EMBO/soda-nel huggingface dataset:
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 6

Figure 6:

FLNAΔECS mice show arterial and cardiac remodeling.

(A) Dorsal aortae were fixed, cross-sectioned and then stained with Elastica van Gieson stain to visualize collagen around the blood vessel. 6 independent mice (5-6 months) of each genotype were used for the analysis. Scale bar: 50 μm.

(B) Plot shows individual data points (black-wt, red-mutant) of adventitial area in both the genotypes. Measurements show increased adventitial area in FLNAΔECS aortae. P value <0.05 was considered significant (Student's t test)

(C) Representative heart sections of a 5-6 months old mice stained with Masson Trichrome shows increased collagen (blue) around coronary vessels (marked by arrowheads) Scale bar: 50 μm.

(D) Quantification shows a significant increase in perivascular fibrosis in FLNAΔECS hearts. Four sections from each heart were measured. Four mice analyzed for each genotype. P value <0.05 considered significant. (mixed model approach)

(E) Representative longitudinal heart sections stained with H&E staining (RV: right ventricle, LV: left ventricle). Black lines represent thickness of interventricular (IVS) and lateral left ventricular wall (LLVH). Scale bar: 1mm.

(F) Scatter graph shows increased ventricular wall thickness in FlnAΔECS mice hearts. Six male mice (6months old) analyzed for each genotype. P value <0.05 was considered significant. (Student's t test)

(G) Heart cross-sections were stained with FITC-WGA (Wheat germ agglutinin) to visualize cell membranes of cardiomyocytes. Scale bar: 20 μm.

(H) Respective cell areas measured by Image J software and represented by box plots for each animal (six per genotype). Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value. P value <0.05 (mixed model approach) was considered significant.
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 7

Figure 7:

Blood velocity profiling in FLNAΔECS and wt mice.

(A) Graph showing the individual blood velocity profile determined by MRI over time in 4 wt mice (shades of grey) and 5 FLNAΔECS mice (shades of red). The mouse shown in dotted purple line is clearly an outlier within the FLNAΔECS cohort. Aortic velocity waveform represents velocity during different stages of cardiac cycle. A significant reduction of velocity was observed at 40ms in the systolic cycle.

(B) Representative wt (top) and FLNAΔECS (below) mouse showing the blood flow just above the aortic valve. Note: Reduced blood flow in FLNAΔECS mouse as depicted by pseudocoloring. The red circle represents the time window at which the images are shown.
Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila (2022)
Figure 1

Fig 1 Discovery of a BH3-only protein, sayonara

A. Alignment of human BIK and fly CG14044. A part of CG14044 can be aligned with a part of BIK that contains the BH3 motif. Φ, Σ, Z and Γ indicate hydrophobic, small, acidic and hydrophilic amino acid residues respectively. The minimal sequence is L-X(3)-G(or A, S)-D.

B. Schematic representation of CG14044.

C. Neighbor-joining phylogenetic tree of Sayonara orthologs from representative ecdysozoan species. Colored circles indicate bootstrap support. Alignment of the region encompassing the L1-X2-X3-X4-X5-D core pattern characteristic of most BH3 sequences is shown (with conserved leucine and aspartic acid residues in white on black background). The BCL2DB (Aouacheria et al., 2019; Rech de Laval et al., 2014) position-specific scoring matrix corresponding to the BH3 motif was used to scan the listed protein sequences for presence of a BH3 motif (green box depicting positive hits). From this profile-based prediction, a BH3 motif can only be inferred in Sayonara sequences from the Brachyceran infraorder Muscomorpha.

D. Ectopic expression of CG14044 with act-Gal4 induces lethality during the larval stage.

E. Ectopic expression of CG14044 in the wing pouch (GFP area indicated by the dotted line) induces caspase activation and DNA fragmentation (TUNEL) in the larval disc, melanization in the pupal wing (arrow head) and a structure defect in the adult wing.

F. Schematic representation of Sayonara WT and the mutant that lacks the BH3 motif.

G. Deletion of the BH3 motif suppresses the effect of Synr on the wing size.

H. Deletion of the BH3 motif suppresses Sayonara-induced caspase activation, which was detected by the caspase sensor GC3Ai.

I. Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant.

J. Schematic representation of Sayonara WT and coiled-coil domain deletion mutants.

K. Deletion of the coiled-coil domains suppresses the effect of Synr to the wing size.

L. Amino acid sequence of the BH3 motif substitution mutant. The four well conserved hydrophobic amino acids were changed to hydrophilic glutamic acid.

