EMBO/soda-nel huggingface dataset:
PARP-1 regulates DNA repair factor availability (2018)
Figure 3

Figure 3. PARP-1 regulates pro-oncogenic transcription factor signaling. (A) Left: Data generated as described above in Figure 2 was utilized for Gene Set Enrichment Analysis (GSEA) Molecular Signature DataBases (MSigDB) KEGG analyses. Cut-off for reporting was a false discovery rate q value of < 0.25, and normalized enrichment scores (NES) are shown, with darker colors indicating more enrichment. Middle: Data generated as described above in Figure 3 was utilized for Gene Set Enrichment Analysis (GSEA) Molecular Signature DataBases (MSigDB) KEGG analyses. Cut-off for reporting was a false discovery rate q value of < 0.25, and normalized enrichment scores (NES) are shown, with darker colors indicating more enrichment. Open circles indicate cell cycle-related hallmarks, closed circles indicate DNA damage repair-related hallmarks. Right: Selected GSEA MSigDB Hallmarks pathways are shown with NES and false discovery rate (FDR). (B) Indicated cell lines were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determine by two-tailed Student's t test where *=p<0.05, **=p<0.01,***=p<0.001, ****=p<0.0001. LNCaP: E2F1, p=0.0159; PCNA, p=0.0217; MCM7, p=4.0936e-6; CCNA2, p=0.0005. C4-2: E2F1, p=0.0074; PCNA, p=0.1258; MCM7, p=3.7471e-5; CCNA2, p=0.0031. (C) ChIP-qPCR after C4-2 cells were treated as depicted in Figure 2. Data are depicted as mean +/- standard deviation of three independent biological experiments. Statistical significance was determined by two-tailed Student's t test where *=p<0.05, ****=p<0.0001. E2F1 ChIP, p=0.4610; PARP-1 ChIP, p=0.1773; Pol II ChIP, p=0.0305; AcH4 ChIP, p=7.4261e-5 (D) Athymic nude mice were injected with C4-2 cell mixed with matrigel. Once tumors became 100mm3, mice were treated with either vehicle control or veliparib. 72 hours later, tumors were harvested, RNA was isolated and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples, +/- standard deviation of three independent xenograft tumors. (E) Prostatectomy tissue (n=6) was cultured as previously described, and treated with either vehicle control or veliparib for six days. RNA was then harvested from the tissues and used for qPCR quantification of the indicated transcripts. Data are depicted as log2 absolute gene regulation of veliparib samples compared to control samples. Each individual tissue is depicted by a separate bar color. Statistical analyses were performed by Wilcoxon signed rank test.
PARP-1 regulates DNA repair factor availability (2018)
Figure 7

Figure 7. PARP-1 regulates DNA repair factor availability and DNA repair competency. (A) Indicated cell lines were transfected with indicated constructs, and treated with veliparib. Cell growth and (B) DDR via γH2AX was assessed. Data represent median +/- standard deviation of independent biological replicates. Control transfected and vehicle control treated cells are set to 1. *=p value<0.05, **=p value<0.01. Statistical significance was determined by two-tailed Student's t test. LNCaP cell growth: Control transfection, p=0.0220; BRCA1 transfection, p=0.67787; BRCA2 transfection, p=0.4676. C4-2 cell growth: Control transfection, p=0.0354; BRCA1 transfection, p=0.1638; BRCA2 transfection, p=0.2519. 22Rv1 cell growth: Control transfection, p=0.0039; BRCA1 transfection, p=0.1085; BRCA2 transfection, p=0.2781. LNCaP γH2AX: Control transfection, p=0.0008; BRCA1 transfection, p=0.9035; BRCA2 transfection, p=0.4685. C4-2 γH2AX: Control transfection, p=0.0009; BRCA1 transfection, p=0.6362; BRCA2 transfection, p=0.4217. 22Rv1 γH2AX: Control transfection, p<0.0001; BRCA1 transfection, p=0.4698; BRCA2 transfection, p=0.4937. (C) Graphical abstract of data presented herein. TF = transcription factor.
Mitochondrial protein biogenesis in the synapse is supported by local translation (2020)
Figure 1

Figure 1 Mitochondrial proteins represent a significant fraction of locally synthesized proteins in synaptoneurosomes

(A) Workflow of the experiment presented in panel (C), depicting synaptoneurosomes (SN) isolation and in vitro stimulation in the presence of radioactive 35S-labelled methionine/cysteine mix.

