EMBO/soda-nel huggingface dataset:
Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning (2018)
Figure 4

Figure 4. Mrc1 inactivation restores CPT resistance in the absence of Tel1.

A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies. (B) Quantitative analysis of Rad53 phosphorylation was performed by calculating the ratio of band intensities for slowly-migrating bands to the total amount of protein. Plotted values are the mean values with error bars denoting SD (n = 3). Statistical analysis: Student"s t-test.; ns, not significant; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001. (C) Nuclear division determined by florescence microscopy.

D Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT at the indicated concentrations.

E Cells were distributed on YEPD plates with or without CPT at different concentrations to determine colony-forming units after 3 days at 25°C. Plotted values are the mean values with error bars denoting SD (n = 3).
Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning (2018)
Figure 5

Figure 5. The lack of Mrc1 function in DNA replication restores CPT resistance in the absence of Tel1.

A Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT at the indicated concentrations.

(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D) or stained with propidium iodide to analyze the kinetics of nuclear division (C, E).

F Exponentially growing cell cultures were serially diluted (1:10) before being spotted out onto YEPD plates with or without CPT.

G Colony-forming units on YEPD plates with or without CPT at different concentrations. Plates were incubated 3 days at 25°C. Plotted values are the mean values with error bars denoting SD (n = 3).
Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning (2018)
Figure 6

Figure 6. The lack of Tel1 allows Mre11-dependent degradation of reversed forks in CPT-treated cells.

A-E G1-arrested cell cultures were released in YEPD supplemented with CPT (50 μM). (A, D) Electron micrographs of a representative reversed fork with the four-way junction magnified in the inset (A) and a three-way replication fork exposing ssDNA at the junction (D). In (D) the black arrow indicates the ssDNA region, which is magnified in the inset. Scale bars: 200nm, 50nm in the insets. (B, C) Frequency of reversed replication forks in CPT (40 minutes, 50 μM CPT). Two independent experiments were performed with very similar results. The results of the individual biological replicates and the number of analyzed molecules are shown in Fig EV4C. (E) Graphical distribution of ssDNA length at the junction (black arrow in (D)) in forks isolated after CPT treatment (40 minutes, 50 μM CPT). Only molecules with detectable ssDNA stretches are included in the analysis. The lines show the median length of ssDNA regions at the fork in the specific set of analyzed molecules. Statistical analysis: Mann-Whitney test; ns, not significant; *, P< 0.05; **, P< 0.01; ****, P< 0.0001. The number of analyzed molecules is in brackets.

F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies.
Acquisition of chromosome instability is a mechanism to evade oncogene addiction (2020)
Figure 1

Figure 1. Non-regressed K and KM tumours show high levels of chromosomal instability. (A) Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).

(B) Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Ns: not significant; * p<0.01, **** p<0.0001 One-way ANOVA, turkey's multiple comparison test. Mean +/- SEM. Exact P values are indicated in Appendix Table S1.

(C) Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.

Data information: K primary (n=4; 74 cells), KM primary (n=5; 84 cells), K non-Regr (n=7; 219 cells), KM non-Regr (n=5; 247 cells).
Acquisition of chromosome instability is a mechanism to evade oncogene addiction (2020)
Figure 2

Figure 2. Recurrent Somatic Copy Number alterations in K and KM non-regressed tumours.

(A) Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).

(B) Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).

(C) Immunostaining of phospho-cMet in non-regressed tumours carrying or not the amplification in chromosome 6. Yellow numbers indicate the total number of cMet positive cells/total number of cells counted. Scale bar 100 µm. Numbers of the K and KM non-regressed tumours are described in Table 1.
Acquisition of chromosome instability is a mechanism to evade oncogene addiction (2020)
Figure 3

Figure 3. cMet amplification is not found in primary tumours.

(A) Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.

(B) Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow).

(C) Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm.

(D) Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.

(E) Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.
Acquisition of chromosome instability is a mechanism to evade oncogene addiction (2020)
Figure 5

Figure 5. cMet amplified non-regressed tumours respond to cMet treatment.

(A) Schematic of the experiment. K and KM animals were set on doxycycline food until mammary tumours developed. Then switched to a normal diet and non-regressed tumours collected. Single cells were then injected into Rag2-/- animals to recapitulate the non-regressed tumours followed by drug treatment or vehicle control treatment.

(B) Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control. Exact P values are indicated in Appendix Table S3.

(C) Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.

(D) Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.

(E) Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.

Data information: (D,E) One-way ANOVA, Tukey's multiple comparison; ** p<0.005; **** p<0.0001. Mean +/- SEM. At least 4 tumors per condition were analyzed and a minimum of 35 Fields of View (FOV) or 12,000 cells were counted for each condition. Exact P values are indicated in Appendix Table S3 and S4, respectively.

Expanded View Figure Legends
Inhibition of NPC1L1 disrupts adaptive responses of drug-tolerant persister cells to chemotherapy (2021)
Figure 2

Figure 2. Upregulation of NPC1L1 supports cell survival in response to cytotoxic stress in MPCs.

