EMBO/soda-nel huggingface dataset:
Myopathy reversion in mice after restauration of mitochondrial complex I (2019)
Figure 2

Fig. 2. Lack of NDUFS3 in skeletal muscle is associated with a mitochondrial myopathy. (A) Representative image of quadriceps from WTFlx and Ndufs3 smKO animals of 8 months of age. Muscle weight from Ndufs3 smKO males (black bar) and wild-type mice (white bar) from 6, and 8 months of age (n=5 each group). Bars represent means ± standard error (SEM). (B) Representative H&E, COX, and SDH staining of quadriceps from WTFlx and Ndufs3-Mlc1f smKO at 8 months old. (C) Cross-sectional area (CSA) of quadriceps muscle fibers. A minimum of 100 fibers/sample were analyzed (n =3/genotype). The percentage of central nuclei in skeletal muscle fibers of quadriceps muscle of WTFlx and Ndufs3 smKO at 8 months (n =3 mice for WTFlx and n=4 mice for Ndufs3 smKO). P values were calculated by Student's t test. (D) Serum lactate levels of WTFlx and Ndufs3-smKO animals at different ages. Ndufs3 smKO male mice (filled black squares) showed increased levels compared to matched WTFlx males (empty black squares). Error bars represent SEM. (E) BN-PAGE In-Gel Activities of Complex I and IV measured in homogenates from quadriceps of wild-type and Ndufs3-Mlc1f smKO animals at 2 and 4 months. (F-G) Western blots of muscle homogenates (quadriceps) of WTFlx and Ndufs3-Mlc1f smKO animals at different ages (2 and 4 months, respectively) using antibodies against PGC-1α, COX1 (complex IV subunit), NDUFS3, VDAC1, SDHA (complex II subunit), and β-actin. (H) Quantification of the western blots in panels F and G. Bars represent means ±SEM of n=4 for each group. P values were determined by Student's t test.
Myopathy reversion in mice after restauration of mitochondrial complex I (2019)
Figure 3

Fig. 3. Replacement of NDUFS3 in 15-18 days old smKO mice prevented the development of myopathy. (A) Representation of the vectors used in this study. WTFlx Ndufs3 cDNA was expressed from rAAV9-Ndufs3. An rAAV9-eGFP expressing viral preparation was used as a control. (B) Experimental design used for rAAV9 administration and analyses. (C) The KO-GFP mouse was visibly smaller and weaker than a KO-NDUFS3 mouse, at 7 months. (D) Females body weight injected with rAAV9-Ndufs3 (heretofore referred to as KO-NDUFS3) mice were similar to WTFlx mice body weight and significantly higher than the rAAV9-eGFP injected mice (KO-GFP) starting at 5 months. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. (E) Males body weight of KO-NDUFS3 mice were similar to WTFlx mice, and significantly different from KO-GFP from 4 months onwards. Statistical analysis was performed by One-way ANOVA followed by a Tukey post-test. (F) Treadmill performance was significantly improved in the KO-NDUFS3 mice when compared to the KO-GFP, from 2 months onwards. (G) Four limb strength was evaluated by the grid hang test. KO-NDUFS3 mice performed significantly better starting at 3 months, when compared to the KO-GFP mice. F) and G) Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. (H) Quadriceps and gastrocnemius wet weight of 6 months old mice was similar between WTFlx and KO-NDUFS3 mice. Quadriceps weight was significantly increased in KO-NDUFS3 mice when compared to KO-GFP injected. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. ns- not significant. (I) Survival curve of Ndufs3 smKO mice injected with rAVV9-Ndufs3 (blue line) and with rAVV9-eGFP (green line). WTFlx mice are depicted in black. rAAV9-Ndufs3 injections extended the survival of the smKO mice. P values were calculated using Log-rank (Mantel-Cox) Test; p=0.0016 (KO-NDUFS3 vs KO-GFP); p=0.0014 (KO-GFP vs WTFlx); n-indicates the number of animals used per group.
Myopathy reversion in mice after restauration of mitochondrial complex I (2019)
Figure 4