(B) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre- and postsynaptic markers, but not glia marker (GFAP) in the SN fraction. Cytosolic markers were depleted, while nuclear markers were barely detected in the SN (n=4 biological replicates, *** p<0.001, ** p<0.01, * p<0.05; one sample t-test; error bars indicate SEM).

(C) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 15-120 min. After labeling, proteins were separated on SDS-PAGE and newly synthesized proteins in SN labeled with [35S] were visualized by autoradiography (left panel). To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol (CAP, 50 μg/ml) or with puromycin (PURO, 3mM) respectively (right panel). After 1 hour after the stimulation, a significant increase in 35S-labelled proteins was observed (n=6 biological replicates, *** p<0.001; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) and the CAP treatment did not affect the overall levels of de novo synthetized proteins (n=6 biological replicates, ns; p=0.220). In contrast, PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes (n=6 biological replicates, ** p=0.0091, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.

(D-F) Results of mass spectrometry (label free quantification - LFQ) analysis identifying proteins with increased abundance in NMDAR-stimulated (20 min) synaptoneurosomes. (D) Volcano plot (Whitney-Mann test, n=4 per group) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. (E) Biological functions of identified proteins annotated using DAVID. Third top functional category identified as mitochondrial ones. (F) Pie-chart showing percentage share of mitochondrial proteins among all identified upregulated proteins.
Mitochondrial protein biogenesis in the synapse is supported by local translation (2020)
Figure 2

Figure 2 Identification of proteins locally synthesized in synaptoneurosomes using high-resolution quantitative mass spectrometry and RNA-sequencing.

(A-D) Results of high-resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes. Volcano plots (Whitney-Mann test, Benjamini Hochberg correction, n=5 per group) showing abundance of identified proteins in NMDAR-stimulated (20 min) synaptoneurosomes as compared to unstimulated using iTRAQ8-plex (A) and TMT10-plex (B) labelling methods. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified, the data is presented on Figure EV2. (C) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation (assembled from all three proteomic analyzes: LFQ, iTRAQ8, TMT10) annotated using DAVID. Second top functional category identified as mitochondrial ones. Mitochondrial proteins constituted 13% (69) of all upregulated proteins. (D) Interactome of identified proteins, mitochondrial proteins shown in orange.

(E) RNA absorbance profiles from polyribosomal fractionation of unstimulated and NMDAR-stimulated (20 min) synaptoneurosomes. Upon NMDAR stimulation, a fraction of mRNAs is shifted towards heavy-polyribosomal fraction, which represents actively translating ribosomes. The fraction of polyribosomes used for RNA-seq experiment is depicted.

(F) Comparison of proteomic data with RNA-seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples. Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA-seq values of polyribosome associated mRNA with total mRNA (y axis). Green dots represent mRNAs encoding proteins upregulated upon stimulation (identified in MS experiments) while orange dots represent mRNAs encoding mitochondrial proteins. Density curves for transcripts encoding mitochondrial proteins (orange) and upregulated proteins (green) detected by the mass spectrometry are present on the right side of the plot. Please note that both classes represent high abundant mRNAs with good ribosome association values. Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red.
Mitochondrial protein biogenesis in the synapse is supported by local translation (2020)
Figure 3

Figure 3 Characterization of mitochondrial respiratory complexes and their activity in synaptoneurosomes

(A) Respiration activity of permeabilized synaptoneurosomes (upper panel) as compared to permeabilized HEK293T (lower panel). Oxygen concentration (blue line) and oxygen consumption (red line) measured using O2k oxygraph are shown.

(B) Blue native gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria.

(C) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN-PAGE followed by Western blot using antibodies specific for: complex I (Ndufb8), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox6a) and complex V (Atp5a). Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria.

