EMBO/soda-nel huggingface dataset:
Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA-damage repair (2022)
Figure 7

Figure 7: DPP9 activity and the BRCA2 N-terminus promote RAD51 foci formation

A. Representative immunofluorescence images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9-SWT cells. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428.

B. Graph showing the number of RAD51 foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9 KO + DPP9WT cells (-Dox). Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51 foci. Each dot represents the number of RAD51 foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).

C-F. Representative images (C, E) and summarizing graph (D, F) showing the number of RAD51 foci per nucleus in HeLa WT cells (C, D) or HeLa DPP9 KD cells (E, F). Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells (C, D). In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51 foci in a single cell, from three (D) or four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-DPP9: RRID:AB_2889071; anti-BRCA2: RRID:AB_2259370.

Expanded View Figure Legends
Induction of autophagy and inhibition of tumorigenesis by beclin 1
 (1999)
figf1

a, Western blot analysis of yeast cell lysates with polyclonal anti-flag
antibody to detect flag-tagged human Beclin 1 expression. Two-hundred micrograms
of protein per lane was loaded. SEY6210, wild-type yeast; JCY3000, SEY6210
disrupted in apg6/vps30; (+) control, lysates of mammalian cells transfected
with flag-beclin 1. b, Autophagicbody formation in yeast deprived
of nitrogen in the presence (+) and absence (-) of 1 mM PMSF. Arrows
denote cells that would be scored as positive in the experiment shown in
c. c, Quantitative effects of apg6/vps30 and beclin 1
transformation on autophagicbody formation in apg6/vps30-disrupted
yeast in the presence of PMSF. Cells with one or more autophagic bodies within
the vacuole were scored as positive (see arrows in b). A minimum of
100 cells was counted for each sample. Results represent the mean (±s.e.m.)
percentage of cells with autophagic bodies within the vacuole for triplicate
samples. Similar results were obtained in five independent experiments.
d, Sorting of vacuolar protein carboxypeptidase Y, (CPY). p2CPY, precursor
form; mCPY, sorted mature form. Mr, relative molecular mass.
Induction of autophagy and inhibition of tumorigenesis by beclin 1
 (1999)
figf2

a–f, Electron micrographs of MCF7.control (clone 38)
(a, b) and MCF7.beclin1 (clone 17) cells (c–
f) grown in nutrient-rich media (a, c) or subjected to 4 h
of serum and amino-acid deprivation (b, d–f). Asterisks
in e and f denote autophagic vacuoles that would be counted
in the experiment shown in g. Autophagic vacuoles were defined as double-membrane
vacuolar structures containing recognizable cytoplasmic contents. Scale bars,
1 µm. g, Quantitative effects of beclin 1 on basal
and nutrient-deprivation-induced autophagy of MCF7 cells. Bars indicate mean
(±s.e.m.) number of autophagic vacuoles per cell for cells growing
in normal media (black), for cells subjected to 4 h of serum and amino-acid
deprivation (grey), and for cells pre-treated for 30 min with 10 mM
3-MA and subjected to 4 h of serum and amino-acid deprivation in the
presence of 3-MA (open). Mean was determined by counting the total number
of autophagic vacuoles in each cell for 100 cells per clone per treatment.
h, Comparison of the rates of degradation of long-lived proteins in MCF7.control
cells (squares, clone 38), MCF7.beclin1 cells (circles, clone 17) and
MCF7.beclin1stop cells (triangles, clone 70), in EBSS + 10% serum and
complete amino acids (open symbols, solid lines) in EBSS alone (closed symbols,
solid lines), and in EBSS + 10 mM 3-MA (closed symbols, dotted lines).
Results are mean (±s.e.m.) of triplicate wells. Similar results were
obtained in three independent experiments. Results shown with these clones
are representative of results obtained with other clones analysed by electron
microscopy in g.
Induction of autophagy and inhibition of tumorigenesis by beclin 1
 (1999)
figf3

