EMBO/soda-nel huggingface dataset:
Synthetic lethal metabolic targeting of cellular senescence in cancer therapy (2013)
f2

a–e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate. b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment. c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.
Synthetic lethal metabolic targeting of cellular senescence in cancer therapy (2013)
f3

a–g, Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TIS lymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-&kgr;B super-repressor I&kgr;B&agr;Dgr;N (SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b. d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in b. f, Expression of the transcription factors Xbp1 (by RT–PCR (PCR with reverse transcription; top); note that the lower band reflects the UPR-related splice form), ATF4, CHOP, phospho-Jun N-terminal kinase (JNK-P-Thr183/Thr185) and total JNK (by immunoblot analysis, with &agr;-tubulin as a loading control; bottom) in lymphoma cells as indicated (n = 3 each, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (&agr;-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P  0.05.
Synthetic lethal metabolic targeting of cellular senescence in cancer therapy (2013)
f4

a, Cellular viability of super-repressor- or empty-vector-infected control;Bcl2 lymphomas treated with ADR or untreated for 5 days, then exposed for 2 days to the inhibitors 2DG or bafilomycin A1, compared to no inhibitor (ut) in vitro. Results represent means ± s.d. (n = 5 each). b, Immunoblot analyses of ATF4, global protein ubiquitination, p62/SQSTM1, and MAP1-LC3-I to MAP1-LC3-II conversion in control;Bcl2 lymphomas with no ADR treatment, in TIS cells, and in TIS cells after exposure to 2DG or bafilomycin A1 in vitro; representative blot with &agr;-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). c, Cleavage (activation) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control;Bcl2 lymphomas as in b; representative blot with &agr;-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). d, Cellular viability of ADR-treated control;Bcl2 lymphoma cells infected with short-hairpin RNAs against either caspase 12 (shCasp12) or caspase 3 (shCasp3) compared to empty vector-infected lymphoma cells treated as in a. Results represent means ± s.d. (n = 4 each). e, TIS cells in primary human acute myeloid leukaemia (AML) samples by SA-&bgr;-gal staining of treated (0.1 µg ml−1 ADR for 5 days) versus untreated cells in vitro. Data represent means ± s.d. (n = 5 each). f, Intracellular glucose (left) and ATP (right) concentrations in senescent AML cells as in e. g, Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a. Results represent means ± s.d. (n = 5 each). h, Colour-coded whole-body fluorescence imaging to visualize green fluorescent protein (GFP)-tagged tumour burden in time-courses of three representative mice harbouring CTX-senescent control;Bcl2 lymphomas at day 5 (see Figure 1a), and two days later, after the administration of 2DG (n = 3 mice), bafilomycin A1 (n = 4 mice) or mock treatment (n = 4 mice) starting from day 5 post CTX (red, highest intensities; blue, lowest intensities). TdT-mediated dUTP nick end labelling (assay) (TUNEL) staining of lymph node sections in situ to visualize apoptosis at day 7. Numbers represent means ± s.d. (n = 3). i, Overall survival of 5 day-CTX-treated control;Bcl2 or Suv-39h1−;Bcl2 lymphoma-bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h (control;Bcl2: n = 12 and Suv39h1−;Bcl2: n = 10), presented as matched pair analyses (P  0.001 for the control; Bcl2 comparison; P = 0.221 for the Suv39h1−;Bcl2 comparison). *P  0.05.
Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors (2020)
Figure 2

Fig.2 Engineering of a bivalent KB-AT-23 sdAb and test of its antithrombin inhibitory activity in vitro

(A) Schematic representation of the bivalent KB-AT-23 sdAb.

(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin. Data represent mean±SD of three experiments.

(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23. Data are representative thrombograms. Detailed information on thrombin generation parameters are provided in Table 2. Antithrombin activity was inhibited by 81.1±6.1% and 54.1±8.4% for the 0.2 mg/ml and 0.1 mg/ml concentration, respectively.
Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors (2020)
Figure 3

Fig.3 Assessment of KB-AT-23 efficacy in haemophilia A mice delivered with the purified sdAb

(A) Schematic representation of the clearance experiment.

(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus. Data represent mean±SD, with n=3 for each data point.