(D) Selected bands (1-8) visible in blue-native gel were excised and analyzed using mass spectrometry (LC-MS). Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands. See Table EV1 for identified peptides.
Mitochondrial protein biogenesis in the synapse is supported by local translation (2020)
Figure 4

Figure 4 Mitochondrial proteins locally synthesized in the synapse are incorporated into respiratory chain complexes

(A) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 1h. Samples were separated using blue-native electrophoresis (BN-PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN-PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.

(B-C) To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) or VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) (C). Additionaly, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3mM) (C). Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * p<0.0145; C, n=5 biological replicates, ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) or VOA (C; n=5 biological replicates, ** p=0.0044, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S-methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n=5 biological replicates, * p=0.0236, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.
Mitochondrial protein biogenesis in the synapse is supported by local translation (2020)
Figure 5

Figure 5 Altered mitochondrial respiration and morphology in Fmr1 KO synapses

(A) Fmr1 KO mice were used as a mouse model of dysregulated local translation. Mitochondria morphology in the brain slices and mitochondria metabolism in synaptoneurosomes was assessed in Fmr1 KO and WT mice.

(B) Equal amounts of synaptoneurosomal samples isolated from mouse brains (Fmr1 KO and wild-type littermates) were permeabilized by saponin and assayed on MitoPlates for 2 hours. Results are presented as the average rate/min/µg of protein, +/- SEM (n=3 per genotype; two-way ANOVA, post-hoc Sidak's multiple comparisons test; *** p< 0.0001).

(C) Mitochondrial O2 consumption rates in SN were measured by high-resolution respirometry in the presence of following respiratory substrates: malate, pyruvate, and glutamate for Complexes I-IV, succinate for Complexes II-IV, glycerol 3-phosphate for mGpDH-IV and ascorbate and TMPD for complex IV, and normalized to protein content. Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD (*p=0.0483 and *p=0.0360 respectively, n=9 per genotype, paired two-tailed t-test). Data are presented as a box-and-whiskers graph (the box extends from 25th to 75th percentiles, central horizontal line is plotted at the median, whiskers show 5th - 95th percentile).

(D) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild-types. Mitochondria are indicated by arrows; presynaptic part in blue, postsynaptic part in green. 3 mice per genotype were analyzed. Scale bars, 0.5 µm. See also Fig. EV3.

Expanded View Figure Legends
A photoconvertible fluorescent reporter to track chaperone-mediated autophagy (2011)
f3

(a) Localization of photoconverted KFERQ–PS–CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum. (b) Percentage of co-localization of KFERQ–PS–CFP2 with the indicated proteins. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (c) Co-localization of photoconverted KFERQ–PS–CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A. (d) Immunofluorescence with antibodies that recognize PS–CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction. (e, f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LC3, CD-M6PR, Rab5 (e) and ubiquitin (f) in mouse fibroblasts maintained in serum-free media. Scale bars, 5 μm.
A photoconvertible fluorescent reporter to track chaperone-mediated autophagy (2011)
f4

(a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ–PS–CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Representative images of cells also immunostained for LAMP-1 are shown. (b, c) Mouse fibroblasts stably expressing KFERQ–PS–CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.
A photoconvertible fluorescent reporter to track chaperone-mediated autophagy (2011)
f6

(a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ–PS–CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Images show representative cells after immunostaining for LAMP-2 (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean+s.e. of three-to-five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for P0.05 (range 0.002–0.03, ANOVA-Bonferroni). (c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ–PS–CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (P=0.002, ANOVA-Bonferroni). (d) Mouse fibroblasts stably expressing KFERQ–PS–CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. (e) Mouse fibroblasts stably expressing KFERQ–PS–CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002–0.0007, t-test). Scale bars, 5 μm.
Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway (2020)
Figure 1

Figure 1. NPCs with centrosome defects delay in mitosis.

(A) Representative whole mount images of WT and Cep63T/T brains at P60. Scale bar = 0.2 cm.

(B) Telencephalon area of WT and Cep63T/T brains. WT littermates N = 4, Cep63T/T N = 7; two-tailed Mann-Whitney t-test.

(C) Representative whole mount images of WT and Sas4cKO brains at P14. Scale bar: 0.2 cm.