Fig. 4. NDUFS3 replacement (at P15-18) normalized the levels of lactate and mitochondrial markers. (A) Western blots of 6 months old quadriceps of WTFlx, KO and KO-NDUFS3 injected mice at 15-18 days old. GFP expression was observed only in the KO-GFP mice. Protein loading was obtained with stain-free gel technology. (B) Quantification of NDUFS3, COXI and SDHA expression levels in female mice showed in A. (C) Western blot of BN-PAGE of quadriceps of 6-month-old mice. We analyzed: CI (NDUFA9, NDUFB8), CIV (COX1), CII (SDHA). (D) Quantification of the BN-PAGE western blots from female mice shown in panel C. (E) BN-PAGE in-gel activity of CI and CIV in quadriceps of 6-month-old mice. (F) Quantification of BN-PAGE in-gel activities in females. (G) Total mtDNA levels in quadriceps from males and females were determined by qPCR using the ratio ND1/18S. Inset shows the appearance of the quadriceps, reflecting normalization of mitochondrial levels after replacement of NDUFS3. (H) Spectrophotometric CI/citrate synthase activity ratio was measured in quadriceps homogenates of 6-month-old males and females. (I) Plasma lactate levels were determined as described in methods. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test.
Myopathy reversion in mice after restauration of mitochondrial complex I (2019)
Figure 5

Fig. 5. Replacement of NDUFS3 in 2-months-old (P60) smKO mice reverted the mitochondrial myopathy. (A) Gene therapy strategy used in in 2-month-old mice. (B) The smKO mice group that received retro-orbital injections at 2 months was tested prior to the rAAV9-Ndufs3 injection showing a significant exercise intolerance attested by the number of falls in the treadmill. The KO-NDUFS3 gradually recovered over time. Individual KO-NDUFS3 mice are color coded. Green triangles represent the animals injected with GFP whereas white triangles represent non-injected mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. (C) The hang test was performed in symptomatic mice which had a significant decrease in four-limb muscle strength and coordination, at 2 months. The KO-NDUFS3 mice showed a significant recovery 3.5 months after injection. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. (D) KO-NDUFS3 muscle weight was similar to WTFlx mice, and it was significantly increased in gastrocnemius when compared to KO-GFP mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. (E) Immunohistochemical analysis with NDUFB8 and COX1 antibodies in quadriceps muscle sections of 6-month-old animals. The expression of NDUFB8 (representative of assembled CI) was recovered in the KO-NDUFS3 mice and COX1 expression was similar to WTFlx animals. (F) COX and COX/SDH activity staining showed that KO-NDUFS3 quadriceps were reverted to levels found in wild-type, at 6 months. n-indicates the number of animals used per group, scale: 100 µm.
Myopathy reversion in mice after restauration of mitochondrial complex I (2019)
Figure 6

Fig. 6. NDUFS3 replacement (at P60) normalized the levels of lactate and mitochondrial markers. (A) Western blots showed NDUFS3 expression in KO-NDUFS3 and GFP expression in the KO-GFP quadriceps samples. PGC-1α expression was normalized in the KO-NDUFS3 mice which was abnormally elevated in the KO-GFP. The same was observed for CIV (COX1), CIII (Core 2) and CII (SDHA). (B) Quantification of western blots showed in A. (C) BN-PAGE western blots showed recovery of Complex I assembly into complex and supercomplexes in KO-NDUFS3 injected mice. CIV and CII were similar to wild-type in the KO-NDUFS3 samples, at 6 months. (D) Quantification of BN-PAGE western blots shown in B. (E) BN-PAGE In-gel CI and IV activities of KO-NDUFS3 mice were also reverted to levels similar to the WTFlx samples (F) Quantification of in-gel activities. (G) Complex I/Citrate synthase activity of KO-NDUFS3 samples measured spectrophotometrically were similar to WTFlx samples and increased in comparison to KO-GFP mice. (H) Total mtDNA levels were determined by qPCR. MtDNA levels in KO-NDUFS3 mice were decreased when compared to KO-GFP mice and not different from the levels in WTFlx mice. Statistical analysis was performed by One-way ANOVA followed by Bonferroni post-test. (I) The serum lactate levels were similar to WTFlx in KO-NDUFS3 samples and significantly decreased when compared to KO-GFP levels. The individual KO-NDUFS3 animals are color coded. Green triangles represent the animals injected with rAAV9-eGFP whereas black triangles represent animals which did not receive an rAAV9-eGFP injection. Statistical analysis was performed by One-way ANOVA followed by Tukey post-test. Data represent means ±SEM.
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 1

