EMBO/soda-nel huggingface dataset:
miR-124-dependent tagging of synapses by synaptopodin enables input-specific homeostatic plasticity (2022)
Figure 9

Figure 9. miR-124-dependent synapse-autonomous HSP at CA3 recurrent synapses.

(A) Experimental design to silence individual presynaptic inputs in recurrent circuits between CA3 pyramidal cells in organotypic hippocampal slices.

(B,C) Whole-cell recordings are used to ensure the functional connectivity between two CA3 pyramidal cells while infusing plasmids encoding GFP-Syp-IRES-TetTx and rescue RFP-SP + BFP-shRNA-SP in the pre- and postsynaptic cells, respectively (B). Slices were fixed 48 h after transfection and processed for confocal microscopy in order to analyze spines contacted by GFP+ terminals vs neighboring spines from the same dendritic branch (C).

(D) Pair recordings from functionally connected neurons during transfection (left) and 24 h after transfection (right). Top traces (black and green) show train of 10 action potentials elicited in the presynaptic cell. Bottom traces show corresponding evoked postsynaptic currents (black, average from 20 sweeps) during transfection but not 24 h after (grey, average from 20 sweeps).

(E) Confocal image showing a pair of pre and postsynaptic CA3 pyramidal neurons transfected with GFP-Syp-IRES-TetTx (green) and rescue RFP-SP (not shown) + SP-shRNA-BFP (grey), respectively. Scale bar: 40 μm.

(F) Higher magnification of dendritic spines from neurons transfected with rescue RFP-SP (magenta) containing wild-type (WT) or mutated (MUT) 3'UTR and BFP-shRNA-SP (grey) and contacted by GFP-Syp+ terminals (green). Arrowheads indicate putative synaptic contacts. Scale bar: 5 μm.

(G) Percentage of SP+ spines in neurons expressing RFP-SP-3'UTR-WT vs -MUT and depending on the apposition or not to a GFP-Syp+ terminal (SP-3'UTR-WT: TetTx-, n = 220, TetTx+, n = 26; SP-3'UTR-MUT: TetTx-, n = 616, TetTx+, n = 25; n indicates the number of spines from 5-7 pairs).

(H) Spine head volume in same conditions as in (G) (SP-3'UTR-WT: TetTx-, n = 26, TetTx+, n = 26 spines from 5 pairs, 4 slices; SP-3'UTR-MUT: TetTx-, n = 25, TetTx+, n = 25; n indicates the number of spines from 7 pairs, 7 slices). ***P < 0.001, *P < 0.05, ns, not significant (Kruskall-Wallis test followed by Dunn's multiple test).

(I) Cumulative probability distributions of spine head volumes for TetTx- and TetTx+ spines from neurons expressing RFP-SP-3'UTR-WT (SP-WT, light gray and light green) or RFP-SP-3'UTR-MUT (SP-MUT, dark gray and dark green).

Data information: Data are represented as mean ± SEM.
A20 controls RANK-dependent osteoclast formation and bone physiology (2022)
Figure 1

Fig. 1 Myeloid-specific A20 deficiency promotes osteoclastogenesis.

(a) Representative micro-CT pictures of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Note the severe osteoporosis in A20myel-KO mice.

(b) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Each dot represents an individual mouse (A20WT, n=10; A20myel-KO, n=8). Data are expressed as mean ± SEM. *, **, *** represent p<0.05, p<0.01, p<0.001, respectively (parametric unpaired t-test).

(c) qRT-PCR mRNA expression analysis of osteoclast-specific genes on RNA isolated from ankles of mice with the indicated genotypes (A20WT, n=9; A20myel-KO, n=10). Data are expressed as mean ± SEM. * and **represent p<0.05, p<0.01, respectively (parametric unpaired t-test).

(d) TRAP staining on osteoclast cultures derived from bone marrow cells isolated from tibia of A20WT and A20myel-KO mice and incubated for 7 days with M-CSF (25ng/ml) and RANKL (100ng/ml). Scale bar, 100 µm.