a, Western blot analysis of flag-tagged beclin-1-transfected
MCF7 clones. C, MCF7.control cells (clone 38); St, MCF7.beclin1stop
cells (clone 70). Mr, relative molecular mass. b,
Western blot analysis of flag-tagged beclin1stop-transfected MCF7 clones.
C, MCF.control cells (clone 38); B, MCF7.beclin1 cells (clone 1).
c, Photomicrographs of MCF7.control cells (left panel, clone 38), MCF7.
beclin1 cells (middle panel, clone 17) and MCF7.beclin1stop cells
(right panel, clone 70) 48 h after seeding at similar densities.
d, Proliferation of MCF7.control cells (clones 37 and 38; closed squares
and circles, respectively, solid lines), MCF7.beclin1 cells (clones
1, 6, 17; open squares, circles, triangles, respectively, solid lines) and
MCF7.beclin1stop cells (clone 70, closed triangles, dotted line). Results
represent mean Absorbance (A) (±s.e.m.) for six wells; similar
results were obtained in three independent experiments. e, Cell viability
of MCF7 clones determined by trypan blue staining. Symbols for each clone
are as in d. f, Clonigenicity in semisolid medium (soft agar)
of MCF7 clones. Results are mean (±s.e.m.) for pooled triplicate wells
from 3–4 independent experiments. g, Tumour formation in NCR
nude mice injected subcutaneously with MCF7 clones. Numbers on top of bar
indicate number of autopsy-confirmed tumours at 8 weeks per number of mice
injected with each clone.
Induction of autophagy and inhibition of tumorigenesis by beclin 1
 (1999)
figf4

a, Western blot analysis of human breast carcinoma cell lines. Results
are from 8 representative cell lines out of a total of 11 analysed. b,
Western blot analysis of matched normal breast and breast carcinoma tissue
from patients with sporadic invasive breast carcinoma. Results are from 7
representative cases out of a total of 17 analysed. N, normal breast tissue;
T, tumour tissue; Mr, relative molecular mass. c,
Immunohistochemical analysis of paraffin-embedded sections of breast tissue
from patient 441 shown in b. Adjacent sections are stained with pre-immune
843 rabbit serum (left column), 843 anti-Beclin 1 serum (middle column) and
anti-cytokeratin (right column). Top row shows low power view of region of
breast containing tumour (T) and normal mammary epithelial duct (N). Middle
row shows higher power view of normal mammary epithelial duct. Bottom row
shows higher power view of tumour. Scale bars, 1 mm.
Natural underlying mtDNA heteroplasmy as a potential source of intra-person hiPSC variability (2016)
Figure 4

Figure 4. Mitochondrial respiration is impaired in hiPSCs-derived cardiomyocytes with GPMs.

(A) In vitro cardiac differentiation protocol with lactate enrichment (from days 14 to 16 after induction) performed in seven isogenic MitoA-derived hiPSCs with different mtDNA genotypes. (B) mtDNA NGS showing the position and mutation load of GPMs in seven isogenic MitoA-derived hiPSCs clones and their differentiated counterparts. The mtDNA genotype is stable during in vitro cardiac differentiation and lactate enrichment. MitoA hiPSCs 57 and 65 (wild-type) are labelled in black, MitoA hiPSCs 61 and 69 (m.3243A>G mutant) are labelled in red and MitoA hiPSCs 58, 59 and 217 (with amplified GPMs in nuclear reprogramming) are labelled in blue.

(C) Quantification of the percentage of mature hiPSCs-derived cardiomyocytes in direct differentiation of hiPSC clones. Representative histograms showing the percentage of the cells expressing mature cardiac markers: myosin heavy chain (MF20), troponin T (cTnT) and troponin I (cTnI) by flow cytometry, in MitoA-57-derived cardiomyocytes (wild-type) and MitoA-69-derived cardiomyocytes (83% m.3243A>G). For complete quantification of cardiac markers in all hiPSCs-derived cardiomyocytes generated.

(D) Immunofluorescence images of cardiac markers (cTnT and MF20) showing similar sarcomere structures in hiPSC-derived cardiomyocytes wild-type (MitoA-57) and mutant (MitoA-69).

(E) Oxygen consumption rate (OCR) in hiPSCs. OCR reported as a % of that in MitoA-57 hiPSC, the control line. MitoA-57 and MitoA-65 hiPSCs wild-type (in black), MitoA-61 and MitoA-69 m.3243A>G mutant (in red), MitoA-58, MitoA-59 and MitoA-217 hiPSCs amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative 3 biological replicates in all samples.

