EMBO/SourceData huggingface dataset:
Figure 3B. Example of bead velocity as a function of time in recycling conditions. The integral of this curve (or area under curve) represents the length of actin polymerized in the comets during the time of the experiment.C. Example of cumulative length of actin polymerized as a function of time.
Figure 4A. Left: Snapshots of actin comet tails in recycling conditions with the indicated ATP concentrations. Right: Tracking of the comet shown in the snapshots. Time is encoded in color.
Figure 5B, C. Test of reaction mix aging without beads. Reaction mix was prepared without beads and left on the bench at room temperature. Then at different time intervals, fresh beads were added to the mix. After 24 hours (right part of the graph), All (fresh) proteins in the assay were added one by one with fresh the beads. Velocity of fresh beads was estimated for those different times. Composition of the reaction mix: 3 µM actin, 6 µM profilin, 90 nM Arp2/3, 15 nM Capping protein, 200 nM cofilin, 400 nM Cyclase Associated Protein (CAP). t = 0: N = 4, n = 494 comets. t = 6 hours: N = 1, n = 65 comets. t = 24 hours: N = 2, n = 46 comets. t = 24 hours + fresh mix: N = 1, n = 32 comets. t = 24 hours + actin: N = 2, n = 108 comets. t = 24 hours + profilin: N = 1, n = 10 comets. t = 24 hours + capping protein: N = 1, n = 12 comets. t = 24 hours + Arp2/3 complex: N = 1, n = 11 comets. t = 24 hours + ADF/cofilin: N = 1, n = 17 comets. t = 24 hours + CAP: N = 1, n = 7 comets. Individual points for each comet are represented with mean and standard deviation superimposed.D. Test of actin monomer aging. Actin monomers were prepared in salt-free motility buffer (Ca-ATP monomers, see Methods) or in salt-free motility buffer with EGTA and MgCl2 (Mg-ATP monomers, see Methods) and left on the bench at room temperature overnight. The day after, aged actin monomers were added to a fresh mixture (without actin monomers) in presence of fresh beads . Velocity of beads was measured in the different conditions. Composition of the reaction mix: 3 µM actin, 6 µM profilin, 90 nM Arp2/3, 15 nM Capping protein, 200 nM cofilin, 400 nM Cyclase Associated Protein (CAP). Ca-ATP actin monomers fresh: N = 1, n = 50 comets. Ca-ATP actin monomers (old) N = 1, n = 46 comets. Mg-ATP actin monomers (fresh): N = 2, n = 121 comets. Mg-ATP actin monomers (old): N = 2, n = 134 comets. Individual points for each comet are represented with mean and standard deviation superimposed.
Figure 1A. Triple RNAscope in situ hybridization showing Engrailed-1 (En1), Choline acetyltransferase (ChAT) and Calbindin-1 (Calb1) expression in the lumbar spinal cord. En1 is expressed in V1 interneurons, dorsal (V1P) and ventral (V1R) to the main ChAT-expressing motoneuron pool. Ventral interneurons correspond to Renshaw cells as shown by Calb1 expression. Scale bar: 500 μm.B. RT-qPCR of RNA from the lumbar enlargement at 4.5- and 16-months of age shows stable En1 expression in WT at both ages and a two-fold reduction of expression in heterozygous mice. Unpaired two-sided t-test. **p<0.005; ***p<0.0005. n=3. Values are mean ± SD.C. Triple staining EN1 IHC (green), En1 RNAscope ISH (red) and ChAT RNAscope (blue) demonstrating the double-staining of En1 mRNA and protein (EN1) in the V1 interneuron population (left panel insets, arrowheads point towards examples of double-stained V1 interneurons), and the presence of EN1 protein in large cells not expressing En1 mRNA (left panel) but expressing ChAT (right panel insets). Scale bar: 500 µm, 30 µm for high magnification insets. D. Left: EN1 (red) detected with the LSBio antibody is localized in ChAT-expressing neurons (green) in the ventral horns of the spinal cord. Right: EN1 signal is lost upon preincubation of the antibody with 1.5 M excess of recombinant hEN1. Scale bar: 50 µm.