(e) qRT-PCR mRNA expression analysis on RNA isolated from bone marrow cultures from A20WT and A20myel-KO mice, incubated with M-CSF (25ng/ml) and RANKL (100ng/ml) for 5 days to induce osteoclastogenesis (A20WT, n=3; A20myel-KO, n=3). Data are expressed as mean ± SEM. ** represents p<0.01 (Kruskal-Wallis one-way ANOVA test between indicated genotypes).
A20 controls RANK-dependent osteoclast formation and bone physiology (2022)
Figure 2

Fig. 2 Osteoclast-specific deletion of A20 in mice induces severe osteoporosis and enhanced osteoclastogenesis.

(a) Levels of TNF and IL-6 in serum of A20WT, A20OC-KO and A20myel-KO mice. Each dot represents an individual mouse (A20WT, n=11; A20OC-KO, n=11 and A20myel-KO, n=14). Data are expressed as mean ± SEM. * represents p<0.05, ** p<0.01, **** p<0.0001, n.s. non-significant (parametric one-way ANOVA between indicated genotypes).

(b) Representative micro-CT pictures of hind legs of 20-30 week-old control (A20WT) and A20OC-KO littermates. Note the severe osteoporosis in A20OC-KO mice.

(c) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20OC-KO littermates. Each dot represents an individual mouse (A20WT, n=6; A20OC-KO, n=9). Data are expressed as mean ± SEM. *, **, *** represent p<0.05, p<0.01, p<0.001, respectively; ns, non-significant (parametric unpaired t-test).

(d) Functional femur strength was determined by the three-point bending test and is expressed as the maximum force (Fmax, Newton) bones can resist before breaking, corrected for femur weight, with higher values mirroring stronger bones. Each dot represents an individual mouse (27 week-old A20WT, n=9; A20OC-KO, n=6). Data are expressed as mean ± SEM. * represents p<0.05 (Non-parametric Mann Whitney test between indicated genotypes).

(e) Representative pictures of TRAP-stained sections from tibia of 20 week-old A20WT and A20OC-KO mice. Red arrows indicate osteoclasts. Scale bar, 500 µm.

(f) Quantification of the number of TRAP-positive cells on sections from tibia of 20 week-old A20WT and A20OC-KO mice. Each dot represents an individual mouse (A20WT, n=5; A20OC-KO, n=6). Data are expressed as mean ± SEM. * represents p<0.05 (Non-parametric Mann Whitney test between indicated genotypes).
A20 controls RANK-dependent osteoclast formation and bone physiology (2022)
Figure 3

Fig. 3 A20 regulates RANK-induced NF-ĸB signalling.

(a) Bone marrow cells isolated from A20WT and A20OC-KO mice were cultured on glass coverslips for 7 days in α-MEM medium supplemented with M-CSF (25ng/ml) alone or with M-CSF and RANKL (100ng/ml). On day 7, osteoclasts were stained with TRAP. Scale bar, 50 µm.

(b) qRT-PCR mRNA expression analysis on RNA from bone marrow cultures isolated from A20WT and A20OC-KO mice and incubated for 6 days with M-CSF (25ng/ml) and RANKL (100ng/ml) (time point 0 represents a 7 day culture with M-CSF alone). (A20WT, n=4-5; A20OC-KO, n=4-5). ** represent p<0.05, p<0.01, respectively (Two-way ANOVA test with Sidak's multiple comparisons between indicated genotypes for each time-point).

(c) qRT-PCR mRNA expression analysis on RNA from BMDM cultures isolated from control (A20WT) mice (n=3) incubated for the indicated time-points with RANKL (100ng/ml). Data are expressed as mean ± SEM.

(d) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF-κB reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and with either an empty vector or a vector expressing wild-type A20. Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency. Data are expressed as the mean of technical triplicates ± SD. **** represent p<0.0001; ns, non-significant (Two-way ANOVA test with Sidak's multiple comparisons between indicated genotypes).

(e) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel-KO mice and stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.

(f) Wild-type and A20 deficient BMDMs were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for RANK, A20, TRAF6, HOIP and Sharpin.

