EMBO/soda-nel huggingface dataset:
E2F1 proteolysis via SCF-Cyclin F underlies synthetic lethality between Cyclin F loss and Chk1 inhibition (2019)
Figure 4

Figure 4. E2F1 mediates DNA replication catastrophe in cyclin F K/O cells treated with Chk1 inhibitors

Percentage (%) of γ-H2AX positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of γ-H2Ax positive cells were plotted as Mean % ± SD.

Percentage (%) of hyper-RPA positive cells in HeLa and CCNF K/O after transfection of the indicated siRNAs treated with Chk1i for 20 hours. Percent of hyper RPA positive cells were plotted as Mean % ± SD.

Percentage (%) of γ-H2AX positive cells in S phase cells by DAPI content after Chk1i (LY2603618) related to experiments presented in Figure 3A.

Relative γ-H2AX intensity in cells transfected with either Empty Vector (EV) or HA-E2F1 treated with Chk1i for 20 hours. γ-H2AX fluorescence in HA positive cells was normalized to the empty vector transfected cells and compared to cells not expressing HA. Related to Expanded View Figure 3F.

HeLa and CCNF K/O cells transfected with the indicated siRNA and treated with 1μM Chk1i (LY2603618) for 20 hours were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. TFIIH served as a loading control.

Data information: (A-D) Data are presented as mean ± SD, with at least 3 independent experiments. p-values (* p<0.05, ** p<0.005, *** p<0.0005) were calculated by paired and two-tailed t test.
E2F1 proteolysis via SCF-Cyclin F underlies synthetic lethality between Cyclin F loss and Chk1 inhibition (2019)
Figure 5

Figure 5. E2F1 is a ubiquitylation substrate of SCFcyclin F degraded at the G2M transition and after checkpoint inhibitors

HeLa and CCNF K/O cells were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. Ponceau S staining was used as a loading control.

U-2-OS cells transfected with non-targeting siRNA (siNC) or with an siRNA targeting Cyc F were lysed using SDS. Whole cell lysates were analyzed by immunoblotting 48h after transfection. H3 was used as a loading control.

Heatmap showing gene set variance analysis (GSVA) score for gene sets related to E2F targets (PID_E2F_PATHWAY, ISHIDA_E2F_TARGETS and HALLMARK_E2F_TARGETS).

HEK293T cells transfected with empty vector (EV), FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were treated with MLN4924 for 4 hours and harvested for immunoprecipitation using FLAG antibodies. Immunoprecipitates were immunoblotted as indicated.

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 in the presence of FLAG-cyclin F WT, FLAG-cyclin F (∆F) and FLAG-cyclin F M309A were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.

HeLa and CCNF K/O cells treated with 10 μM Chk1i (LY2603618) for the indicated time points (h=hours) were harvested and lysed using SDS. Indicated protein were resolved by SDS page and detected by WB. H2B served as a loading control.

HEK293 cells transfected with empty vector (EV) or HA-E2F1 were treated with Chk1i for 20 hours and harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.

Quantification of immunoprecipitation results of Figure 5G from three independent experiments. Data from 3 independent experiments were plotted with Mean % ± SD. *< 0.05 p-value and calculated by two-tailed paired t-test.

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 were treated with Chk1i for 20 hours and siRNA of cyclin F were indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.
E2F1 proteolysis via SCF-Cyclin F underlies synthetic lethality between Cyclin F loss and Chk1 inhibition (2019)
Figure 6

Figure 6. A mutant of E2F1 lacking a CY motif is not ubiquitylated by cyclin F and promotes DNA replication stress and cell death after Chk1 inhibitors

HEK293T cells transfected with HA-E2F1 Wild Type (WT) and HA-E2F1 lacking a CY motif (∆RxL) harvested for immunoprecipitation using HA antibodies. Immunoprecipitates were immunoblotted as indicated.