(F) Oxygen consumption rate in hiPSCs-derived cardiomyocytes at day 19 of cardiac differentiation. OCR reported as % of that in MitoA-57-derived cardiomyocytes, the control line. MitoA-57 and MitoA-65 derived cardiomyocytes wild-type (in black), MitoA-61 and MitoA-69 derived cardiomyocytes mutants m.3243A>G (in red), MitoA-58, MitoA-59 and MitoA-217 derived cardiomyocytes with amplified GPMs (in blue). Error bars are mean ± SD and OCR data are representative of two independent in vitro cardiac differentiation experiments, with at least 3 biological replicates in each sample, and significance established using Student’s t-test.
MITRAC15/COA1 promotes mitochondrial translation in a ND2-ribosome nascent chain complex (2019)
Figure 1

Figure 1. MITRAC15 interacts with distinct intermediates of complex I and complex IV.

A Solubilized mitochondria from wild type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum. Eluates were analysed by SDS-PAGE and western botting. Total 1.5 %, Eluate, 100 %.

B Wild type mitochondria were solubilized and subjected to 2D-BN/SDS-PAGE and western blotting with indicated antibodies.

C Solubilized mitochondria from MITRAC12FLAG- or ACAD9FLAG-expressing cells were subjected to anti-FLAG immunoisolation and native elution. Eluates were analysed by 2D-BN/SDS-PAGE and western blot analysis. Total 0.8 % (MITRAC12FLAG isolation), 0.35 % (ACAD9FLAG isolation), Eluate 100 %.

D Solubilized mitochondria from MITRAC15FLAG-expressing cells were first subjected to native anti-FLAG immunoisolation (Total 0.1 %, Eluate 10 %, lane 4). Purified complexes were applied to anti-MITRAC12 immunoisolation (Eluates 100 %, lane 5). All samples were analyzed by SDS-PAGE and immunoblotting.
MITRAC15/COA1 promotes mitochondrial translation in a ND2-ribosome nascent chain complex (2019)
Figure 2

Figure 2. MITRAC15 interacts with mitochondrial ribosomes and isolates preferentially ND2.

A Mitochondrial translational products were labelled with [35S]methionine and anti-FLAG immunoisolation was performed as described in Fig. 1D. Eluates were analyzed by SDS-PAGE and digital autoradiography. Total 1.5 %, Eluate 100 %.

B Solubilized mitochondria from MITRAC15FLAG- or TIM21FLAG-expressing cells were subjected to anti-FLAG immunoisolation and analyzed by 2D-BN/SDS-PAGE and western blotting. MITRAC15FLAG isolation: Total 0.1 %, Eluate 100 %. TIM21FLAG isolation: Total 0.2 %, Eluate 66 % (TIM21FLAG decoration), 33 % (MITRAC15, ACAD9, MITRAC12, COX1 decoration).

C Solubilized mitochondria from MITRAC15FLAG- or C12ORF62FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.4 %, Eluate 100 % (MITRAC15FLAG isolation), 70 % (C12ORF62FLAG isolation).

D Solubilized mitochondria from ACAD9FLAG-or MITRAC15FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.5 % (ACAD9FLAG isolation), 0.4 % (MITRAC15FLAG isolation) Eluate 100 %. Asterisk (*) indicates TIM21 signal. ACAD9/ACAD9FLAG could not be separated with the used gel system, both proteins are detected with ACAD9 antiserum.
MITRAC15/COA1 promotes mitochondrial translation in a ND2-ribosome nascent chain complex (2019)
Figure 3

Figure 3. MITRAC15 affects ND2 translation.

A A knockout cell line of MITRAC15 (MITRAC15-/-) was created in HEK293T cells by using CRISPR/Cas9 technology. Alignment of respective mutated region compared to wild type shows premature STOP codons (*) in MITRAC15-/-.

B Mitochondria from wild type and MITRAC15-/- cells were isolated and subjected to steady-state protein analyses by western blotting.

C Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates).

D Complex I and citrate synthase activity were measured in WT and MITRAC15-/- cells (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).

E Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).