Figure 2A. Analysis of the number of En1+, Calb1+ and ChAT+ neurons (triple RNAscope). At 4.5 months, WT and En1-Het mice show no difference in the number of cells expressing En1, Calb1 or ChAT. Unpaired two-sided t-test. n=5-6. Data information: The analysis of neuron number in A were performed once. Values are mean ± SD.C. Cresyl violet and ChAT staining at the lumbar level show no medium size (200-299 µm2) cell loss (γMNs) and a decrease of about 50% in the number of large size (>300 µm2) cells (αMNs). Unpaired two-sided t-test. **p<0.005; ****p<0.0001. n=5. Data information: The direct comparison of Cresyl violet and ChAT in C were performed once. Values are mean ± SD.D. Lumbar level Cresyl violet staining shows that, in contrast with small and medium size neurons (interneurons and γMNs, left and center panels), large size neurons (αMNs, right panel) undergo progressive death first measured at 4.5 months. The values represent the average number of cells per ventral horn. For the small neurons (100-199 µm2), there was no main effect, two-way ANOVA for repeated measures for treatment group: F(1,43)=0.0017, p=0.968, ns. For the medium sized neurons (200-299 µm2) two-way ANOVA for repeated measures for treatment group: showed no main effect F(1,43)=2.085, ns. For the large neurons (>300 µm2), two-way ANOVA for repeated measures showed a significant main effect F(1,43)=59.99, p<0.0001. Post-hoc comparisons were performed by unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=5 to 6. Data information: The longitudinal studies in D were performed once. Values are mean ± SD.E. Compared to WT mice, En1-Het mice experience gradual strength loss. This loss is observed with the forepaw grip strength (left panel), the inverted grid test (center panel and the hindlimb extensor reflex test (right panel). Strength loss is first observed between 2 and 3 months, thus before measurable αMN cell body loss. Two-way ANOVA showed significant main effects for grip strength (F(1,136)=19.18, p<0.0001), inverted grid test (F(1,103)=143.1, p<0.0001) and extensor score (F(1,103)=10.1, p<0.0001). Comparisons were made by unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n=4 to 20. Data information: The longitudinal studies in E were performed once. Values are mean ± SD.
Figure 3A. NMJs of En1-Het and WT mice show similar numbers of AChR clusters (left panel) and late-occurring (15.5 months) decrease in endplate area (center panel) and percentage of perforated endplates (right panel). The number of AChR clusters seems to decrease between 2 and 3 months, but two-way ANOVA showed no significant genotype effect (F(1,47)=0.1291, ns). There was a significant main effect for endplate area (F(1,47)=5.778, p=0.0202) and for the percentage of perforated endplates (F(1,47)=13.82, p=0.0005). Post hoc analysis revealed significant genotype differences at 15.5 month of age. Unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (**p<0.005). n=4 to 8. Data information: The longitudinal study in A was performed once. Values are mean ± SD.B. Left panel illustrates the use of Alexa Fluor 488-conjugated α-bungarotoxin (α-BTX, in green) and of neurofilament and synaptic vesicle glycoprotein antibodies (2H3 and SV2A, in red) to evaluate the percentage of fully occupied endplates (>80% occupancy). The right panel shows that the % of fully occupied endplates decreases progressively in the En1-Het mouse, starting between 3 and 4.5 months of age. Scale bar: 50µm. Two-way ANOVA showed a significant main effect (F(1,47)=45.45, p<0.0001). Unpaired two-sided t-test with equal SD comparing WT with En1-Het at each time point (*p<0.05; **p<0.005). n=4 to 8. See details of analysis in the Methods section. Data information: The longitudinal study in B was performed once. Values are mean ± SD.
Figure 4C. Left panel illustrates that expressing the scFvEN1, but not scFvMUT, abolishes EN1 staining by LSBio anti-EN1 antibody in ventral horn ChAT+ cells and right panel quantifies this inhibition 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (n=5 mice per group, ***p<0.0005, ****p<0.0001). Scale bar: 100 µm.E. Six months following infection (7-month-old mice), extracellular EN1 neutralization does not modify the number of AChR clusters, nor the endplate surface area, nor the percentage of perforated endplates. In contrast, the percentage of fully occupied endplates is diminished (right end panel). 1-way ANOVA followed by Tukey corrected post-hoc comparisons. (**p<0.005). n=5 per treatment.F. Six months following infection, extracellular EN1 neutralization does not globally modify the total neuron number of cells at the lumbar level (left panel). A separate analysis of small (100-199 µm2), medium (200-299 µm2), and large (>300 µm2) neurons demonstrate a specific (p<0.0557) loss of the latter category (αMNs). 1-way ANOVA followed by Tukey corrected post-hoc comparisons. N=5 per treatment.