(g) BMDMs from A20WT and A20myel-KO mice were stimulated with RANKL (100ng/ml) for the indicated time periods and immunoblotted to visualize p100 processing to p52. Actin is shown as a loading control.
A20 controls RANK-dependent osteoclast formation and bone physiology (2022)
Figure 4

Fig. 4 A20 regulates RANK-induced NF-ĸB signalling and osteoclast formation via its ubiquitin binding domains.

(a) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transfected with an NF-κB luciferase reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and plasmids encoding different A20 variants: A20WT, A20DUB (C103A mutation), A20ZnF4 (C624A/C627A), A20ZnF7 (C775A/C779A), and A20ZnF4ZnF7 (C624A/C627A/C775A/C779A). Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency. Data are expressed as the mean of technical triplicates ± SD. **** represent p<0.0001; ns, non-significant (Two-way ANOVA test with Sidak's multiple comparisons between indicated genotypes).

(b) qRT-PCR mRNA expression analysis on RNA from BMDM cultures isolated from control (A20WT), A20KO, A20ZnF4ZnF7 and A20DUB mice incubated for the indicated time-points with RANKL (100ng/ml). Data are expressed as mean ± SEM. *, **, *** and **** represent p<0.05, p<0.01, p<0.001 and p<0.0001, respectively; ns, non-significant (Two-way ANOVA test with Sidak's multiple comparisons between indicated genotypes for each time-point).

(c-d) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice, stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.

(e) BMDMs isolated from control (A20WT), A20ZnF7, A20ZnF4ZnF7 and A20DUB mice were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.

(f) Wild-type BMDMs were pretreated for 1h with HOIPIN-8 (50µM) or left untreated and stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.

Expanded View Figure legends
Mitochondria are required for pro-ageing features of the senescent phenotype (2016)
Figure 1

Figure 1 - Mitochondria are key factors for the development of pro-ageing features of cellular senescence.

A) Scheme illustrating the experimental design: Parkin-expressing and Control MRC5 fibroblasts were irradiated with 20Gy X-ray and 2 days after were treated with 12.5µM CCCP for 48h. At day 4 after irradiation, CCCP was removed and cells were kept in culture with normal serum supplemented medium. Cells were harvested at days 10 or 20 after irradiation (6 or 16 days after CCCP treatment, respectively) for senescent phenotypes analysis;

B) Western blots showing absence of mitochondrial proteins from the different mitochondrial complexes: NDUFB8 (complex I), SDHA (complex II), UQCRC2 (complex III) and MTCO-1 (complex IV), in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data is representative of 3 independent experiments;

C) Cellular oxygen consumption rates (OCR) in senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data were obtained using Seahorse XF24 analyzer and shows mean±S.D. n=4 technical repeats (representative of 2 independent experiments);

D) Representative 3D EM pictures of senescent (20 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts with or without CCCP. Mitochondria are in red, the nucleus in blue;

E) Representative images of cell size, Sen-β-Gal activity (blue cytoplasmic staining), macro-H2A foci and SDHA immunofluorescence in proliferating and senescent (10 days after 20Gy X-ray) Parkin- expressing MRC5 fibroblasts;

F) Quantification of ROS levels (DHE fluorescence), Sen-β-Gal positive cells and Senescence-associated heterochromatin foci (SAHF) observed by DAPI in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

G) Representative western blots showing p21 and p16 expression in proliferating and senescent (10 days after 20Gy X-ray) Control and Parkin-expressing MRC5 fibroblasts. Data are representative of 3 independent experiments;

H) Secreted protein measured in a cytokine array (RayBiotech) of a variety of inflammatory proteins in proliferating and senescent (10 days after 20Gy X-ray) Parkin-expressing MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.
Mitochondria are required for pro-ageing features of the senescent phenotype (2016)
Figure 3

Figure 3 - PGC-1β-dependent mitochondrial biogenesis downstream of the DDR modulates cellular senescence.