Fig. 1: ADAM10 is required for NrCAM shedding in primary neurons. (A) Schematic diagram of NrCAM's domain structure and sequential proteolytic processing. NrCAM is firstly cleaved by furin, then by ADAM10 and finally by the γ-secretase. The red antibody indicates the binding region of the N-terminal NrCAM antibody. ICD (intracellular domain), CTFf (C-terminal fragment created by furin cleavage), CTFα (C-terminal fragment created by ADAM10 cleavage). (B) Detection of soluble, cleaved NrCAM (sNrCAM) and full-length, mature NrCAM (mNrCAM) and soluble APPα (sAPPα) in neuronal supernatants and lysates (prepared from E16 neurons), treated with GI254023x (5 µM), or solvent for 48h. (C) Detection of sAPPα, sNrCAM and mNrCAM in ADAM10 cKO neuronal supernatants and lysates at 7 days in vitro (DIV7). The neurons prepared from floxed ADAM10 (ADAM10 fl/fl) mice were infected with a lentivirus encoding improved Cre recombinase (iCre) or a control GFP lentivirus at DIV2. Conditioned media were collected for 48 h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 2

Fig. 2: NrCAM is a γ-secretase substrate: HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 3

Fig. 3: ADAM10 is controlling surface expression of NrCAM and NrCAM-mediated neurite outgrowth. (A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. (B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown. (C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 4

Fig. 4: Acitretin increases APP but not NrCAM shedding in primary neurons and AD patients. (A) Wt neurons were kept in culture until DIV5, then the cells were treated with acitretin (4 µM) or vehicle control for 48h, as described earlier (Tippmann et al, 2009). (B) Densitometric quantifications of the Western Blots are shown. Given are mean +/- the standard error of the mean (*** p < 0.001; **** p < 0. 0001, two-sided students t-test n = 9-20). The mean levels of solvent treated cells were set to 1. (C) Detection of sNrCAM and human serum albumin (hSA) in AD patients CSF that had been treated with acitretin (30 mg/d) or placebo for 30 days (n = 9) (Endres et al, 2014). (D) Densitometric quantification of the Western Blots in (C). sNrCAM/hSA ratios were calculated, and then the treatment to baseline ratios (t/b) for every patient were calculated. Two-sided student's t-test (n = 9). Given are mean +/- the standard error of them mean. Representative Western Blots are shown.
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 5

Fig. 5: Neuronal stimulation by NMDA increases ADAM10-mediated NrCAM shedding. (A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies. (B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 6

Fig.6: Mass spectrometric analysis of acitretin CSF samples. Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.
NrCAM is a marker for substrate-selective activation of ADAM10 in Alzheimer's disease (2019)
Figure 7

Fig.7: TSPAN15 has opposite effects on ADAM10-mediated NrCAM and APP shedding. HEK293 cells were transiently transfected with hNrCAM, FLAG-tagged hTSPAN15, either plasmids or the empty vector. Detection of sAPPα and sNrCAM in the conditioned media and ADAM10 in the cell lysate. Densitometric quantification of the Western Blots are shown (** p < 0.01), (*** p < 0.001), (**** p < 0.0001), two-sided students t-test, or one-way ANOVA with post hoc Dunnett's test (n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.