F Pulse radiolabeling after re-expressing MITRAC15FLAG in MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01; unpaired t-test).
MITRAC15/COA1 promotes mitochondrial translation in a ND2-ribosome nascent chain complex (2019)
Figure 4

Figure 4. MITRAC15 interacts with nascent chains of ND2.

A Mitochondrial translation products were radiolabelled with [35S]methionine after siRNA-mediated depletion of ACAD9 in wild type and MITRAC15-/- cells. Cell lysates were analyzed by SDS-PAGE and digital autoradiography. For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal. (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01 ***p < 0.001; unpaired t-test).

B FLAG-immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG-expressing cells after siRNA-mediated down regulation of ACAD9 and pulse radiolabeling. SDS-PAGE followed by autoradiography was used to analyze eluates. Relative signal intensity of ND2, ND3 and ND4L were normalized to ATP6 (total) or isolation efficiency (eluate). Total 2 %, Eluate 100 %. Error bars indicate SEM from three biological replicates; **p < 0.01; unpaired t-test.

C After C12orf62 down regulation, [35S]methionine labelling was performed. During labeling mitochondrial translation was inhibited with puromycin (pur) (2 µg/ml). MITRAC15FLAG interacting nascent chains were isolated by FLAG-immunoprecipitation from cell lysates. Eluates were subjected to SDS-PAGE, analyzed by digital autoradiography. Total 2 %, MITRAC15FLAG eluates 100 % (in case of C12ORF62FLAG, 25% of eluate was loaded as a standard). Red asterisks, accumulating nascent chains of ND2. The accumulating nascent chains of ND2 of lane 12 is presented in a magnification. F1-F3 mark nascent chains of COX1 [18].

D HEK293T cells were pulse labelled with [35S]methionine in the presence of puromycin (2 µg/ml). During puromycin treatment, cells were incubated for 5 min and 10 min in medium lacking [35S]methionine. Anti-FLAG immunoisolations were performed as described and eluates analyzed as described in Fig. 4C. Total 1 %, Eluates 100 %. The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % (error bars indicate SEM from three biological replicates; *p < 0,05; paired t-test). Red asterisks, accumulating nascent chains of ND2.
MITRAC15/COA1 promotes mitochondrial translation in a ND2-ribosome nascent chain complex (2019)
Figure 5

Figure 5. ACAD9 is not present during ND2 translation.

A/B Radiolabeling with [35S]methionine, anti-FLAG immunoisolation and sample analyses were performed as described in Fig. 4C. B Depletion of ACAD9 by siRNA was performed as in Fig. 4A. Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.

C Identified crosslink between murine ACAD9 and ND2 (spectrum in Fig EV1). ND2: Red - crosslinked peptide, green - C-terminal helix, Yellow - crosslinked Lys319. ACAD9: Orange - crosslinked Lys504. Sequences of identified crosslinked peptides from mouse and the homologous sequences from the human proteins are presented (yellow: crosslinked lysine).

Expand view figure legends
A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein (2021)
Figure 1

Figure 1: Electroporated-antibody dependent neutralization assay (EDNA) for coronavirus. (A) A titration of anti-MHV polyclonal serum was added either directly to media (extracellular) or electroporated into L929 cells (intracellular), then cells were infected with MHV-A59. The kinetics of cell growth was monitored by measuring total cell area.

(B) Phase-contrast images of cells at 48h post-infection. Scale bar = 200µm.

(C) At 48h post-infection, the viability of cells infected with MHV-A59 in the presence of intracellular or extracellular antiserum was determined by ATP luminescence assay. Increasing doses of antiserum results in increased cell survival.

(D) Kinetics of cell growth following MHV-A59 infection in the presence of a titration of electroporated anti-N antibody.

(E) Phase contrast images of cells at 48h post-infection. Scale bar = 200 µm.

(F) Quantification of cell viability by ATP luminescence assay at 48h post-infection in the presence of electroporated anti-N or anti-GFP antibodies.

(G&H) WT or TRIM21 KO L929 cells were infected with MHV-A59 in the presence of electroporated polyclonal antiserum (G) or anti-N antibody (H) and quantified by cell area 48h later. Co-electroporation of Protein A/G with anti-N antibody into WT cells mimics the TRIM21 KO phenotype.