A) Representative images of colony assays of wild-type and PGC-1β -/- MEFs grown at 3% or 21% O2 (10 days after seeding 5,000 cells/well). Data are representative of 3 independent experiments;

B) Effect of 3% or 21% O2 and X-ray irradiation (at 3% O2) on the percentage of Ki67 (at day 6) and Sen-β-Gal positive cells (at day 10) and number (N) of 53BP1 foci (at day 6) in wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

C) Representative images of Sen-β-Gal activity (blue cytoplasmatic staining), Ki-67 and 53BP1 foci in proliferating and senescent wild-type and PGC-1β-/- MEFs (scale bar=10µm);

D) mRNA expression of PGC-1β CXCL-1 and p16 in proliferating and senescent (10 days after 10Gy X-ray) wild type and PGC-1β-/- MEFs. Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

E) Effects of overexpression of PGC-1β on the percentage of Ki67 and Sen-β-Gal positive cells, number (N) of 53BP1 foci and percentage change of mitochondrial mass (NAO intensity) in proliferating and senescent (2 days after 10Gy X-ray) MEFs cultured at 3% O2. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.
Mitochondria are required for pro-ageing features of the senescent phenotype (2016)
Figure 4

Figure 4 - mTORC1 integrates DDR signals towards mitochondrial biogenesis during cellular senescence.

A) Representative western blot of mTORC1 activity measured by phosphorylated p70S6K (T389) from 6 to 72 hours after 20Gy in MRC5 fibroblasts. Data are representative of 3 independent experiments;

B) Representative western blots of the mitochondrial proteins TOMM20, NDUFB8 (complex I), SDHA (complex II), UQCR2 (complex III) and MT-CO1 (complex IV) following 20Gy irradiation with or without 100nM Rapamycin treatment in MRC5 fibroblasts. Data are representative of 4 independent experiments;

C) Effect of 100nM rapamycin on mitochondrial mass (measured by NAO fluorescence) 2-4 days following replication exhaustion (RS) or genotoxic stress (generated by X-ray irradiation, Etoposide, Neocarcinostatin (NCS), H2O2 or telomere dysfunction (TRF2BM)) in a variety of cell lines. Data are mean from 3 independent experiments per cell line or treatment;

D) (top) Representative transmission electron microscopy (T.E.M.) micrographs of proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. Mitochondria are labeled in pink. Scale bar=2µm; (bottom left and middle) Quantification of mitochondrial volume fraction (%Vv) and mitochondrial number per cross section in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM rapamycin. T.E.M. mitochondrial analysis are mean±S.E.M of 24 electron micrographs per condition; (bottom right) mtDNA copy number analysis by q-PCR in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

E) Representative western blots showing expression of phosphorylated p70S6K (T389) and AKT (S473), the mitochondrial protein NDUFB8 and the DDR downstream target p21 in MRC5 fibroblasts treated with or without 10µM of the ATM inhibitor KU55933 and in fibroblasts from a patient with Ataxia Telangiectasis (AT) at different time points after 20Gy X-ray. Data are representative of 3 independent experiments (ATM inhibitor) and 1 experiment (AT patient);

F) Western blots showing the effect of the ATM inhibitor KU55933 on γH2A.X, AKT phosphorylation and p21 expression in MRC5 fibroblasts after 20Gy X-ray. Data is from one experiment;

G) Effect of rapamycin and/or ATM inhibitor (KU55933) on mitochondrial mass (NAO intensity) in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Asterisks denote statistical significant P<0.05 One-way ANOVA.
Mitochondria are required for pro-ageing features of the senescent phenotype (2016)
Figure 5

Figure 5 - mTORC1 activation promotes ROS-dependent DDR and contributes to the senescent phenotype via PGC1-β dependent mitochondrial biogenesis.

A) ROS levels (DHE intensity) and mean number (N) of γH2A.X foci after mTOR knockdown (72 hours) in proliferating and senescent (2 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

B) ROS levels (DHE intensity) and mean number (N) of γH2A.X foci in proliferating and senescent (3 days after 20Gy X-ray) MRC5 fibroblasts treated with or without 100nM Rapamycin and/or 2.5mM of the antioxidant NAC. Data are mean±S.E.M of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

C) (top) Scheme illustrating the experimental design: human MRC5 fibroblasts were irradiated with 20Gy X-ray and treated at day 3 after IR with 10µM of the ATM inhibitor (ATMi) KU55933 and/or 100nM Rapamycin (Rap); (bottom) Effect of single or combined inhibition of ATM and mTORC1 on the mean number (N) of γH2A.X foci, Sen-β-Gal activity and p21 expression in proliferating and senescent (10 days after 20Gy X-ray) MRC5 fibroblasts. Data are mean±S.E.M of n=3 independent experiments. Western blots are representative of 3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