Figure 5A. Mice were tested for strength at three months of age, before the onset of αMN loss, but when strength has already decreased in En1-het mice as measured in the forepaw grip strength, inverted grid, and extensor reflex tests (left side of each graph). The next day, the En1-het mice were separated into two groups. One group received buffer and the other group recombinant hEN1 (1 µg in 5 µl), injected at the L5 level. One and a half months later (4.5 months of age) En1-het mice injected with hEN1 have recovered normal strength, in contrast with non-injected mice or mice injected with buffer. Unpaired two-sided t-test used at three months of age. ****p<0.0001. For 4.5-month comparisons, 1-way ANOVA followed by Tukey corrected post-hoc comparisons. ***p<0.0005; ****p<0.0001. n=9 to 31
Figure 6B. Based on the results shown in A, a new experiment was performed with a second injection 12 weeks after the first injection at 3 months of age. Again, the injection at three months of age restored strength and in mice receiving the second injection strength was maintained an additional ten weeks or more compared to mice receiving a single injection. The three strength graphs demonstrate a positive effect of the second injection with values intermediate between those measured in WT mice and En1-Het mice with a single injection. The percentage of fully occupied endplates and the number of αMNs are back to wild type values in En1-Het mice injected twice. Two-way ANOVA revealed significant main effects for grip strength (F(2,81)=25.47, p<0.0001), inverted grid (F(2,91)=51.96, p<0.0001) and extensor score (F(2,104)=30.42, p<0.0001). At all times groups were compared by Unpaired T-test with equal variances through 12 weeks. After, the groups were compared by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). For the endplate analysis and αMNs, groups were compared by 1-way ANOVA followed by Tukey corrected post hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001). n= 4 to 10.
Figure 7A. Top left panel shows that 24 hours after intrathecal injection (1 μg in 5µL) at the L5 level of 2-month-old mice, hEN1 (red) can be primarily visualized in ventral horn ChAT+ cells (green). Arrowheads show hEN1 internalized by MNs. Scale bar: 100 µm. Top right panels show the progressive accumulation and clearance of hEN1 in ventral horn MNs, with a peak between 6 and 24 hours. EN1 was revealed by the 86/8 antibody allowing for the visualization of exogenous EN1 only (scale bar: 100µm). Bottom panels show the quantification of EN1 in γMNs and αMNs with the 86/8 (two left panels) and the LSBio (two right panels) antibodies. Since the LSBio sees both endogenous and exogenous EN1, the increase is only 3-fold, but qualitatively, the results are very similar, demonstrating rapid internalization and clearance of the exogenous protein and allowing one to calculate a half-life of 24 hours. 1-way ANOVA followed by Tukey corrected post-hoc comparisons (*p<0.05; **p<0.005; ***p<0.0005). When not significant, p values are shown. n=3. Values are mean ± SD. Data information: The internalization experiments were performed once with each antibody.
Figure 8B. Immunohistochemical staining shows the presence of high amounts of p62/SQTSM1 in ChAT+ cell bodies of 3-month-old mice. Scale bar: 50 µm.C. Intensity measurements demonstrates that the mean of SQTSM1/p62 expression increases with age in WT γMNs (left) and αMNs (right). However, comparing WT and En1-Het shows a significant difference only at 3 months and not later. Unpaired two-sided t-test. (*p<0.05; **p<0.005; ***p<0.0005, ****p<0.0001). Data information: this experiment was done once. Values are mean ± SD. Between 151 and 1215 neurons and 3 to 5 mice were analyzed for each condition.D. Mean intensity of SQTSM1/p62 expression is increased in γMNs (left) and αMNs (right) in mice expressing scFvEN1 6 months after virus injection (7-month-old mice) demonstrating that EN1 extracellular neutralization increases SQTSM1/p62 expression. Expression of the mutated antibody (scFvMUT) does not increase p62 expression. Unpaired two-sided t-test. (*p<0.05; **p<0.005; ***p<0.0005, ****p<0.0001). Data information: this experiment was performed once. Values are mean ± SD. Between 448 and 759 neurons and 5 mice were analyzed for each condition.
Figure 9A. Left panel: Injection and analysis protocol. Right panel: muscle strength analysis demonstrating that hEN1 injection at 1 month prevents muscular strength decrease observed in 3-month-old En1-Het mice. One-way ANOVA followed by two-sided t-test. (*p<0.05, ****p<0.0001). N=6-10. Data information: This experiment was performed once. Values are mean ± SD.B. Left panel: Increased p62/SQTSM1 staining in 3-month-old ChAT+ MNs from control En1-Het is abolished by hEN1 injection at 1 month. Right panel: quantification of p62/SQTSM1 staining in γMNs and αMNs of control and hEN1-injected 3-month-old En1-het mice. One-way ANOVA followed by two-sided t-test. (**p<0.01, ****p<0.0001). Between XXX neurons in YY mice were analyzed in this experiment. Data information: This experiment was performed once. Values are mean ± SD. Between 111 and 303 neurons and 4-5 mice were analyzed for each condition.