(C) Schematic representation of the tail vein transection (TVT) assay performed in F8-/- mice. Ø 2.3mm: size of the diameter of the tail at the location of the TVT; 0.5mm: depth of the cut performed on the mouse tail.

(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25%-75% quartiles, the line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Tukey's correction for multiple comparisons.
Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors (2020)
Figure 5

Fig.5 Long term safety and efficacy of KB-AT-23 delivered via AAV vectors in haemophilia A mice

(A) Scheme representing the study design. FVIII deficient mice (FVIII -/-, n=6-9/group) were administered with 2 doses (1x1010 or 1x1012 vg/mouse) of AAV vector. Control mice were injected with PBS. Red symbols represent timing of blood collection.

(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.

(C) Measurement of D-dimers (D2D) plasma concentration 27 weeks post vector administration. Data are presented as mean ±SD (n=6-9/group).

(D) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD.

(E) TVT test on males and females (n=4-5 per group) performed 29 weeks post vector administration. The volume of blood loss (µL) during 30 minutes observation time is reported. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25-75% quartiles; the central line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons.

(F) Vector genome copy number (VGCN) per cell in liver samples collected at sacrifice (n=8/group). Data represent mean±SD.
Single-domain antibodies targeting antithrombin reduce bleeding in hemophilic mice with or without inhibitors (2020)
Figure 6

Fig.6 Assessment of KB-AT-23 efficacy in haemophilia B mice with or without inhibitors

(A) Scheme representing the study design. FIX deficient mice (FIX -/-, n=5/group) were administered with factor IX (FIX, 40 µg/mouse) in the pre-immunization phase and with AAV vectors (2x1011 vg/mouse) in the treatment phase. Control mice were injected with PBS. Red symbols represent timing of blood collection.

(B) Table reporting the different mice groups of the study.

(C) Measurement of anti-hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS (n=5/group) at different time points. Data represent mean±SEM.

(D) Representative western blot analyses on plasma samples (n=4) collected from animals 8 weeks post-AAV injection.

(E) Antithrombin activity in plasma samples collected from mice at different time points. Data are presented as mean±SD (n=3-4 per datapoint). Data were analyzed in a 2-way ANOVA with Tukey's correction for multiple comparisons. ***p<0.01 ****p<0.001 (2-way ANOVA with Tukey's multiple comparison test)

(F) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max.

G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.
During autophagy mitochondria elongate, are spared from degradation and sustain cell viability (2011)
figf2

(a–d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS–PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times. (e) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS–PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS–PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments. (h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS–PAGE and immunoblotted using the indicated antibodies. (i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.
During autophagy mitochondria elongate, are spared from degradation and sustain cell viability (2011)
figf3

(a) Pseudocolour-coded images of EPACI–camps FRET from real-time imaging of wild-type MEFs transfected with EPACI–camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates
F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments. (c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS–PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8. (d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and
Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition). (f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition). (h) Representative confocal micrographs of mitochondrial morphology of
Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).
During autophagy mitochondria elongate, are spared from degradation and sustain cell viability (2011)
figf4

(a–c) MEFs of the indicated genotype were treated as indicated, counted and 2.7×105 cells were lysed. Lysates were separated by SDS–PAGE and immunoblotted using the indicated antibodies. (d) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype. One representative experiment of five independent repetitions performed as in a–c is shown. (e) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS–PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.
During autophagy mitochondria elongate, are spared from degradation and sustain cell viability (2011)
figf5

(a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments. (c) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments. (d) Mitochondrial ATP measured
in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value. (e,f) Cells of the indicated genotype were starved for the indicated times. Viability was determined by flow cytometry as the percentage of annexin-V- and propidium iodide (PI)-negative events. Data represent mean ± s.e.m. of five independent experiments. (g) MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments. (h) Cells of the indicated genotype were starved for the indicated times. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments. (i)
Drp1−/− MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated. Cell death was determined by flow cytometry as the percentage of YFP- and propidium-iodide-positive events. Data represent mean ± s.e.m. of four independent experiments. (j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.
During autophagy mitochondria elongate, are spared from degradation and sustain cell viability (2011)
figf6