M. Amino acid substitution at the BH3 motif or the coiled-coil domain suppresses the effect of Synr on the wing size.

Data information: Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Scale bars, 100 μm in E (cleaved caspase) and H, 20 μm in E (TUNEL) and 500 μm in E (pupae and adult wing).
Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila (2022)
Figure 2

Fig 2 Synr interacts with Debcl and Buffy both genetically and biochemically.

A-D. The Synr-induced wing structural defect is suppressed by knockdown of Debcl, Buffy or DCP1.

E. Quantification of the adult wing size with RNAis for apoptotic genes.

F. Synr-induced caspase activation is suppressed by knockdown of Debcl.

G. GC3Ai signals are suppressed by knockdown of Debcl or Buffy (quantification is shown in Fig EV2H).

H-I. Lysates from HEK293T cells that express Flag-Debcl or Flag-Buffy were incubated with GST or GST-Synr bound to glutathione-Sepharose. Flag-Debcl or Flag-Buffy is pulled down with GST-Synr but not with GST.

J. Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag-Debcl to GST-Synr.

K. Quantification of the GST-pulldown assay shown in J.

L. Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding of Flag-Buffy to GST-Synr.

M. Quantification of the GST-pulldown assay shown in L.

N. Amino acids that were replaced with BPA are shown. Tyrosine124 is within the BH3 motif and phenylalanine141 is in a close proximity to the BH3 motif. Tyrosine340 is for a negative control.

O. Debcl can be photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. BPA at Y340 was used as a negative control. Red arrowheads indicate covalently crosslinked products.

P. Buffy is also photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. Red arrowheads indicate covalently crosslinked products.

Q. GST-Synr pulls down both Flag-Buffy and HA-Debcl simultaneously, suggesting a possibility of complex formation among Synr, Buffy and Debcl.

R. HA-Debcl is detected after two series of immunoprecipitation. The first immunoprecipitation was performed for Flag-Buffy, followed by Flag peptide-mediated elution and secondary precipitation for Myc-Synr. HA-Debcl can be detected after this sequential immunoprecipitation.

Data information: Statistical significance was determined using one-way repeated measures ANOVA with Dunnett's post hoc test. Scale bars, 500 μm in A and 50 μm in F and G
Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila (2022)
Figure 3

Fig 3 Feedback loop between apoptosis and autophagy

A. Ectopic expression of Synr induces autolysosome accumulation, which was detected by Lysotracker, in the wing disc.

B. Ectopic expression of Synr also induces autophagosome accumulation, which was detected by mCherry-atg8a. The dotted square regions are magnified in the right pictures.

C. Synr is colocalized with mCherry-Atg8a (arrowheads) in the wing disc and the salivary gland at 112h AEL.

D-F. Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr.

G. Atg2 knockdown suppresses Synr-induced cell death, which was detected by TUNEL staining.

H-I. Synr-induced caspase activation is suppressed by inhibition of autophagy (atg2, atg8a RNAis) in a similar manner to apoptosis inhibition (Debcl, Dcp-1 RNAis).

J. Synr-induced autolysosome accumulation is suppressed by Dcp-1 knockdown.

Data information: Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Scale bars, 100 μm in A and H, 50 μm in B and J, 10 μm in C and G, 500 μm in D.
Evidence for existence of an apoptosis-inducing BH3-only protein, sayonara, in Drosophila (2022)
Figure 4

Fig 4 Endogenous function of Synr

A. Inhibition of Synr suppresses the wing defect that is induced by p53, a potent apoptosis inducer.

B. Inhibition of Synr suppresses p53-induced caspase activation.

C. Inhibition of Synr in the midgut enterocytes suppresses starvation-induced caspase activation in the midgut.

D. Inhibition of Synr in the midgut enterocytes enhances resistance to starvation.

E. The synr mutant also exhibits enhanced resistance to starvation.

F. A proposed model of Synr-induced cell death. Synr, which functions together with Buffy and Debcl, induces initial caspase activation, which leads to feedback loop between apoptosis and autophagy.

Data information: Statistical significance was determined using a two-tailed unpaired t-test (B), one-way ANOVA with Holm-Sidak's multiple comparisons test (C) and a log-rank (Mantel-Cox) test (D-E). Scale bar, 500 μm in A

Expanded View Figure legends
Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 1

Figure 1. CDC50A is expressed at synapses.

