EMBO/soda-nel huggingface dataset:
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 3

Inhibition of the fission machinery or of Bnip3 partially protects from muscle loss. (A) RNAi‐mediated knockdown of Fis1 showed by western blot assay. MEF cells were transfected with vectors expressing four different Fis shRNAs. For in vivo experiments Fis (4) was used. Efficiency of protein knockdown is indicated in Supplementary Table S2. (B) Blockade of Fis1 and of Bnip3 by RNAi reduces muscle loss during fasting. Adult skeletal muscles were transfected with specific shRNAs for mouse Fis1 (oligo 4) and mouse Bnip3 or co‐transfected and 8 days later mice were starved for 48 h. Cross‐sectional area (CSA) of transfected fibres, identified by GFP, was measured as described earlier (*P0.01) n=450. (C) RNAi of Fis1 and Bnip3 significantly prevents MuRF‐1 and atrogin‐1 promoters activation during starvation. TA muscles were transfected with either 5 kb of the MuRF‐1 promoter (left panel) or 3.5 kb of atrogin‐1 promoter (right panel) with or without shRNAs vectors against Fis and Bnip3. In fasting experiments, after 7 days from transfection mice were starved for 24 h. A renilla‐luciferase construct (pRL‐TK) was co‐transfected to normalized for transfection efficiency. The activation of MuRF‐1 and atrogin‐1 promoters was determined by firefly activity and normalize for the renilla. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05). (D) Muscle atrophy induced by FoxO3 is reduced by d.n.DRP1. Adult skeletal muscles were transfected with HA‐tagged c.a.FoxO3 with or without d.n.DRP1 and examined after 2 weeks. CSA of transfected fibres, identified by anti‐HA immunofluorescence, was measured as described earlier (*P0.001) n=400. (E) Blockade of the fission machinery by d.n.DRP1 or Bnip3 inhibition by RNAi reduces FoxO3‐mediated autophagosome formation. Animals were transfected with YFP‐LC3, c.a.FoxO3 in presence or absence of d.n.DRP1 or shRNAs against Bnip3. Twelve days later, muscles were collected and analysed for fluorescent vesicles formation (*P0.001) n=450. (F) FoxO3‐dependent activation of MuRF‐1 and atrogin‐1 is significantly reduced by inhibition of the fission machinery. MuRF‐1 promoter (left panel) or atrogin‐1 promoter (right panel) was co‐transfected into adult TA muscle with or without c.a.FoxO3 and in the presence or absence of d.n.DRP1. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 4

Overexpression of DRP1/Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo. In vivo imaging of the mitochondrial network was performed in muscles expressing Bnip3, an atrophy‐related gene under FoxO3 control, and DRP1/Fis1 proteins. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and either mock vector, Bnip3, HA‐DRP1, Fis1 or DRP1/Fis1. Seven days later (for Bnip3) or 12 days later (for DRP1 and Fis1), muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Muscles transfected with mtM13‐YFP and mock vector showed the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria (upper panel‐IMF). Scale bar: 20 μm.
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 5

Expression of pro‐fission proteins and Bnip3 induces muscle atrophy. (A) Adult TA muscles were transfected with either DRP1, Fis1, DRP1 and Fis1 or mock vector. Muscles were collected 12 or 21 days after transfection and cross‐sectional area of transfected fibres, identified by immunofluorescence, was measured as described earlier (*P0.001) n=400. (B) Bnip3 or Bnip3 l expression for 7 days causes a 20% reduction of the cross‐sectional area. TA muscles were transfected with Bnip3, Bnip3l or mock vector. Cross‐sectional area of transfected fibres, identified by immunofluorescence, was analysed (*P0.001) n=500. (C) Quantification of LC3‐positive autophagic vesicles in adult muscles co‐tranfected with YFP‐LC3 and DRP1, Fis1 or DRP1/Fis1 expression plasmids or mock vector. (D) Mutations in BH3 domain of Bnip3 (Bnip3BH3mut) do not affect BNIP3‐mediated autophagy. Adult muscles were co‐transfected with YFP‐LC3 and Bnip3 or Bnip3BH3mut expression plasmids and LC3‐positive vesicles were quantified. (E) Bnip3‐mediated atrophy is not modified by mutating the BH3 domain (Bnip3BH3mut). Adult TA muscles were transfected with Bnip3 or Bnip3BH3mut expression plasmids and cross‐sectional area was quantified 7 days later and plotted as % of controls (*P0.001) n=440.
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 6