HEK293T cells were cotransfected with MYC-tagged ubiquitin and HA-E2F1 or HA-E2F1 ∆RxL lacking the CY motif as indicated (+). HA-E2F1 was immunoprecipitated with an anti-HA resin under denaturing conditions and immunoblotted.

HeLa cells stably expressing HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Cycloheximide (CHX) at 50 μg/ml for the indicated minutes (min)

Quantification of E2F1 half-life in Figure 6C

HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter were treated with Chk1i for the indicated hours (h). Indicated protein were resolved by SDS page and detected by WB. H3 was used as a loading control.

Relative survival (%) of HeLa cells stably expressing an Empty Vector (EV), HA-E2F1 WT and HA-E2F1 ∆RxL under the control of a retroviral promoter measured using rezasurin and compared to EV treated with Chk1i at the indicated concentrations.

Data information: (D, F) Data are presented as mean ± SD, with at least 3 independent experiments. p-values (* p<0.05, ** p<0.005) were calculated by paired and two-tailed t test.

Expanded View Figure Legends
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death (2016)
Figure 1

Figure 1. GSDMDNterm induces the formation of a large plasma membrane pore

A Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post transfection and the percentage of GFP-positive cells was determined 16 h later by flow cytometry. B LDH release from HEK293T cells transfected with the pRetroX TETOne3G-eGFP plasmid only (vector) or pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD. At 24 h post transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells. C–D. PI staining of and LDH release from HEK293T cells transfected pRetroX TETOne3G-eGFP harbouring the N-terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng/ml doxycycline for 8 h and the level of PI staining (C) or LDH release (D) was determined. E–F. PI staining of and LDH release from LPS-primed primary BMDMs infected with log-phase S. typhimurium for the indicated timepoints in the presence of PEGs of the indicated molecular weight (numbers on the X-axis, 30 mM final concentration).* p<0.05 as determined by student’s t test. Graphs show mean and s.d. of quadruplicate wells. Data are representative of at least three independent expeirments.
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death (2016)
Figure 2

Figure 2. GSDMDNterm localizes to cellular membranes after inflammasome activation

A. Immunoblot analysis of cleaved GSDMD in culture supernatants and full length GSDMD, cleaved GSDMD and α-tubulin in the cell lysates of immortalized LPS-primed WT macrophages left uninfected (NS) or infected for 10–40 minutes with log phase S. typhimurium (MOI=50). B. Schematic representation of the subcellular fractionation shown in C. C. Fractionation and immunoblot analysis for GSDMD, Na+K+ ATPase, VDAC (Voltage-dependent anion channel), HDAC1 (Histone deacetylase 1) and GAPDH (Glycerinaldehyd-3-phosphat-Dehydrogenase) of WT macrophages infected for 10 and 20 minutes with log phase S. typhimurium (MOI=50). Fractionation was carried out as described in the Material and Methods section and equivalent amount of protein was loaded per lane. D. Extraction of cleaved GSDMD from isolated membranes of WT macrophages infected for 10 minutes with log phase S. typhimurium (MOI=50). Extraction was carried in variable conditions as described in the Material and Methods section.
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death (2016)
Figure 3

Figure 3. GSDMDNterm targets liposomes after caspase-1 cleavage.

A. Human GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. The protein was cleaved in a time-dependent manner into two bands of 31 kDa (GSDMDNterm) and 22 kDa (GSDMDCterm). B. Cross-linking experiment of full-length and cleaved GSDMD. GSDMD at a concentration of 2 μM was incubated at room temperature with 5 nM caspase-1. After enzymatic cleavage, GSDMDNterm is highly cross-linked by DSS, resulting in the gel-impenetrating species highlighted by the arrow. GSDMDCterm is not cross-linked. C. GSDMD at a concentration of 1 μM was incubated at room temperature with 5 nM caspase-1 and liposomes composed of 4 mM DMPC or polar lipid extract derived from E. coli. After 2 h, the lipid fraction (L) was separated from the supernatant (S) by ultracentrifugation at 4C for 1 h at 120,000g.
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death (2016)
Figure 4