Table 1: Significantly changed proteins in AD patients treated with acitretin:

Table 1: Significantly changed proteins in AD patients treated with acitretin: This table contains all proteins with a t-test significance p-value of less than 0.05 (not corrected for multiple testing) and a change of the protein level of more than 20 % compared to placebo-treated controls. ADAM10 substrates are marked in bold.

Expanded view figure legends
B1-type cyclins control microtubule organization during cell division in Arabidopsis (2021)
Figure 3

Figure 3. Embryo sac development is controlled by CYCB1 members.

A. DIC images of abnormal embryo sacs in cycb1 mutant combinations. Red arrowheads indicate the visible nuclei in Col-0 embryo sacs (central and egg cells) and the corresponding structures in the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant. Scale bars: 20 μm.

B. Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations (n = 202-459 embryo sacs per genotype). Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. WT = wildtype.

C. DIC images of embryo sacs 3 DAP with wildtype pollen (female x male). Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant as a female donor, while the control Col-0 x Col-0 cross exhibits a developing embryo. Scale bars: 20 μm.

D. Quantification of seed abortion in different cycb1 mutant combinations. Graph represents the average seed abortion rate per plant ± SD of three biological replicates, n = 463-579 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).

E. Silique pictures of cycb1 triple and quadruple mutants. White arrowheads indicate early aborted ovules. Scale bars: 500 μm.
B1-type cyclins control microtubule organization during cell division in Arabidopsis (2021)
Figure 4

Figure 4. Pollen development is affected by mutations in the CYCB1 group.

A, B. DAPI staining of pollen in cycb1 mutants, including pollen configurations found in cycb1;1-/- cycb1;2+/- cycb1;3+/- cycb1;4-/- mutants (B). Scale bars: 5 μm.

C. Quantification of DAPI-stained pollen configurations in different cycb1 mutant combinations, n = 420-616 pollen grains per genotype. Different letters indicate significant differences in the proportion of abnormal pollen (uni- and bicellular) in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.

D. Alexander staining of mature pollen indicating pollen viability. Scale bars: 5 μm.

E. Quantification of Alexander-stained pollen viability, n = 403-498 pollen grains per genotype. Different letters indicate significant differences in the proportion of dead pollen in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.

Data information: Red arrowheads indicate dead pollen, while white arrowheads indicate bicellular pollen.
B1-type cyclins control microtubule organization during cell division in Arabidopsis (2021)
Figure 5

Figure 5. The double cycb1;1 cycb1;2 mutant has abnormal microtubule arrays.

A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm.

D. Quantification of wildtype (WT), double and misplaced PPBs. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.

E. Quantification of PPBs with prominent perinuclear microtubules (MTs). Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett's multiple comparisons test (** P < 0.01 and **** P < 0.0001).

F. Quantification of spindles with lagging chromosomes. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (** P < 0.01 and ns = non-significant).
B1-type cyclins control microtubule organization during cell division in Arabidopsis (2021)
Figure 6

Figure 6. Both cycb1;1 cycb1;2 and cycb1;2 cycb1;3 have abnormal phragmoplasts and extended spindle stages.

A. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). Scale bars: 5 μm.

B. Quantification of wildtype (WT) and abnormal phragmoplasts. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.

C. Quantification of the different mitotic stages in roots of the different genotypes. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.
B1-type cyclins control microtubule organization during cell division in Arabidopsis (2021)
Figure 7

Figure 7. CYCB1-CDKB2;2 complexes are the most active and are able to phosphorylate a MT-nucleation factor.

A. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2.

B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2.

C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein.

D. Time-lapse of confocal microscope pictures of root meristematic cells tagged with GFP-GIP1 in Col-0 (top panel) and cycb1;1 cycb1;2 (bottom panel). GIP1 localizes at the nuclear polar caps, followed by co-localization with microtubules at the spindle and phragmoplast arrays. In cycb1;1 cycb1;2 double mutants, GIP1 exhibited an abnormal localization, being found at the spindle (magenta arrowheads) and phragmoplast (white arrowheads) midzones, which are normally devoid of the protein. Scale bars: 5 μm.

Expanded View Figure legends