Fis1K148R induces mitochondrial fission but does not cause muscle atrophy and preserves mitochondrial function. (A) In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1K148R. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and Fis1K148R. Twelve days later muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved. (B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001). (C) hFisK148R‐transfected muscles were collected 12 and 21 days after transfection. Cross‐sectional area of transfected fibres, identified by anti‐myc immunofluorescence, was quantified (n=550). (D) Fis1‐dependent activation of MuRF‐1. MuRF‐1 promoter was co‐transfected into adult TA muscle with or without Fis1 or Fis1K148R. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least five independent experiments±s.e.m. (*P0.05).
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 7

Mitochondrial fission and dysfunction contribute to muscle atrophy through AMPK. (A) Left panel: mitochondrial fission activates AMPK. HEK293 cells were transfected with a mock vector (control), Bnip3, DRP1, Fis1 or co‐transfected with DRP1 and Fis1. Cells were collected 24 h later and lysates were analysed by immunoblotting. Right panel: immunoblotting analysis shows AMPK phosphorylation in EDL and soleus muscles during muscle atrophy. P‐AMPK/AMPK ratio analysed by densitometric analysis is indicated below the panels. (B–E) Inhibition of AMPK blocks Bnip3‐ and DRP1/Fis1‐mediated atrophy. (B) Adult TA muscles were transfected with Bnip3 together with d.n.AMPK or mock vector and cross‐sectional area was measured (*P0.001) n=480. (C) Adult TA muscles were transfected with DRP1 and Fis1 together with d.n.AMPK or mock vector. Twelve days later muscles were collected and cross‐sectional area was quantified (*P0.001) n=400. (D, E) RNAi‐mediated knockdown of AMPK α 1 and α 2 inhibits Bnip3‐ and DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with Bnip3 or DRP1 and Fis1 and pSuper vectors expressing shRNAs against lacZ (control) or AMPK. Twelve days later, TA muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.
Mitochondrial fission and remodelling contributes to muscle atrophy (2010)
Figure 8

AMPK activation amplifies FoxO3‐dependent muscle atrophy. (A, B) AMPK activation induces expression of atrophy‐related genes in C2C12 myotubes. Myotubes were treated for 6 h with 1 mM AICAR. (A) Quantitative PCR analysis was performed in triplicates using specific oligonucleotides (see Supplementary Table 1) (*P0.01). (B) Immunoblotting analysis shows that AICAR treatment causes phosphorylation of AMPK and the downstream target ACC but does not affect phosphorylation of AKT and FoxO3. (C) FoxO3 binding to atrogin‐1 and MuRF‐1 promoters, as determined by ChIP, is induced by AICAR treatment. (D) RNAi‐mediated knockdown of FoxO3 inhibits DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with DRP1 and Fis1 and pSuper vectors expressing shRNAs against either lacZ (control) or FoxO3. Twelve days later, muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.
AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model (2022)
Figure 1

Fig. 1 : DGKk expression is altered in FXS, and FMRP is required for translation control via its N-terminal region.

A) Western blot analysis of lysates from cerebellum of control and FXS patients ''FXS'' (n = 4). Extract of pCI-hDGKk-transfected Hela cells (hDGKk) was used as antibody specificity control and size marker. Representative images of immunoblots probed with antibodies against the indicated proteins are shown. GAPDH was the loading control. Quantification of Western blots is shown on right. Protein amounts of DGKk are normalized to GAPDH and presented as fold change relative to control. Bars and error bars represent means ± SEM. P value was calculated using an unpaired two-tailed t-test. *P < 0.05.

B) Schematic map of DGKk constructs used for the transfection experiments and subsequent vector preparations. The different domains of the protein are indicated and represented at scale (repeated EPAPE, Pleckstrin Homology PH domain, phorbol ester/diacyl glycerol binding C1 domain, catalytic split C4 a and b domains, putative PDZ binding motive, HA-tag, grey bars interval 1kB), 5' and 3' UTR regions are represented with blue line, 3'UTR not at scale (3.8 kB).