A, volcano plot showing expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR or MCF-7ADR. Dotted lines represent the screening criteria (|fold change| ≥ 2, P ≤ 0.05).

B, Venn diagram indicating the overlaps of expressed genes in (A).

C, volcano plot revealed 109 consistently expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR and MCF-7ADR. Dotted lines represent the screening criteria (|fold change| ≥ 2, P ≤ 0.05).

D, heat map based on scoring rank indicated consistently expressed genes identified by RNA-seq of MCs VS. MPCs in Du145TXR and MCF-7ADR.

E, qRT-PCR analysis of NPC1L1 in MCs and MPCs of Du145TXR or MCF-7ADR. Student's t test was used to analyze statistical differences. Mean with ± SD.

F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR.

G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.

H-I, colony formation assay and quantification of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents. One-way ANOVA was used to analyze statistical differences.

Data information: Results are representative of three independent experiments.
Inhibition of NPC1L1 disrupts adaptive responses of drug-tolerant persister cells to chemotherapy (2021)
Figure 5

Figure 5. NRF2-mediated transcription contributes to upregulation of NPC1L1 in MPCs.

A, immunoblotting of NRF2 and Keap1 in MCs and MPCs of Du145TXR or MCF-7ADR cells.

B, immunofluorescence analysis of NRF2 in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 hours. Scale bar, 10 μm.

C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours.

D, the profiles of predicted binding sequence of NRF2 in NPC1L1 promotor by JASPAR.

E, ChIP-qPCR analysis of NRF2 binding on the NPC1L1 proximal promoter in MCs and MPCs of Du145TXR or MCF-7ADR cells. Student's t test was used to analyze statistical differences. Mean with ± SD.

F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD.

H-I, cell viability of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD.

J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences.

Data information: Results are representative of three independent experiments.
Inhibition of NPC1L1 disrupts adaptive responses of drug-tolerant persister cells to chemotherapy (2021)
Figure 6

Figure 6. Ezetimibe synergizes with chemotherapy to eradicate MDR cancer cells and prevent recurrence in vivo.

A-C, Du145TXR-derived xenograft male nude mice received the indicated treatments. (A) Images, (B) tumor volumes and (C) weights measured at the indicated time points are shown. Scale bar, 1 cm. The red dotted line indicates cured tumors. Two-way ANOVA was used to analyze statistical differences in (B). One-way ANOVA was used to analyze statistical differences in (C). Mean with ± SD.

D-E, immunohistochemical staining of Ki67 in indicated groups. (D) Representative images and (E) relative immunohistochemical scores are shown. Scale bar, 50 μm. Student's t test was used to analyze statistical differences. Mean with ± SD.

F-G, immunohistochemical staining of cleaved-caspase 3 (CC3) in indicated groups. (F) Representative images and (G) relative immunohistochemical scores are shown. Scale bar, 50 μm. One-way ANOVA was used to analyze statistical differences. Mean with ± SD.

H-I, (H) schematic of tumor recurrence model establishment and subsequent treatment. (I) Corresponding images and isolated tumors are shown. The red dotted line represents cured tumors.

Data information: in B, C, E and G, data were obtained from 4-5 tumors per group.

Expanded View Figure Legends
Plant photoreceptors and their signaling components compete for COP1 binding via VP-peptide motifs (2019)
Figure 2

Figure 2 - High-affinity cooperative binding of activated full-length UVR8 to the COP1 WD40 domain is mediated by its UV-B-activated β-propeller core and its C-terminal VP-peptide motif.

A. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation).

B. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.

C. The domain organization of Arabidopsis UVR8. It consists of a UV-B-sensing β-propeller core (residues 12-381) and a long C-terminus containing the VP motif. Constructs and peptides used and their residue endings are indicated.

D. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.

E. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.

F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.
Plant photoreceptors and their signaling components compete for COP1 binding via VP-peptide motifs (2019)
Figure 4

Figure 4 - Many COP1 substrates and interacting photoreceptors contain VP-peptides that can bind the COP1 WD40 domain.

A. Table summarizing affinities (Kd, dissociation constant) of VP-containing peptides versus the COP1 WD40 domain (WT) and COP1Lys422Ala as determined by ITC. All values are μM (± standard deviation).

B. A sequence comparison of VP-peptide motifs that interact with the COP1 WD40 domain. The VP core is colored in red and the anchor residues in orange. The peptide sequences are numbered on a register where the V in the VP is 0.

C. (Top panel) Superposition of the VP-peptide binding modes of the HY5 (green), COL3 (orange), and STO (gray) peptides depicted in ball-and-stick representation when bound to the COP1 WD40 domain as observed in their respective x-ray crystal structures. (Bottom panel) A comparison of the HY5, COL3, STO, CRY1, and UVR8 peptides highlight the chemically diverse anchor residues (orange) and their variant positions.