(a) Blue-native electrophoresis analysis of ATPase dimerization and activity. Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4% digitonin and proteins were separated by blue-native PAGE. ATPase activity was measured in-gel (top) and ATPase levels were measured by immunoblotting for the indicated antibody (bottom). (b,c) Quantitative analysis of the ratio of levels (b) and activity (c) between dimeric and monomeric forms of ATPase, assessed by densitometric analysis of gels from experiments performed as in a. Data represent mean ± s.e.m. of five independent experiments. (d) Experiments were carried out as in a, but with cells of the indicated genotype.
FET family fusion oncoproteins target the SWI/SNF chromatin remodeling complex (2019)
Figure 1

Figure 1. Identification of proteins bound to recombinant FET-NTD baits.

A. The three normal RNA-binding FET proteins consist of N-terminal repetitive and structurally disordered domains (NTD: N-terminal domain), central RNA-binding domains (RRM: RNA recognition motifs), zinc finger domains (ZF) and degenerated repeat regions (RGG: RGG repeat regions). The GST-tagged FET-NTD baits shown were used for pulldown experiments and represent the shortest parts commonly present in the FET fusion oncoproteins. The FET binding motif is a conserved sequence required for complex formation between the three FET proteins. Amino acid numbers are indicated.

B. Schematic illustration of a representative FET fusion protein (FUS-DDIT3 Type II) and its parental proteins. DBD: DNA-binding domain, LZ: leucine zipper domain. Type I and Type II show locations of the two most common MLS fusion breakpoints in FUS.

C. Coomassie staining of SDS-PAGE-separated pulldown samples with/without cell extracts (C.E) using GST-tagged FET-NTDs as baits. Sepharose with bound GST was included as control. Background from recombinant protein baits (shown by white arrowheads) as well as smaller partial recombinant products is visualized in the left panel (C.E -). Several high-molecular weight proteins are retained by the FET-NTDs (C.E +). Black arrowheads indicate protein bands and gel parts analyzed by mass spectrometry (see also Table EV1).

D. Immunoblot analysis (IB) of pulldown samples with FET-NTD baits with/without cell extracts (C.E). Antibodies against ARID1A, BRG1 and BAF60A were used for detection of SWI/SNF components. Input samples diluted 1:5 were included as a control.

E. In situ proximity ligation assays (PLA) using antibodies against BRG1, ARID1A, DDIT3 and C-terminal parts of normal FUS show protein complexes containing FUS/BRG1, FUS/ARID1A, FUS-DDIT3/BRG1 and FUS-DDIT3/ARID1A as red fluorescent spots in nuclei of MLS cell lines 2645-94 and 402-91. C-terminal parts of FUS are not present in the FUS-DDIT3 fusion protein and normal DDIT3 is not expressed in these cell lines. Merged images also include DAPI nuclear counterstain in blue. Combinations of primary antibodies are used to detect interactions (left panel). In control experiments (right panel), one primary antibody is omitted to evaluate the background fluorescent signals. Scale bars = 10 μm.
FET family fusion oncoproteins target the SWI/SNF chromatin remodeling complex (2019)
Figure 2

Figure 2. Analysis of the binding between FUS-NTD and SWI/SNF core components.

A. Schematic illustration of FUS‑NTD‑GST truncation constructs. Amino acid numbers are indicated.

B. Immunoblot analysis (IB) of GST-pulldown samples with deletion mutants of FUS‑NTD‑GST as baits with/without cell extract (C.E). ARID1A and BRG1 were used as tracers of SWI/SNF binding. Coomassie staining of recombinant proteins are shown in the lower panel. Amino acid numbers are indicated.

C. Immunoblot analysis (IB) of FUS-NTD‑GST pulldown samples of cell extracts (C.E.) after treatment with RNase or DNase, using antibodies against ARID1A and BRG1.

D. Immunoblot analysis (IB) of SWI/SNF components (ARID1A, BRG1, BAF170, BAF155, BAF60A, SS18 and BAF47) and full-length FET proteins (EWSR1 and FUS) remaining on sepharose-bound FUS-NTD‑GST pulldown baits after increasing stringency washes with 100, 150, 250, 500 or 1000 mM NaCl. Note loss of BRG1 at NaCl concentration > 250mM.