C-D) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-mDGKk-HA or non-transfected (NT) and pre-transfected 24h before with siRNA control (siC) or against FMRP (siFMR1). GAPDH was used as a loading control. For quantification, the DGKk and FMRP signals were normalized against GAPDH signal and presented relative to the signal for siC treated cells (C). Immunoblots and quantification of lysates from Cos-1 cells transfected with pCI (empty plasmid), pCI-HA-∆N-DGKk, or pCI-mDGKk-HA, and pre-treated with siRNA control (siC) or against FMRP (siFMR1) (D). Quantifications as in C. Each point represents data from an individual culture, and all values are shown as mean ± SEM. P values were calculated using one-way ANOVA with Tukey's multiple comparison test. ****P < 0.0001; ns, P > 0.05.
AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model (2022)
Figure 2

Fig. 2 : ∆N-DGKk localizes and impacts cellular signaling similarly to full-length protein.

A) HeLa cells transfected with indicated plasmids expressing mouse (m) or human (h) DGKk were analyzed by immunofluorescence confocal microscopy for HA tag (red) and Dapi staining (blue). ∆C1-hDGKk construct bears deletion of phorbol ester C1 domain (328-449).

B) Immunoblots and quantification of lysates from Cos-1 cells transfected with plasmid pCI-HA-∆N-DGKk, untransfected (NT), or plasmid pCI control, with the indicated amount of plasmid (µg) and after 24h, incubated with puromycin (20µg/ml, 30 min) to measure rates of protein synthesis. GAPDH was used as a loading control. 0 indicates no puromycin treatment, -/+ indicates treatment without or with DGK inhibitor (DGKi) 3 µM R59022 and 0.2 µM R59949 at 6 µM, 15 min. Densitogram of puromycin incorporation is presented as change relative to mock transfected conditions. Each point represents data from an individual culture, and all values are shown as mean ± SEM. P values were calculated using one-way ANOVA with Tukey's multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001.

C) Representative immunofluorescence staining of cortical neuron cultures transduced at 8 DIV (days in vitro) with 10e9 VG/ml of AAVRh10-GFP or AAVRh10-∆N-DGKk and assessed after 5 days using anti-MAP2 and anti-HA for ∆N-DGKk or direct 488 nm excitation for GFP. DAPI was used to visualize nuclei on merged images. Scale bar, 40 µm.

D) Representative immunoblots of lysates from WT and Fmr1-KO cortical neurons transduced with AAVRh10-∆N-DGKk (AAV ∆N-DGKk), at the indicated titers (VG/ml culture volume) and quantification of phosphorylation and total levels of eIF4E. GAPDH was used as a loading control. For quantification, the phospho-protein signal was normalized first against total protein signal and is presented relative to the signal for WT culture. Each point represents data from an individual culture, and all values are shown as mean ± SEM . P values were calculated using one-way ANOVA with Tukey's multiple comparison test. **P < 0.01.
AAV-delivered diacylglycerol kinase DGKk achieves long-term rescue of fragile X syndrome mouse model (2022)
Figure 3

Fig. 3 : ∆N-DGKk expression in vivo with AVV vectors corrects abnormal cortical phosphatidic acid level of Fmr1-KO mice.

A) Scheme of timeline analyses performed.

B) Viral titers (viral genome copy VG per cell) determined by qPCR in cortical, hippocampal and rest of brain areas of Fmr1-KO mice treated with saline solution (Vehicle), AAVPHP.eB-∆N-DGKk (PHP.eB), AAVRh10-∆N-DGKk (Rh10) 8 weeks after injections. Data are mean ± SEM. Each dot represents an individual mouse.

C) Immunoblots and quantification of ∆N-DGKk protein in lysates from brain areas of mice treated as in B. GAPDH was used as a loading control. Densitogram of ∆N-DGKk expression is presented as change relative to vehicle conditions. Data are expressed as median with interquartile range with minimum and maximum values and analyzed (n = 5).