Figure 4. GSDMDNterm causes liposome permeability by pore formation

A–I. Dye release time courses from liposomes as a percentage of maximal release. A. Five different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli polar lipid extract were incubated with GSDMD concentrations of (nM): 520, 260, 130, 100, 65 (colored dark to light orange). The time point of 20 min is highlighted by a vertical dashed line. B. Five different reactions, where 130 nM of GSDMD and 400μM 6-carboxifluorescein-loaded liposomes prepared with E. Coli extract polar lipid were incubated with caspase-1 concentration of (nM): 15, 8, 5, 2.5, 1.2 (colored dark to light blue). The time point of 20 min is highlighted by a vertical dashed line. C. Three different reactions, where 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes prepared with porcine brain total lipid extract were incubated with GSDMD concentrations of (nM): 520, 260,100 (colored dark to light green). D. Dye release at 20 minutes reaction as a function of GSDMD (dark orange) and caspase-1 (dark blue) concentrations. Errors bars for three independent experiments are shown. E. Two different sets of reactions, where wild type GSDMD (dark to light orange) and the mutant GSDMDI104N (dark to light blue) were independently incubated at the concentrations of 260, 130 and 65 nM with 5 nM caspase-1 and 400 μM 6-carboxifluorescein-loaded liposomes. The time point of 60 minutes is highlighted by a vertical dashed line. F. Dye release at 60 minutes of reaction as a function of GSDMD wild type (dark orange) and GSDMDI104N (dark blue) concentration. Error bars for three independent experiments are shown. G–I. Dye release from 400 μM liposomes loaded with the 6-carboxifluorescein-derivates FD-20, FD-40 and FD-150, with variable Stokes diameters, as indicated. 130 nM of GSDMD and 5 nM caspase-1 were incubated with the liposomes. For each experiment A–I, a representative from three independent experiments are shown. The corresponding raw data is shown in Appendix Figure S5.
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death (2016)
Figure 5

Figure 5. Visualization of GSDMDNterm pores in liposomes by cryo-electron microscopy

A–C. Cryo-EM micrographs of GSDMDNterm pores in E. coli extract polar lipids liposomes. The micrographs were acquired at protein/lipid molar ratios of 1/1000, 1/500 and 1/100, respectively. Black arrows indicate ring-shaped structures corresponding to oligomeric GSDMDNterm pore forms. Scale bars = 80 nm. D. Proteoliposome with protein/lipid molar ratio of 1/100 at higher magnification. Scale bar = 80 nm.
GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death (2016)
Figure 6

Figure 6. Characterization of GSDMDNterm pores by atomic force microscopy

A. AFM topograph of GSDMDNterm bound to lipid membranes of E. coli extract polar lipids. Overview topograph showing arc-, slit- and ring-like GSDMD oligomers. B–E. High-resolution AFM topographs and height profiles of GSDMDNterm oligomers (red curves). Height profiles were measured along the red lines in the topographs. Dashed zero lines (0 nm) indicate the surface of the lipid membrane; heights of <−4 nm indicate that the oligomers formed transmembrane pores. Topographs were taken in buffer solution (150 NaCl, 20 mM Hepes, pH 7.8) at room temperature. The experiment was reproduced at least three times using independent liposome and GSDMD preparations. Scale bars of topographs correspond to 100 nm (A) and 50 nm (B–E) and of height profiles 10 nm. The full color range of the AFM topographs correspond to a vertical scale (height) of 20 nm. F–H. Analysis of GSDMDNterm oligomers bound to lipid membranes of E. coli extract polar lipids. F. GSDMDNterm oligomers assembled into arcs, slits and rings leading to transmembrane (TM) pores or not transmembrane aggregate (n = 218). G. Height of GSDMDNterm oligomers protruding from the lipid membrane. Bars represent mean and error bars represent s.e.m. (n = 218). H. Distribution of the diameters of rings formed by GSDMDNterm oligomers. The average distribution was 21.2 ± 5.6 nm (n = 164; average ± s.d.) and the bin size was 3 nm.
The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy. (2016)
Fig 2

Apoptotic induction of bortezomib.