(I) L929 Cells were infected with MHV-A59 for 4h, then cyclohexamide (CHX) was added to block further viral protein synthesis. After 5h, cells were electroporated with anti-N or anti-GFP antibodies and left for a further 3h before being western blotted for cellular N protein levels (*denotes a non-specific band).

Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.
A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein (2021)
Figure 2

Figure 2 N-antibodies inhibit SARS-CoV-2 replication intracellularly.

(A&B) Vero cells OE ACE2 and TMPRSS2 were infected with SARS-CoV-2 in the presence of IgG or anti-N antibodies added directly into media or electroporated into cells. Viral replication was then determined by RT-qPCR (A) or plaque assay (B). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication (*** = p < 0.0002).

(C&D) As with A&B, except with a titration of electroporated anti-N antibodies.

(E) Western-blot of 293T cells and 293T ACE2 OE/TRIM21 KOs alone or reconstituted with empty vector (EV) or an endogenous-promoter driven TRIM21 vector (T21)(Zeng et al., 2019). (F&G) 293T ACE2 OE/TRIM21 KO cells reconstituted with EV or TRIM21, electroporated with IgG or anti-N antibodies, and infected with SARS-CoV-2. Viral replication was then determined by RT-qPCR (F) or plaque assay (G). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication only in TRIM21-reconstituted cells (* = p < 0.05).

Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way (A&B) or two-way (F&G) ANOVA.
A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein (2021)
Figure 3

Figure 3 EDNA detects intracellular neutralization activity in SARS-CoV-2 convalescent sera.

(A) Capillary-based protein detection (Jess) of antibodies against SARS-CoV-2 antigens in convalescent sera.

(B) Quantification of antigen-specific antibodies in convalescent sera.

(C) 293T cells OE ACE2 infected with SARS-CoV-2 in the presence of electroporated convalescent sera. There are statistically significant differences in the ability of serum from different individuals to inhibit viral replication intracellularly (** = p< 0.005, * = p< 0.05).

(D) 293T ACE2 OE/TRIM21 KOs reconstituted with either empty vector (EV) or TRIM21-expressing vector (TRIM21) infected with SARS-CoV-2 in the presence of electroporated convalescent sera. Intracellular neutralization of viral replication is only observed in cells reconstituted with TRIM21 (** = p< 0.005).

Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way (C) or two-way (D) ANOVA.
A functional assay for serum detection of antibodies against SARS-CoV-2 nucleoprotein (2021)
Figure 4

Figure 4 SARS-CoV-2 convalescents make potently neutralizing N antibodies whose activity correlates with the number of active N-specific T cells.

(A) Capillary-based protein detection (Jess) of N-antibodies in convalescent sera, quantified by chemiluminescence.

(B) Quantification of SARS-CoV-2 intracellular neutralization by electroporated convalescent sera using EDNA and RT-qPCR (as % of PBS condition).

(C) Correlation between N-antibody titre, as calculated by Jess, and intracellular neutralization (fold decrease in SARS-CoV-2 replication), as measured by EDNA, in convalescent sera from multiple individuals.

(D) Intracellular neutralization of SARS-CoV-2 by titrated serum from two strongly N-antibody positive individuals.

(E) ELISpot measuring IFNγ production in T cells from SARS-CoV-2 convalescents upon stimulation with a peptide library covering N-protein.

(F) Correlation between N-specific T cell activity and intracellular neutralization activity.

(G) Correlation between N-specific T cell activity and N-antibody titre.

Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical analysis was performed using a semilog non-linear fit (F&G).
Attenuation of pattern recognition receptor signaling is mediated by a MAP 2 kinase kinase kinase (2015)
Figure 1

Figure 1. Flagellin receptor FLS2 co-immunoprecipitates with MKKK7. Immunoprecipitation was performed with GFP-binding protein immobilized on magnetic beads using extracts of Arabidopsis seedlings, expressing either YFP-MKKK7 or Lti6B. Seedlings were treated with 1 μM flg22 for the indicated time. YFP-MKKK7 and Lti6B-GFP were detected with an anti-GFP antibody while FLS2 was detected with an FLS-specific antibody. Upper panel shows (co)-immunoprecipated proteins, lower panel shows input levels of protein. Arrowheads indicate the position of proteins of interest.