Figure 1(C) Cell morphology and SA-β-Gal activity of mouse or NMR fibroblasts 12 or 21 days after DXR treatment. Arrowheads indicate dying cells. The number in the upper left corner indicates Hoechst-positive nuclei. Scale bar, 100 μm.Time-course analysis of mouse or NMR fibroblasts after DXR treatment quantification of BrdU-positive cells (%) (E)Time-course analysis of mouse or NMR fibroblasts after DXR treatment qRT-PCR analysis of the expression of INK4a (F) normalized to ACTB mRNA levelsqRT-PCR analysis of the expression of INK4a (J) in shINK4a-transduced NMR fibroblasts at 21 days after DXR treatment.quantification of Annexin V-positive cells (%) (K) in shINK4a-transduced NMR fibroblasts at 21 days after DXR treatment.
Figure 2(B) qRT-PCR analysis of the expression of INK4a at 12 days after INK4a or mock transduction, normalized to ACTB mRNA levels.(C) Cell morphology and SA-β-Gal activity of mouse or NMR fibroblasts 12 days after INK4a transduction. Arrowheads indicate dying cells. ​​The number in the upper left corner indicates Hoechst-positive nuclei. Scale bar, 100 μm.(F) Western blot analysis of RB in mouse or NMR fibroblasts at 12 days after INK4a transduction ﻿(p, phospho-specific antibody). ACTIN was used as a loading control. The arrowhead indicates a non-specific band.(I) SA-β-Gal activity in the adherent living cell population and floating dead cell population in mouse or NMR fibroblast cultures at 12 days after INK4a transduction. Scale bar, 100 μm. (J) Quantification of SA-β-Gal-positive cells in the adherent living cell population and floating dead cell population after the same treatment as in I.
Figure 3(C and D) Quantification of SA-β-Gal-positive cells (%) (C) and Annexin V-positive cells (%) (Annexin V+/PI- as early apoptotic and Annexin V+/PI+ double-positive as late apoptotic) (D) in NMR-fibroblasts transduced with different forms of SV40 Large T antigen (LT, LT∆, and LTK1) and INK4a. OE; overexpression.(F) Quantification of SA-β-Gal-positive cells (%) at 21 days after DXR treatment in NMR-fibroblasts transduced with different forms of SV40 Large T antigen (LT, LT∆, and LTK1). (G) Quantification of Annexin V-positive cells after the same treatment as in F. Annexin V+/PI- cells were counted as early apoptotic cells and Annexin V+/PI- cells as late apoptotic cells.
Figure 4(C) Quantification of PI-positive cells in NMR and mouse fibroblasts 18 h after 6 h of treatment with the indicated doses of hydrogen peroxide (H2O2). Data are expressed as the mean ± SD from n = 3 biological replicates.
Figure 5(B) Left, representative images of lipid peroxidation (green) staining in NMR fibroblasts at 12 days after INK4a-transduction. Scale bar, 50 μm. Right, quantification of signal intensity of lipid peroxidation staining. Data are expressed as the mean ± SD from n = 3 biological replicates.(C) Quantification of PI-positive cells in NMR fibroblasts treated for 24 h with N-acetyl L-cysteine (NAC), Trolox, or Tempol at 20 days after INK4a transduction (%). Data are expressed as the mean ± SD from n = 6 biological replicates except for Trolox and Tempol (n = 4).(D) Western blot of monoamine oxidase (MAO)-A and MAO-B in mouse or NMR fibroblasts at 20 days after INK4a transduction. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO-A or -B/ACTIN intensity (n = 3 average).(G) Western blot of MAO-A and MAO-B in mouse or NMR fibroblasts at 21 days after doxorubicin (DXR) treatment. ACTIN was used as a loading control. Numbers below the gel images indicate quantification of MAO-A or -B/ACTIN intensity (n = 3 average).
Figure 6(B) qRT-PCR analysis of INK4a expression in the skin, muscle, and white adipose tissue (WAT) of 6-week-old mice (young; 6 w), 1-year-old mice (middle-aged; 1 y), 1-year-old NMRs (young; 1 y), and 15-year-old NMRs (middle-aged; 15 y).(D) SA-β-Gal activity (SA-β-Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 2, 7, 14, and 21 days after Bleo administration.Time-course analysis of mice or NMR lungs after Bleo administration: qRT-PCR analysis of the expression of INK4a normalized to ACTB mRNA levels (F)Time-course analysis of mice or NMR lungs after Bleo administration ; quantification of TUNEL-positive cells (%) (G).
