EMBO/SourceData huggingface dataset:
Figure 4(B) Metabolomic analysis focused on strains with over-expression or deletion of the AYT1 gene shows a significant reduction or accumulation, respectively, in acetyl-glutamate compared to the matched control strain of each mutant. Only metabolites changing significantly in at least one of the two conditions are presented. D-EIP is D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate.(D) A growth assay in a condition that does not induce high levels of acetyl-CoA in peroxisomes (glucose) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital. In stationary phase, the over-expression (OE) of AYT1 grows to a lower density than the control strain, plausibly due to burdening of peroxisomal functions. (E) A growth assay in a condition expected to elevate acetyl-glutamate levels in peroxisomes (oleate as a sole carbon source and glutamate as a nitrogen source) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control. Data information: The growth assays in D and E were done in three biologically independent replicates and error bars were plotted. Note that some error bars are shorter than the symbol, hence are not visible in the graph.(G) A β-oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon- or 18 carbon-free fatty acids in media supplemented with 0.5% glucose shows a significant reduction in β-oxidation activity compared to the wild type and the two single mutants, suggesting an overlapping role for Fsh3 with Lpx1.This assay was done in three biologically independent replicates. Bars represent the mean and error bars represent the standard deviation.
Figure 5(E) Western blot analysis of Fbp1-HA levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1 targeting) demonstrates that two hours after the transition from glucose to ethanol (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA levels, while for Δpex3 and Δpex5 cells Fbp1-HA levels remain low. Antibodies were used against the HA tag (for Fbp1) and Actin as a loading control.
Figure 3(G) Size-exclusion chromatography profiles and corresponding SDS-PAGE of the indicated samples. Two mutant RZZ complexes containing mutations in ROD L110F-L119F-L120K or I191M are indicated respectively as FFK and I/M. Note that in the bottom SDS-PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker. For all other shown SDS-PAGE gels, the marker precedes the first fraction.
Figure 4(B) Coomassie and ProQ Diamond-stained gels on mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM.(D) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM reversine, 3.3 µM nocodazole, and 10 µM MG132 for 1 hour and imaged while in mitosis. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed. (E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm.(F) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.(G) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole for an additional hour. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed.(H) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G. Red lines indicate median with interquartile range.(I) Levels of CENP-E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly. Three biological replicates were performed. (J) Scatter dot plots representing normalized total area of the CENP-E and Zwilch signals, normalized to the RNAi negative control, in the indicated number of cells from the experiment shown in panel I. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.
Figure 5(E) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS-PAGE separation of reactions. Data information: Two technical replicates for panels , E
Figure 6(D) Heat-induced mChRZZGFPSF and MPS1-induced rings of mChRZZmChSF have similar diameters. As shown in panel B, heat- and MPS1-induced rings of mChRZZmChSF also have very similar dimensions. Data information: Experiments in D were carried out once.
Figure 7(A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction. Two technical replicates were performed.(D) Representative images of fixed HeLa cells electroporated with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly by RNAi. Spindly localization was detected with an antibody against the C-terminal region of Spindly. Corona expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI to stain for DNA. Scale bar: 10 µm. Experiments were replicated three times.(E) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (G) Kinetochore intensities were quantified like in panel E. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.
Figure 1A. Schematic representation of the experimental protocol. Abbreviations: LCM, laser capture microdissection. B, C. RT-PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Abbreviations: Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; -, PCR no template control; NT, RT no template control. F. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. Abbreviations: MB, molecular beacon; Tm, MB melting temperature.H. Quantification of the expression levels of pre-mir-181a-1 using the 2-ΔCt method and U6 as normalizer from small total RNA fraction (<~150 nt). Each data point corresponds to one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm.M. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 354 puncta, 32 axons (MB), 337 puncta, 32 axons (cy5-pre-miR-181a-1). n=5 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon.N. Representative image of RGC axons. White, red and blue arrows indicate, respectively, co-localized, single MB, and single pre-miR-181a-1 puncta. Scale bars: 5µm.
Figure 2B. Representative image of a single distal RGC axon from MB-electroporated retina. Growth cone wrist and central domain are indicated with white arrow and star, respectively. Dashed white line delineates the axon. Abbreviations: MB, molecular beacon. Scale bars: 5µm. C. Illustrative kymograph. Scale bars: 5µm. D. Frequency distribution (in percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta along the RGC axon shaft. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.E. Average velocity of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; antero, anterograde movement; retro, retrograde movement.F. MSD data for MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles were fitted with an anomalous diffusion model and α thus calculated (red). Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiment. Values are mean ± SEM. Abbreviations: MB, molecular beacon.G. MSD alpha coefficient distribution for each single MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particle. Each data point corresponds to one particle. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range.H. Relative frequency distribution (percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles. Each data point corresponds to one independent experiment. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.J. Representative kymographs before Nocodazole (top panel; - Noco) and 30 min after 2.4μM Noco bath-application (bottom panel; +Noco). Stationary puncta in both panels are indicated with black arrows. Scale bars: 5µm.K. Frequency distribution (in percentage) of MB punctum speed. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco); 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.L. Frequency distribution of MB punctum speed. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco), 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.