A,C) Bortezomib treated chondrosarcoma cells showed a significantly higher level of caspase 3/7 activity than untreated cells. Untreated control cells served as reference value (ratio = 1). B,D) Cleavage of caspase-3 was detected after 24 h of bortezomib treatment by flow cytometry. The y-axis denotes cell counts and the x-axis represents fluorescence intensity of the APC antibody. The black filled histogram represents untreated control cells, the striated histogram represents 5 nM, and the checkered histogram shows 10 nM bortezomib treated cells.
The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy. (2016)
Fig 3

Analysis of the mitochondria-caspase dependent pathway.

A,B) Relative gene expression analysis of the pro- and anti-apoptotic markers Bax, Bak, Bcl-2, and Bcl-xl in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h (n = 10). C) The human apoptosis antibody protein array revealed the downregulation of the heat shock proteins HSP27, HSP60, and HSP70 as well as HTRA, Livin, p27, and p53 in both cell lines. D) Western blot analysis showed an upregulation of cytochrome C in the cytoplasmic fraction after bortezomib treatment.
The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy. (2016)
Fig 4

Analysis of TRAIL/death receptor pathway.

A,B) Relative gene expression analysis of IGF1R, Fas, and the death receptors TRAILR-1 and TRAILR-2 after bortezomib treatment with the respective IC50 concentrations for 48 h. Untreated control cells served as reference value (ratio = 1). C) The human apoptosis antibody protein array supported the important role of the IGF binding proteins (IGFBP-1 to IGFBP-6), the TNF receptor family, and the TRAIL receptors in both cell lines. D) Western blot analysis showed the downregulation of Fas and TNF-R1.
The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy. (2016)
Fig 5

Bortezomib induces autophagy in human chondrosarcoma cells.

A) Relative gene expression and B) western blot analysis of whole cell lysates for the expression of the autophagy markers Atg 5/12 and Beclin in Cal-78 and SW-1353 cells treated with the respective IC50 values of bortezomib for 24 h. C) Western blot analysis for the expression of LC3BI-II. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II. D) Quantification of the relative LC3B protein expression.
The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy. (2016)
Fig 6

LC3B immunostaining.

A) Effect of bortezomib treatment on LC3B immunostaining of Cal-78 and SW-1353 cells. Double immunolabeling with DAPI (blue) and anti-LC3B (green) antibodies was performed as described in the methods section. The lysosomal protease inhibitors E64d and pepstatin A (Inh) blocked the autophagic flux and inhibited the degradation of LC3B-II (bar: 20 μm). B) Cal-78 and SW-1353 cells were treated either with bortezomib alone or in combination with the lysosomal protease inhibitors E64d and pepstatin A (Inh; striated bars) and cell viability was analysed by the MTS assay. Untreated cells were measured as controls (light grey; n = 8, mean ± S.D.).
Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway (2021)
Figure 1

Figure 1.

RNF4 promotes resolution of SUMO-modified DNMT1 DPCs

A. HeLa cells released from double thymidine synchronization in early S phase were treated with 5-azadC for 30 min, washed and collected at the indicated times. Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies.

B. U2OS cells treated as in (A) were pre-extracted and immunostained with DNMT1 antibody. DNMT1 foci formation was analyzed by quantitative image-based cytometry (QIBC) (red bars, mean; >1500 cells analyzed per condition). Data are representative of three independent experiments. See also Fig. EV1A.

C. As in (B), except that cells were pre-treated with the proteasome inhibitor MG132 and/or SUMOi for 30 and 15 min, respectively, before exposure to 5-azadC (red bars, mean; >1900 cells analyzed per condition). Data are representative of three independent experiments.