D. ITC assays between the COL3 VP-peptide (left) and CRY1 VP-peptide (right) versus the COP1 WD40 and COP1Lys422Ala, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): COL3 - COP1 (2240 μM in 195 μM); COL3 - COP1Lys422Ala (1500 μM in 175 μM); CRY1 - COP1 (750 μM in 75 μM); CRY1 - COP1Lys422Ala (1500 μM in 175 μM). The inset shows the dissociation constant (Kd). The stoichiometries of binding (N) for COL3 - COP1 = 0.75 ± 0.2; COL3 - COP1Lys422Ala = 0.90 ± 0.2; CRY1 - COP1 = 0.99 ± 0.3; CRY1 - COP1Lys422Ala = 0.98 ± 0.1 (All measurements ± standard deviation).

E, F. The crystal structure of the (E) COL3 peptide and (F) CRY1 peptide bound to the COP1 WD40 domain. The peptides are depicted in ball-and-stick representation. Selected residues from COP1 are depicted in gray in stick representation.
Plant photoreceptors and their signaling components compete for COP1 binding via VP-peptide motifs (2019)
Figure 5

Figure 5 - COP1Lys422Ala shows a delayed flowering phenotype under long days and suggests that CO - CRY2 may compete for the COP1 VP-binding site.

A. Representative image of wild-type (Ws), cop1-5/Pro35S:YFP-COP1, cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala and cop1-5/Pro35S:YFP-COP1Trp467Ala transgenic lines grown for 39 days in long day conditions.

B. Representative image of individual wild-type (Ws), co-11, cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala plants grown for 39 days in long days conditions.

C. Quantification of flowering time. Means and SD are shown (n = 14).

D. Number of rosette and cauline leaves at flowering. Means and SD are shown (n = 14).

E. Immunoblot analysis of CRY2 and actin (loading control) protein levels in wild-type (Col), cry2-1, wild-type (Ws), cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala seedlings grown for 4 days in darkness.

F. ITC assays between the CO VP-peptide (left) and CRY2 VP-peptide (right) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): CO - COP1 (2240 μM in 195 μM); CO - COP1Lys422Ala (1200 μM in 120 μM); CRY2 - COP1 (750 μM in 70 μM); CRY2 - COP1Lys422Ala (3000 μM in 175 μM). The stoichiometries of binding (N) for CO - COP1 = 1.03 ± 0.00; CO - COP1Lys422Ala = 0.89 ± 0.03; CRY2 - COP1 = 1.04 ± 0.20; CRY2 - COP1Lys422Ala = 1 (fixed) (All measurements ± standard deviation).

G, H. Binding kinetics of the (G) full-length activated CRY2 and (H) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of protein injected are shown in red, with their respective heterogenous ligand binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - CRY2 (highest concentration 14 μM), CRY2NC80 (highest concentration 450 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.

Expanded View Figure Legends
A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters (2022)
Figure 1

Figure 1. Gid4 and Gid10 expression is regulated by the GID E3 ligase

Yeast two-hybrid interactions between SR-Gal4 activation domain (AD) and Gid5-DNA binding domain (DBD). Growth on -His-Ade+Aureobasidin A (AbA) is indicative of an interaction between the two test proteins. Spots represent 1:5 serial dilutions.

Strep-Tactin pull down of GIDAnt (strep-tagged at Gid8 C-terminus) probing binding of Gid1057-292 and Gid4117-362 to the complex visualized with Coomassie-stained SDS-PAGE. The experiment was performed with WT and C-terminal deletion (∆C) of the substrate receptors (∆289-292 (FEIA) and ∆359-362 (FEFA) for Gid10 and Gid4, respectively) and WT and mutant (mut, Gid5W606A/Y613A/Q649A) GIDAnt.

Tetracycline reference-based chase performed during transition from ethanol to glucose media with wildtype, ∆Gid4 and ∆Gid10 strains, and in wildtype and ∆Gid4 strains overexpressing (OE) Gid10. Points represent mean, error bars represent standard deviation (n>3 biological replicates).

Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3x-FLAG-Gid10 that were grown in SD complete at 30°C (no stress), SE complete for 19 hours (EtOH), SD complete for 1 hour following 19 hour ethanol treatment (EtOH rec.), 42°C for 1 hour (HS), SD complete supplemented with 0.5M NaCl for 1 hour (OS), or SD-N for 1 hour (-N) were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.

Lysates from yeast strains expressing endogenously tagged 3xFLAG-Gid4 or 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG (two exposures from the same gel are shown) and αPGK.

Lysates from wildtype and ∆Gid2 yeast strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.

Lysates from wildtype, Gid5W606A,Y613A,Q649A, and Gid10∆C strains expressing endogenously tagged 3xFLAG-Gid10 that were grown at 42°C for one hour, and then returned to 30°C for the indicated timepoints were run on an SDS-PAGE gel and immunoblotted with αFLAG and αPGK.