Figure 3B. Representative axon where MB-labeled pre-miR-181a-1 (red) and CD63-eGFP-labeled vesicles (green) are co-trafficked (white arrows). Abbreviations: MB, molecular beacon. Scale bars: 5µm.C. Representative kymographs. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon. Scale bars: 5µm.D. Composite kymograph shown in (C) where the individual traces where drawn and color coded. Yellow trajectories represent co-trafficking MB-labeled pre-miRNA (red) and CD63-eGFP-labeled vesicle (green). Abbreviations: MB, molecular beacon. Scale bars: 5µm.E. Representative time-lapse depicting MB-labeled pre-miR-181a-1 (red arrow) and CD63-eGFP-positive vesicle (green arrow) co-trafficked along the axon shaft to the growth cone (delineated with dashed white lines) wrist (white arrowhead) and central domain (white star). Scale bars: 5µm.F. Frequency (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 253 puncta, 22 axons (MB+), 306 puncta, 22 axons (CD63+). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.G. Frequency distribution (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 92 puncta, 22 axons (anterograde), 78 puncta, 22 axons (retrograde). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.I. Representative image of an electroporated and dissociated RGC. Abbreviations: CD63, CD63-eGFP. Scale bars: 5µm.K,L. Fluorescence intensity of MB (K) or CD63-eGFP (L) measured in axons and growth cone central domains. Each data point corresponds to one axon or a growth cone central domain. Total number of axons analyzed: 16. n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon; GC, growth cone.
Figure 5A. Schematic representation of the experimental protocol. B. Representative Xenopus growth cones stained with anti-Dicer and anti-Ago2. White dashed lines delineate growth cones. Scale bars: 5µm. C. Representative E13.5 mice brain cross-section (dashed red line in the schematic) comprising the optic nerve (ON) stained with anti-neurofilament and anti-HA antibodies to detect RGC axons and (FLAG-HA2)-Dicer, respectively. Note the absence of HA signal in wild type (WT) mice. The white dashed line delineates the ON. Zoom of the triple stained ON (right panel): Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se (arrowheads). n=3 independent experiments. Abbreviations: WT, wild type; HA, (FLAG-HA2)-Dicer; E13.5, embryonic day 13.5; ONH, optic nerve head; ON, optic nerve. Scale bars: 30µm.D. Representative P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.
Figure 6Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Sema3A (B) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=3 (B) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Slit2 (C) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=4 (C) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.
Figure 7B. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. C. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 45 brains (co-MO), 52 brains (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino. D. Schematic representation of the experimental paradigm. Concentrations used: 0.5μg/μL pCS2-mCherry or pCS2-eGFP plasmids; 250μM miR-181-MO or control MO cocktail; 50μM miR-181 or control mimics (top). Representative images of RGC axons within the optic tectum. White arrows indicate axons targeted both by MO (green) and miRNA mimics (red) (bottom). Abbreviations: co-MO, control morpholino. Scale bars: 20µm. E. Quantification of misprojecting axons. The number reported on the bars are the total number of co-electroporated axons. Note how miR-181 mimics rescued aberrant misprojection of morphant axons in vivo. Each data point corresponds to one independent experiment. Total number of axons and brains analyzed: 96 axons, 4 brains (co-MO+ctrl mimics), 101 axons, 4 brains (miR-181-MO + ctrl mimics), 134 axons, 4 brains (miR-181-MO+miR-181 mimics). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino; ns, not significant. F. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. G. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 21 brains (co-MO), 31 brains (miR-181-MO). n=3 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino. I. Frequency (in percentage) of the amount of time embryos spent on black background. Each data point corresponds to one embryo. Total number of embryos analyzed: 43 embryos (co-MO), 45 embryos (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.
Figure 9B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range.D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant.G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III.
Figure 1Left graph reports the tumor volume in NSG mice injected with control (shCTR) and antimiR-9 FaDu cells followed for up to 35 days (n=5 mice/group). Data represent the mean (±SD) and unpaired t-test was used to verify the statistical significance at each time point. On the right, typical images of explanted tumors formed by control (shCTR) and antimiR-9 FaDu cells at necropsy.Typical images of Hematoxylin&Eosin (H&E - upper images) and Ki67 expression (Ki67 - bottom images) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in H. 4X and 20X magnification are shown. Right graph reports the percentage of Ki67 positive cells. Each dot represents one tumor. Data are expressed as mean (±SD) and unpaired t-test was used to verify the statistical significance.
Figure 2qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.Graph reporting the cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and unpaired t-test was used to verify the statistical significance per each dose.
Figure 3qRT-PCR analyses of Sp1 expression in control (shCTR) and antimiR-9 FaDu cells transiently transduced with PGK-miR-9 or control vector as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. qRT-PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR-9 or control vector. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control (shCTR) or antimiR-9 FaDu cells. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.