D) Representative coronal brain sections processed for detection of ∆N-DGKk using immunohistochemistry on Fmr1-KO mice treated with indicated treatment, 8 weeks post-injections, counter stained with eosin hematoxylin. Adjacent sections were immunolabelled with NeuN. 3 mice per genotype were processed. The sections shown are between Bregma levels -1.50 mm and -1.80 mm. Scale bar is 2 mm. Magnifications of regions of cortex (c), hippocampus (h), and striatum (s) are shown in side panels, scale bar 200 µm.

E) Total phosphatidic acid (PA) level measure by mass spectrometry in cortex of WT mice treated with saline solution (WT-S) and Fmr1-KO mice treated with saline (Fmr1-S), AAVPHP.eB-∆N-DGKk (Fmr1-PHP.eB), AAVRh10-∆N-DGKk (Fmr1-Rh10) 8 weeks after injections. Data are expressed as mol % of total lipids and represented as median with interquartile range with minimum and maximum values. Statistics: one-way ANOVA and Tukey's multiple comparisons test, n=8 individual animals, except for WT-S and Fmr1-S n=11. **P < 0.01, ***P < 0.001.

F) Total diacylglycerol (DAG) level in cortex measured as in E).
ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification (2020)
Figure 1

Figure 1. A CRISPR knockout screen identifies novel genes that arrest the proliferation of cells with centrosome amplification.

(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(B) Schematic overview of the screen design.

(C) Schematic showing the procedure for a CRISPR/Cas9 positive selection screen to identify gene knockouts that increase the proliferation of cells with extra centrosomes.

(D) Top hits that emerged from the screens ranked by MaGeCK FDR value. Blue hits are genes required for centriole duplication or stability. Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes.

(E) Hits with known role in centriole duplication or stability.

(F) Candidate hits responsible for arresting the proliferation of cells with centrosome amplification. Purple hits correspond to PIDDosome genes. Red hits correspond to downstream effectors. Green hits are novel regulators.

(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.

(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.

Data information: Asterisks indicate statistically significant differences between measurements (*: P<0.05; **: P<0.01; ***: P<0.001, ****: P<0.0001). Statistics for all Figures were calculated using a two-tailed Student's t-test.
ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification (2020)
Figure 2

Figure 2. Distal appendage proteins are required to recruit PIDD1 to the mature parent centriole.

(A) Top, widefield image showing the localization of CEP164 and PIDD1 at the mature-mother centriole. Bottom: Representative 3D STORM image of the same centriole showing PIDD1 localization. STORM image colors correspond to the Z-depth with red being the closest to the coverslip and blue being the most distant.

(B) 3D STORM image of PIDD1 at the mature mother centriole. The overlaid mask represents the inner and outer diameters of the PIDD1 signal.

(C) Inner and outer diameter measurements for distal appendage proteins and PIDD1. Measurements in the shaded region were previously reported in (Bowler et al., 2019). Data displayed as box and whisker plots, where the box represents the upper and lower quartile and the whiskers represent the s.d. Scale bars: 1 μm for all wide-field images of centrioles and 500 nm for STORM images. Data acquired across n ≥6 cells.

(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(E) Schematic depicting the hierarchy of recruitment of distal appendage proteins.

(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.

(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

Data information: Asterisks indicate statistically significant differences between measurements (**: P<0.01; ***: P<0.001, ****: P<0.0001). Statistics for all Figures were calculated using a two-tailed Student's t-test.
ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification (2020)
Figure 3

Figure 3. ANKRD26 is required to arrest cell proliferation in response to centrosome amplification.

(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm.

(E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m.

(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells.

(I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m.

Data information: Asterisks indicate statistically significant differences between measurements (**: P<0.01; ****: P<0.0001). Statistics for all Figures were calculated using a two-tailed Student's t-test.
ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification (2020)
Figure 4

Figure 4. The coiled-coil domain of ANKRD26 is required for PIDD1 recruitment to the centriole and PIDDosome activation following centrosome amplification.

(A) Top: schematic representation of PIDD1 and its various cleavage products. Bottom: HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.

(B) HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.

(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes.

(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.

(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes.

Data information: Asterisks indicate statistically significant differences between measurements (*: P<0.05; **: P<0.01; ***: P<0.001, ****: P<0.0001). Statistics for all Figures were calculated using a two-tailed Student's t-test.