D. HeLa cells stably expressing GFP-DNMT1 were treated or not with 5-azadC for 30 min, collected and subjected to GFP immunoprecipitation under denaturing conditions, and immunoblotted with indicated antibodies.

E. HeLa/GFP-DNMT1 cells transfected with indicated siRNAs were processed as in (D).

F. Immunoblot analysis of soluble and chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs, left untreated or exposed to 5-azadC for 30 min, and collected at the indicated times.

G. U2OS cells transfected with indicated siRNAs were treated with 5-azadC for 30 min and processed for QIBC analysis of DNMT1 foci counts as in (B) (red bars, mean; >5600 cells analyzed per condition). Data are representative of three independent experiments.

H. Representative images of U2OS cells transfected with indicated siRNAs followed by mCherry-RNF4 expression plasmid. Cells were treated with 5-azadC in the presence or absence of SUMOi, fixed 2 h later, pre-extracted and co-immunostained with DNMT1 and SUMO2/3 antibodies. Scale bar, 5 μm.

I. Immunoblot analysis of soluble and chromatin-enriched fractions of HeLa cells treated with 5-azadC and/or SUMOi as indicated.

J. GFP-tagged DNMT1 from extracts of HeLa/GFP-DNMT1 cells treated or not with 5-azadC was immobilized on GFP-Trap agarose, subjected to stringent washing to remove proteins non-covalently bound to GFP-DNMT1 and incubated with recombinant HA-ubiquitin, E1 and E2 (UbcH5a) enzymes and RNF4 proteins (STUbL reaction) at 37 °C for 1 h. Samples were then subjected to immunoblotting to assay for RNF4-dependent STUbL activity towards GFP-DNMT1.
Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO-RNF4 pathway (2021)
Figure 4

Figure 4.

DPCs on duplex DNA do not activate the DNA damage checkpoint

A. Outline of experimental set up to monitor DNMT1 DPC levels in S phase and mitotic cells.

B. HeLa cells released from double thymidine block in early S phase were pulse-labeled for 30 min or not with 5-azadC in the presence of SUMOi in late S phase. Following 5-azadC removal, cells were incubated with SUMOi and nocodazole, and mitotic cells were collected by shake-off, as outlined in (A). Soluble and chromatin-enriched fractions were immunoblotted with indicated antibodies.

C. U2OS cells transfected with indicated siRNAs and then treated as in (B) were collected in late S phase or mitosis (M), subjected to stringent pre-extraction and immunostained with DNMT1 antibody. DNMT1 foci formation was quantified by QIBC analysis (red bars, mean; >7400 cells analyzed per condition). Data are representative of three independent experiments.

D. HeLa cells transfected with indicated siRNAs and synchronized in early S phase by double thymidine block were pulse-labeled with 5-azadC in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.

E. HeLa cells transfected with indicated siRNAs were treated with 5-azadC and/or SUMOi in late S phase as outlined in (A). Cells were then collected at the indicated times after 5-azadC withdrawal and analyzed by flow cytometry. Data are representative of three independent experiments. Proportion of cells with G2/M DNA content is indicated.

F. HeLa cells transfected with indicated siRNAs were pulse-labeled or not with 5-azadC for 30 min in late S phase according to the experimental setup in (A). Following 5-azadC withdrawal cells were incubated with nocodazole, collected at the indicated times and immunoblotted with indicated antibodies.

G. HeLa cells transfected or not with indicated siRNAs were synchronized in early S phase by double thymidine block, released and pulse-labeled 5,5 h later with 5-azadC for 30 min in the presence or absence of SUMOi. Following 5-azadC removal, cells were subjected to live-cell imaging analysis, and the duration from late S phase to mitotic entry (nuclear envelope breakdown (NEBD)) was quantified (red bars, median; at least 83 cells, pooled from three independent experiments, were analyzed per condition; **p<0.01, ns: not significant, Mann-Whitney test).

H. Immunoblot analysis of siRNA-treated HeLa cells that were exposed to indicated genotoxic agents and collected 1 h later.