(D) Telencephalon area of WT and Sas4cKO brains. WT littermates N = 8, Sas4cKO N = 7; two-tailed Mann-Whitney t-test. Black circles represent Nestin-Cre+ animals.

(E) Representative images of E14.5 WT, Cep63T/T and Sas4cKO cortices stained with antibodies against α-Tubulin (green), γ-Tubulin (red) and DAPI (blue). Magenta arrows indicate dead cells; white and yellow arrows indicate cells with bipolar and monopolar spindles, respectively. Insets showing zoomed in view of 2 representative cells. Dashed lines showing the orientation of the cleavage plane relative to the ventricular surface. Scale bar = 25 μm.

(F) Graph showing the percentage of bipolar, monopolar and multipolar cells at the ventricular surface of E14.5 cortices. WT n = 201 cells, N = 3 embryos; Cep63T/T n = 312 cells, N = 3 embryos; Sas4cKO n = 296 cells, N = 3 embryos; #, chi-square test with *, post-hoc analysis, comparisons are made to WT.

(G) E14.5 cortices stained with antibodies against Ki67 (red) and phosphorylated-Histone H3 (PH3, yellow). Insets showing zoomed in view of cells in the ventricular zone (VZ) and sub-ventricular zone (SVZ). Scale bar = 100 μm.

(H) Ratio of mitotic (PH3+) to cycling (Ki67+) cells in E14.5 cortices. WT N = 5, Cep63T/T N = 3, Sas4cKO N = 3; two-tailed Mann-Whitney t-test.

(I) Protocol for the establishment of primary NPC cultures for live imaging.

(J) Representative images from live-cell movies of Nuclear-mCherry+ WT, Cep63T/T and Sas4cKO NPCs. The timing of mitosis began at nuclear envelope breakdown (NEBD) and finished at nuclear envelope reformation. White arrows track the mitotic events of the mother cell from NEBD to the generation of daughter cells following cytokinesis.

(K) Plot showing the duration of mitosis in WT, Cep63T/T and Sas4cKO NPCs. Triangles represent individual cells; triangles of the same color represent cells derived from the same embryo. Circles represent the average mitotic duration of cells from each embryo. WT n = 202 cells, N = 4 embryos; Cep63T/T n = 141 cells, N = 3 embryos; Sas4cKO n = 138 cells, N = 3 embryos; two-tailed Welch's t-test.

Data information: All data represent the means ± S.E.M. *P < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001 and not significant indicates P > 0.05.

See also Figure EV1.
Centrosome defects cause microcephaly by activating the 53BP1-USP28-TP53 mitotic surveillance pathway (2020)
Figure 2

Figure 2.­­ Prolonged mitosis leads to an increase frequency of cell death in the progeny of NPCs.

(A-C) Graph showing the mitotic duration of NPCs and the fate of their progeny in primary cultures generated from E14.5 embryos. Each bar represents one cell; its height represents the amount of time the mother cell spent in mitosis; bar color represents the fate of the progeny. Dashed line is set at 30 mins. WT n = 330 cells, N = 4 embryos; Cep63T/T n = 260 cells, N = 3 embryos; Sas4cKO n = 223 cells, N = 3 embryos.

(D-F) Percentage of proliferating, arrested and apoptotic progeny within each group of the indicated mitotic duration, calculated from data shown in A-C; groups with n < 20 cells were excluded from this quantification; #, chi-square test with *, post-hoc analysis, within each genotype, comparisons are made to the 0-30min group.

(G) Genome-wide copy number plots of single cells sequenced from dissociated dorsolateral telencephalons of E14.5 embryos. Individual cells are represented in rows with copy number states indicated by colors; arrows on the zoomed in view (right) indicate aneuploid cells. WT n = 38 cells, N = 2 embryos; Cep63T/T n = 40 cells, N = 2 embryos; Sas4cKO n = 42 cells, N = 2 embryos.

(H) Percentage of aneuploid cells from the single-cell sequencing data shown in G.

Data information: All data represent the means ± S.E.M. *P < 0.05; ** < 0.01; *** < 0.001; and not significant indicates P > 0.05.

See also Figure EV2.