(A) Western Blot shows developmental expression of CDC50A in isolated synaptosomes from mouse brains. The quantification was normalized to the CDC50A expression of P7. GAPDH was used as an internal control. P1, postnatal day 1; 1M, 1 month; 1Y, 1 year. N = 4 for technical replicates.

(B) IHC for CDC50A and RIM1 on cultured neurons at DIV21. The left panel shows CDC50A is expressed in both neuronal soma and processes. The right panel exhibits CDC50A colocalize to pre-synapses alongside neuronal processes. MAP2, Microtubule-associated protein 2, was used to identify neuronal cells and trace dendritic processes. RIM1 is an active zone protein, which marks pre-synapses. Left scale bar = 50 µm; right scale bar = 2 µm.

(C) Quantification of the percentage of RIM1+ pre-synapses colocalizing to CDC50A. N = 3 for biological replicates.

(D) Single cell RNAseq in the retina revealed the percentage of RGCs that express the members of flippase family and their chaperon gene Cdc50a in mice (Macosko et al., 2015).

(E) IHC for CDC50A and vGluT2 in the developmental dLGN at P14, P21 and P30. The left panels show the overview of immunostaining in the dLGN, scale bar = 200 µm. The right panels are the zoomed-in images, scale bar = 5 µm. Arrows indicate the colocalized CDC50A and vGluT2.

(F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. The result shows that a significantly higher percentage of CDC50A+/vGlut2+ synapses at P21 and P30 than P14. N = 6 for biological replicates.

Data information: All data are presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 by one-way ANOVA with Tukey's multiple comparisons test.

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Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 2

Figure 2. CDC50A expression is dependent on neuronal activity.

(A) A scheme showing experimental timeline when neuronal inhibitors were added to and removed from the cultured neurons. A cocktail of TTX, D-AP5 and NBQX was added to neurons at DIV 21.

(B) Quantitative PCR of Cdc50a during neuronal inactivation and recovery. Neurons without any treatment were used as controls. N = 4 for biological replicates, mean ± SEM, ** p < 0.01. **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.

(C) IHC for CDC50A. CDC50A protein intensities were reduced after neuronal inactivation (day 7), and was recovered after removing neuronal inhibitors (day 13). Scale bar, 10 µm.

(D) Relative CDC50A intensities of neurons. N = 5 for biological replicates, mean ± SD, * p < 0.5, **** p < 0.0001 by one-way ANOVA with Holm-Sidak's multiple comparisons test.

(E) CDC50A expression at RGC inputs after 5-day consecutive TTX treatment. Scale bar, 10 µm.

(F) Quantification of the percentage of CDC50A+/vGlut2+ synapses. N = 5 for biological replicates, mean ± SD, **** p < 0.0001 by Student's test.
Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 3

Figure 3. Cdc50a knockdown induces PS exposure at synapses.

(A) Immunostaining of CDC50A on Cdc50a knockdown neurons and controls. Accell Cdc50a siRNA pool was used to knock down Cdc50a in neurons for 7 days. CDC50A expression alongside neuronal processed was reduced after Cdc50a siRNA treatment. Scale bar, 10 µm.

(B) Quantification of relative CDC50A fluorescence intensity in Cdc50a-RNAi and controls. N = 4 for biological replicates, mean ± SD, **** p < 0.0001 by Student's t-test.

(C) Live neurons were imaged with or without Cdc50a siRNA treatment under a confocal microscope. Propidium iodide (PI) was used to label dead cells. In controls, live neurons exhibit a few weak pSIVA signals. However, in Cdc50a-RNAi neurons, more and stronger pSIVA signals were observed alongside neuronal processes. Scale bar, 20 µm.

(D) IHC of Cdc50a-RNAi neurons exhibited pSIVA colocalized with synapsin I, a general synapse marker. The dotted square was zoomed-in shown on the right side. Arrows indicated where pSIVA colocalized with synapsin I. Scale bar, 5 µm.

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Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 4

Figure 4. Cdc50a depletion causes PS exposure at synapses.

(A) AAV2 constructs containing 5'-inverted terminal repeats (ITR), U6 promoter, shRNA sequences, CMV promoter, hChR2-mCherry and 3'-ITR. Cdc50a shRNA was used to knock down Cdc50a. Scrambled shRNA was used as controls.

