days with (+) or without (-) Dox. Representative images are shown.

C RT-qPCR expression analysis of selected miRs in U87FO cells grown 48 h in the presence (+) or absence (-) of Dox (n = 3; mean ± SD). Expression levels were normalized using U6 RNA as control. Expression levels in non-induced cells were set as 1.

D EGFR, ZEB1, and GAPDH immunoblots in the same cells as in panel C. Representative images are shown.

E EGFR, ZEB1, and GAPDH immunoblots in uninduced U87FO cells (-) and in cells transfected with LNA oligos targeting miR-200a-200b-429 (LNA 200) and miR-146b (LNA 146b), or with a non-targeting LNA (C), and induced with Dox for 48 h (+Dox). Representative images are shown.

F MTS cell proliferation assay of uninduced or Dox induced U87FO cells, transfected with either a non-targeting LNA (LNA C), or LNA oligos targeting the miR-200a-200b-429 cluster (LNA 200), miR-146b (LNA 146b), or both (LNAmix) and analysed at 24, 48 and 72h (n = 3; mean ± SD).

G Migration assay of U87FO cells transfected with non-targeting control LNA oligos (LNA C) or with LNA oligos targeting miR-200a-200b-429 (LNA 200); cells were either untreated (-) or treated with Dox for 24 h (+Dox); (n = 3; mean ± SD).
SCFFbxw5 targets kinesin-13 proteins to facilitate ciliogenesis (2021)
Figure 1

Figure 1: Comprehensive substrate screening on protein microarrays (protoarray®) identifies 161 candidate substrates of SCFFbxw5

A. Coomassie-stained SDS-polyacrylamide gel electrophoresis (PAGE) of different proteins used in the assay. Cul1 was obtained using a split and co-express system (in which the C- and N-terminal domains are co-expressed as individual proteins and therefore run as two distinct bands at around 50 kDa) followed by in vitro neddylation. SCFFbxw5 complexes were prepared by mixing equimolar amounts of Fbxw5/Skp1 and Cul1~Nedd8/Rbx1 sub-complexes. Numbers left of the gel indicate molecular weight marker in kilo-Dalton (kDa, same accounts for all following gels and blots).

B. Workflow and example of a sub-array of the protoarray screen. Protein microarrays containing more than 9000 human proteins spotted in duplicates were incubated with 15 µM FITC-labelled ubiquitin (Ub), 100 nM E1 (Uba1-His6), E2s (0.5 µM each of UbcH5b and Cdc34) and 0.15 µM SCFFbxw5 for 1.5 hours at 37°C. Right panel shows overlay of a selected sub-array probed with (red) or without (green) SCFFbxw5 complexes. White box marks the established substrate Sas6.

C. Comparison of protoarray signal intensities of candidate substrates probed with or without SCFFbxw5 complexes. Sas6, Sec23b and MCAK are marked as red dots (other published substrates (e.g. Ask1, Eps8) were not among the 9000 proteins spotted on the array). Note that axes have different scaling.

D. Cellular components GO analysis of identified substrates using DAVID webtool with protoarray proteins as background.

E. Validation of individual targets by manually curated in vitro ubiquitylation experiments. HA-tagged (hemagglutinin) candidate proteins were purified from Hek293T cells via anti-HA immunoprecipitation (IP) followed by HA-peptide elution. Candidates were incubated with 20 µM His6-Ubiquitin, 170 nM E1, E2s (0.5 µM each of UbcH5b and Cdc34) and 5 mM ATP in the presence or absence of 0.1 µM SCFFbxw5 for 2 hours at 37°C. Substrates were detected via SDS-PAGE followed by western blotting using anti-HA antibodies for detection.

Data information: Source data are presented in Dataset EV1.