Figure 7(B) SA-β-Gal activity (SA-β-Gal, blue; nuclei, green) and TUNEL staining (TUNEL, red; nuclei, blue) in lungs of mice or NMRs at 21 days after Bleo administration, with or without Phe are shown.qRT-PCR analysis of INK4a expression normalized to ACTB mRNA levels (D) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.quantification of TUNEL-positive cells (%) (E) in mouse or NMR lungs at 21 days after Bleo administration, with or without Phe.
Figure 3B. qRT-PCR expression analysis of lncMN2-203 isoform, lncMN2-202/204, miR-325-3p and miR-384-5p embedded miRNAs in WT, ∆SP1 and ∆SP2 EB6. Data, that are expressed as mean (error bars represent SD), are normalized over Atp5o mRNA levels (for lncRNA isoform quantification) or over snoRNA U25 levels (for miRNA quantification) and are reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).C. Cytofluorimetric analysis of cells from dissociated EB6. The right diagram reports the cell distribution (percentage of the peak) according to GFP levels, from WT (gray line), ∆SP1 (purple line) and ∆SP2 (blue line) EB6. In the histogram aside, the percentage of GFP+ cells in WT, ∆SP1 and ∆SP2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).D. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆SP mutants (clone 1 and clone 2). The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Data are expressed as mean and error bars represent SD. Scale bar=100 mm. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).
Figure 1(A) Timelapse TIRF microscopy images showing an EL4-MyD88-GFP cell interacting with a IL-1 functionalized SLB. MyD88-GFP assembles into puncta that over time cluster and coalesce at the cell:SLB interface. Scale bar, 2 µm.(C) TIRF and brightfield microscopy images of EL4-MyD88-GFP cells incubated for 30 mins with IL-1 functionalized SLBs formed off grid and on 1 and 2.5 µm grids. In the presence of a 1 and 2.5 µm grids, Myddosomes only coalesce within individual corrals and do not form multi-complex clusters. Scale bar, 5 µm.(D) Time series showing Myddosome formation in an EL4 cell interacting with both continuous and 2.5 µm gridded partitioned SLBs. t = 0s denotes the start of cellular observation. Scale bar, 5 µm.(F) Quantification of MyD88-GFP puncta maximum fluorescence intensity normalized to GFP from cells stimulated off and on 2.5 or 1 µm grids, at a ligand density of 10 IL-1/µm2. Violin plots show the distribution of average max puncta intensities from individual cells across replicates. Data points superimposed on the violin plots are the averages from independent experimental replicates. The average max MyD88 puncta intensity (mean ± SEM): for off grid is 7.5 ± 1.8 GFPs, n = 8 biological replicates, with 88304 MyD88-GFP puncta from 161 cells measured in total across all replicates; for 2.5 µm grids is 5.2 ± 1.7 GFPs, n = 3 biological replicates, with 13164 MyD88-GFP puncta from 31 cells measured in total across all replicates; for 1 µm grids is 2.3 ± 0.1 GFPs, n = 8 biological replicates, with 126600 MyD88-GFP puncta from 254 cells measured in total across all replicates. Bars represent mean ± SEM.(G) Widefield images showing RelA localization in unstimulated EL4 cells and EL4 cells stimulated by SLB formed on and off grids. EL4 were fixed 30 min after addition to IL-1-functionalized SLBs and stained for RelA (magenta); DAPI stained nuclei (blue). Scale bar, 10 µm.(H) Quantification of RelA nucleus to cytoplasm ratio. Violin plots show the distribution of measurements from individual cells. Data points superimposed on the violin plots are the averages from independent experiments. The RelA nucleus to cytoplasm ratio of single cells marked with X in panel (G) are superimposed on the violin plot. RelA nucleus to cytoplasm ratio off grids, on 2.5 and 1 µm grids, and unstimulated conditions are 0.45 ± 0.01, 0.34 ± 0.02, 0.32 ± 0.02, 0.21 ± 0.03 (mean ± SEM) respectively. The p value are * = 0.0133 and ** = 0.0027. Bars represent mean ± SEM (n = 3 to 5 biological replicates, with a total of 18370, 3627, 2988 and 965 cells measured off grids, on 2.5 µm and 1 µm grids, and unstimulated conditions respectively). Statistical significance is determined using unpaired two-tailed Student's t-test.