(B) An experimental diagram showing 1-month old mice were intravitreally injected with AAV2 control and shRNA viruses and collected 17 days later.

(C) Confocal images of dLGN after AAV2 injection. The dLGN was outlined with the dotted line. MCherry reveals axons and synapses of AAV2-infected RGCs. Left scale bar, 50 µm. Right scale bar, 5 µm.

(D) Representative IHC images of CDC50A expression at synapses. Scale bar, 5 µm. Arrows indicate VGluT2+ RGC inputs.

(E) Relative CDC50A expression at mCherry+ synapses in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test.

(F) PSIVA staining in the dLGN. Exposed PS was detected by pSIVA on RGC inputs in Cdc50a-RNAi mice. Scale bar, 5 µm. Arrows indicate pSIVA+/mCherry+ RGC inputs.

(G) Quantification of the percentage of pSIVA+/mCherry+ RGC inputs in total mCherry+ RGC inputs. N = 4 for biological replicates, mean ± SEM, **p < 0.01 by Student's t-test.
Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 5

Figure 5. Cdc50a depletion causes aberrant synapse loss.

(A) An experimental diagram showing 1-month old mice were intravitreally injected with AAV2 and their brains were collected 1 month later.

(B) IHC images showed vGluT2+ signal was dramatically reduced in the outlined area of mice received Cdc50a shRNA viruses, but not in controls. Scale bar, 100 µm. d: dorsal, m: medial.

(C) Single-plane confocal images of vGluT2+ synapses in the dLGN. Dotted line divided the mCherry+ and mCherry- areas. Scale bar, 20 µm.

(D) Quantification of vGlut2+ synapse density in the AAV-infected areas and the neighboring non-infected areas of Cdc50a RNAi mice and controls. N = 5 for biological replicates, mean ± SD, **** p< 0.0001 by by one-way ANOVA with Holm-Sidak's multiple comparisons test.

(E) IHC for vGluT2 and mCherry in the shell and core regions of the dLGN. Scale bar, 100 µm.

(F) Maximum intensity z-projection images of vGluT2+ synapses in the core region of Cdc50a RNAi mice and controls. Right panel showed enlarged synapses in the boxed region. Scale bar, 20 µm.

(G) Quantification of vGluT2+ synapse density. N = 3 for biological replicates, mean ± SD, * p < 0.05 by Student's t-test.

(H) Maximum intensity z-projection images of vGluT2+ synapses in the shell region. Enlarged images (right panel) shows large "complex encapsulated" boutons. Scale bar, 20 µm.

(I) Quantification of vGluT2+ synapse density. N = 3 for biological replicates, mean ± SD, Student's t-test.
Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 6

Figure 6. Cdc50a depletion induces synapse loss.

(A) A schematic diagram of intravitreal AAV2 injection to 1-month-old mice followed by CTB488 and CTB-647 injection after 29 days. The brains were collected one day later.

(B) Confocal images of RGC inputs traced by CTB488 and mCherry. CTB488+ RGC inputs in the Cdc50a-RNAi area overlapped with vGluT2. Scale bar, 20 µm.

(C) Quantification of colocalization among CTB488, mCherry and vGluT2 described ~98% CTB488+/mCherry+ RGC inputs were vGluT2+. N = 7 for biological replicates, mean ± SD.

(D) Images of CTB488 and CTB647 labeling of RGC inputs in the dLGN. Scale bar, 20 µm.

(E) Quantification of CTB488+ inputs (non-infected areas) versus CTB647+ inputs (infected areas) in Cdc50a RNAi mice and controls. N = 3 for biological replicates, mean ± SEM, **** p < 0.0001, two-way ANOVA with Sidak's multiple comparisons test.
Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 7

Figure 7. CDC50A knockdown results in aberrant synaptic engulfment by microglia.

(A) The confocal images, 3D-rendered images and orthogonal views of microglia engulfing vGluT2+ synapses in controls and Cdc50a-RNAi mice. Scale bar, 10 µm.

(B) Quantification of engulfed vGluT2+ synapses in microglia. % Engulfment of vGluT2+ synapse = Volume of vGluT2+ synapse inside microglia / Volume of microglia. N = 5 for biological replicates.

(C) Quantification of engulfed mCherry+ materials in microglia. N = 5 for biological replicates.

(D) Quantification of colocalized vGluT2+/mCherry+ synapses within microglia. N = 5 for biological replicates.