D) ROS levels (DHE intensity) in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β-/- MEFs (top) and PGC-1β overexpressing MEFs (bottom). Data are mean±S.E.M. of n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

E) (top) Representative images and quantification of immunofluorescence staining against FLAG-PGC-1β (red) and 53BP1 (green) in proliferating and senescent (2 days after 10Gy X-ray) PGC-1β overexpressing MEFs cultured at 3% O2, with or without 250µM of the antioxidant Trolox (scale bar=10µm). Data are mean±S.E.M, n=3 independent experiments; Asterisks denote statistical significant P<0.05 One-way ANOVA.

F) (top) Effect of overexpression of mutated Rheb (N153T) and (bottom) effect of 100nM rapamycin on the mean number (N) of 53BP1 foci in proliferating and senescent (3 days after 10Gy X-ray) wild-type and PGC-1β -/- MEFs. Data are mean±S.E.M of n=3 independent experiments (at least 125 cells were analyzed per condition). Asterisks denote statistical significant P<0.05 One-way ANOVA.
Synthesis and degradation of FtsZ quantitatively predicts the first cell division in starved bacteria (2018)
Figure 9

Figure 9: Experimental modulation of parameters support the FtsZ limitation model.

Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate (f = 0.38 mmol/g/h). Induction level corresponds to the amount of doxycycline (max - 50 ng/uL, half - 10 ng/uL, and zero 0 ng/uL) added at the onset of starvation. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line.

Lag time reduced with synthesis levels of titrated FtsZ in the wild-type strain at f = 0.31-0.32 mmol/g/h.

Lag time increased with titrated synthesis of ClpX in wild-type cells at f = 0.43-0.45 mmol/g/h.

Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate (f = 0.38 mmol/g/h). Induction level corresponds to the amount of doxycycline (max - 50 ng/uL, half - 10 ng/uL, and zero 0 ng/uL) added at the onset of starvation. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line.

Lag time reduced with synthesis levels of titrated FtsZ in the wild-type strain at f = 0.31-0.32 mmol/g/h.

Lag time increased with titrated synthesis of ClpX in wild-type cells at f = 0.43-0.45 mmol/g/h.

Expanded View Figures
Targeting the SHP2 phosphatase promotes vascular damage and inhibition of tumor growth (2021)
Figure 1

Figure 1. EphrinB2, TIE2 and SHP2 phosphorylation in mouse tumor vessels.

(A,B) Representative B16F10 tumor established in a syngeneic mouse showing that tumor vessels, identified by CD31 immunostaining, are generally p-EphrinB+ (scale bars: 500μm in A; 50μm in B).

(C) CD31+ endothelial cells lining a vessel are mostly p-EphrinB+ whereas NG2+ perivascular cells are mostly p-EphrinB-; cell nuclei are identified by DAPI; confocal images (scale bar: 10μm).

(D) EphrinB2 is detected in cell lysates of 4T1, LLC1 and HUVEC, but not in B16F10 cells; representative of 3 immunoblotting.

(E) EphB4 depletion in B16F10 cells; immunoprecipitation/immunoblotting; control: no infection; pLKO: infection with control lentivirus; shEphB4: infection with silencing lentivirus.

(F) B16F10 tumors induced by inoculation of EphB4-depleted B16F10 cells contain reduced levels of EphB4 compared to control pLKO-infected B16F10; representative immunoblotting (of 16 mice); TRP2: tyrosine-related protein 2 (TRP2).

(G) CD31+ vessels in EphB4-depleted tumors are generally p-EphrinB+; scale bar: 200μm.

(H) Ang-2 dose and time-dependently activates p-EphrinB, p-TIE2 and p-AKT in HUVEC; immunoblotting results; NA3VO4: sodium orthovanadate.

(I) Tumor vessels in B16F10 tumors are mostly p-TIE2 (Tyr992)+ and pSHP2(Tyr542)+; confocal images (scale bars: 200μm).