Data information: All data are mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by Tukey's multiple comparisons test.
Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure (2021)
Figure 8

Figure 8. Microglial GPR56 is required for CDC50A knockdown-induced synapse loss.

(A) IHC images for vGluT2 staining in the dLGN of microglial Gpr56 conditional knockout mice (Gpr56fl/fl; Cx3cr1cre/+) and controls (Gpr56+/+; Cx3cr1cre/+). Both mice were intravitreally injected with AAV2 carrying Cdc50a shRNA. The yellow squares indicated the AAV2 non-infected areas and the dotted outlines indicated the AAV2 infected areas. Scale bar, 200 µm.

(B) Confocal images of vGluT2+ synapses in the AAV2 non-infected areas and infected areas of the dLGN. Scale bar, 20 µm.

(C) Quantification of vGluT2+ synapses in the non-infected areas and infected areas of microglial GPR56 conditional knockout mice and controls. N = 4 for biological replicates, mean ± SD, * p<0.05, **** p < 0.0001 by two-way ANOVA with Sidak's multiple comparisons test.

Expanded View Figure Legends
Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20 (2018)
Figure 1

Figure 1 Calcineurin is required for efficient cyclin B2 and XErp1 degradation at meiotic exit.

A) Scheme for the preparation of CSF extract.

B) CSF extract was treated with DMSO, CsA, buffer or His-CnA420-508 at the indicated concentrations. Meiotic exit was induced by calcium addition and samples were taken at the indicated time points. Samples were immunoblotted for cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin.

C) CSF extract was treated with DMSO or CsA. Both reactions were divided and supplemented with the indicated amounts of calcium or H2O. After 20min, samples were taken and immunoblotted for cyclin B2 for which a low and high exposure is shown. The cyclin B2 membrane was stripped and reprobed for α-tubulin.

D) CSF extract was supplemented with Myc-XErp1 wt IVT at the indicated dilutions. An empty IVT reaction not expressing Myc-XErp1 served as control. All reactions were divided and treated with DMSO or CsA. Calcium was added to all reactions and samples were taken at the indicated time points, treated with λ-phosphatase and immunoblotted for XErp1, the Myc-tag, cyclin B2 and α-tubulin. Asterisks indicate unspecific bands.

E) CSF extract was supplemented with Myc-XErp1 wt IVT. The reaction was divided and treated with DMSO or CsA. Both reactions were treated with calcium, samples were taken at the indicated time points and as indicated incubated with λ-phosphatase. Samples were immunoblotted for XErp1 and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.
Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20 (2018)
Figure 2

Figure 2 Calcineurin affects the phospho-regulation of XErp1.

A) Schematic representation of the XErp1 variants and their characteristics used in this study.

B) CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT and as indicated treated with DMSO or CsA. Both reactions were treated with calcium and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag, cyclin B2 and α-tubulin. A pseudocoloured representation of the Myc immunoblot is shown in Fig EV2A. Arrow marks the meiotic phosphorylation state of Myc-XErp1. Asterisk indicates unspecific bands.

C) CSF extract was supplemented with Myc-XErp1 DSG- DSA- ZBR- (S33N S38N S284N S288N C583A) IVT that was either wild-type or mutated to alanine at the p90RSK-target sites S335, T336 and S342 (Rsk3A). Both reactions were divided and treated with DMSO or CsA. Calcium was added and samples were taken at the indicated time points. Samples were immunoblotted for the Myc-tag and cyclin B2. The cyclin B2 membrane was stripped and reprobed for α-tubulin. Asterisk indicates unspecific bands.

D) CSF extract was treated with Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT. An empty IVT reaction not expressing XErp1 was used as control. The extracts were treated with DMSO or CsA as indicated. Calcium was added, samples were taken at the indicated time points and immunoblotted for cyclin B2, pSer335 XErp1 and α-tubulin. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisks indicate unspecific bands.

E) Myc-XErp1 CaMKII- ZBR- (T195A C583A) IVT was in vitro phosphorylated by recombinant PKA and isolated by α-Myc immunoprecipitation. The immunoprecipitate was supplemented with calcium and treated with recombinant hs_CaN and calmodulin as indicated. Samples were taken at the indicated time points and immunoblotted for the catalytic calcineurin subunit CnA and pSer335 XErp1. The pSer335 XErp1 membrane was stripped and reprobed for the Myc-tag. Asterisk indicates the IgG heavy chain.