EMBO/soda-nel huggingface dataset:
Fxr1 regulates sleep and synaptic homeostasis (2020)
Figure 5

Figure 5. Fxr1 protein expression is decreased during sleep deprivation.

A, Schematic representation of sleep deprivation (SD) experiments. Zeitgeber time 0 (ZT0) corresponding to lights ON.

B-E, Western blot analysis of (B) p845GluA1 and GluA1 expression (S n=9, SD n=10), (C) p880GluA2 and GluA2 expression (S n=9, SD n=8), (D) Fxr1 expression (S n=11, SD n=10), and (E) Fmrp expression (S n=11, SD n=10) in the prefrontal cortex of sleeping and sleep deprived mice. Student's t-test *p < 0.05, **p < 0.01, ***p < 0.001.

F-H, RNAseq measurement of mRNA for (F) Fxr1, (G) Fxr2, (H) Fmr1 during SD. n=3 in each condition, Student's T-test *p<0.05.

Data information: Error bars are ± SEM.
Fxr1 regulates sleep and synaptic homeostasis (2020)
Figure 6

Figure 6. Fxr1 blocks increase of synaptic strength during sleep deprivation.

A, Schematic representation of viral injection and sleep deprivation (SD) experiments. Zeitgeber time 0 (ZT0) corresponding to lights ON.

B-D, Cumulative probability plots of mEPSCs amplitude (500 events per cell) and representative examples of mEPSCs (left panel) recorded from brain slices of S and SD mice. (B) Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), (C) Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and (D) Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice).

E, F, mEPSC (E) mean amplitude and (F) frequency of cortical neurons of S or SD mice. Control neurons (Ctrl/S n=8 cells/4 mice, Ctrl/SD n=8 cells/4 mice), Fxr1 overexpressing (Fxr1/S n=7 cells/5 mice, Fxr1 over/SD n=9 cells/5 mice), and Gsk3 sKO (Gsk3sKO/S n=6 cells/3 mice, Gsk3sKO/SD n=7 cells/4 mice). Student's T-test *p<0.05.

G, Representative examples of the current-voltage relationship of evoked EPSC amplitude recorded from S (top panel) and SD (bottom panel) mice.

H, Summary bar graphs showing rectification index of control (S n=12 cells/5 mice, and SD n=10 cells/5 mice), Fxr1P overexpressing (S n=9 cells/4 mice, and SD n=14 cells/4 mice) and Gsk3 sKO (S n=9 cells/3 mice, and SD n=9 cells/3 mice) neurons. One way ANOVA with Bonferroni's Multiple Comparison *p < 0.05.

Data information: Error bars are ± SEM.
Fxr1 regulates sleep and synaptic homeostasis (2020)
Figure 7

Figure 7. Neuronal translatome regulation by Fxr1 during sleep deprivation.

A, Schematic representation of the experimental design. Zeitgeber time 0 (ZT0) corresponding to lights ON.

B, Immunostaining for GFP and HA in Ctrl and Fxr1 mouse brain slices. Arrows indicate presence and arrowheads indicate an absence of GFP tagged Fxr1 granules.

C, Quantification of colocalization of GFP and HA labeled neurons (GFP+HA+ 71.3%±3.7, n=3, GFP-Fxr1+HA+ 79.7%±2.8, n=3).

D, Enrichment of differentially expressed transcripts from Ctrl/S vs Ctrl/SD comparison in GO:BP. Top 10 clusters with the most number of enriched pathways (nodes) are shown.

E, SynGO enrichment for synaptic localization and function of differentially expressed transcripts from Ctrl/S vs Ctrl/SD comparison.

F, Venn diagram showing overlap of differentially expressed transcripts between Ctrl/S vs Ctrl/SD and Ctrl/SD vs Fxr1 overexpression (Fxr1)/SD comparisons.

G, Heat map showing transcripts that have bidirectional expression changes between Ctrl/S, Ctrl/SD, and Fxr1/SD conditions.

H, Enrichment of commonly affected transcripts between Ctrl/S vs Ctrl/SD and Ctrl/SD vs Fxr1/SD comparisons in GO:BP.

I, SynGO enrichment for synaptic localization and function of commonly affected transcripts between Ctrl/S vs Ctrl/SD and Ctrl/SD vs Fxr1over/SD comparisons.

Data information: SynGO Localization: 1-Synapse, 2-Postsynapse, 3-Postsynaptic density, 4-Postsynaptic specialization, 5-Postsynaptic ribosome, 6-Presynapse, 7-Presynaptic ribosome, 8-Postsynaptic cytosol, 9-Postsynaptic density intracellular compartment, 10-Presynaptic active zone, 11-Integral component of postsynaptic density membrane.

SynGO function: 1-Process in the synapse, 2-Synapse organization, 3-Protein translation at presynapse, 4-Protein translation at postsynapse, 5-Trans-synaptic signaling, 6-Synapse adhesion between pre- and post-synapse, 7-Metabolism, 8-Structural constituent of synapse, 9-Chemical synaptic transmission, 10-Synapse assembly, 11-Regulation of synapse assembly, 12-Regulation of modification of postsynaptic actin cytoskeleton, 13-Postsynaptic cytoskeleton organization, 14-Axo-dendritic transport, 15-Dendritic transport, 16-Structural constituent of postsynapse, 17-Postsynaptic specialization assembly.

Expanded View Figure Legends
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 1

Figure 1. Kinetochore-localized Bub1 is required for the centromeric accumulation of TOP2A in mitosis

A-E U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance.

F, G U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. Example images are shown (F). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 500 chromosomes in 15 cells (G).

H-K U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H). The immunofluorescence intensity ratios of centromeric Bub1/ACA (I), centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells.

L U2OS cells stably expressing CB-GFP or CB-Bub1-K-GFP were treated as in (H). Example images of the immunofluorescence staining are shown.

Data information: Means and error bars representing S.D. are shown (B-D, G and I-K; unpaired t test). Scale bars, 10 μm. See also Figure EV1.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 2

Figure 2. Bub1 kinase activity is necessary for TOP2A localization at mitotic centromeres

A, B U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA, and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A (A). Lysates of nocodazole-arrested mitotic cells were immunoblotted (B). * represents non-specific bands.

C-F U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. Example images are shown (C). The immunofluorescence intensity ratios of centromeric H2ApT120/ACA (D), centromeric TOP2A/ACA (E), and centromeric TOP2A/arm TOP2A (F) were determined on around 450 chromosomes in 15 cells. Means and error bars representing S.D. are shown (D-F; unpaired t test).

Data information: Scale bars, 10 μm. See also Figure EV2.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 3

Figure 3. Bub1-mediated H2ApT120 is sufficient to recruit TOP2A to centromeres and to a non-centromeric chromatin region

A U2OS cells were subjected to immunofluorescence staining. Example images of mitotic cells at various stages are shown.

B U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining. Example images of anaphase cells are shown.

C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2.

D U2OS-LacO cells transiently expressing EGFP-LacI, EGFP-LacI-Bub1-K (WT or D946N) were subjected to immunofluorescence staining. Arrows point to the transgene loci.

E, F MBP-TOP2A (429-1531)-10xHis was subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP, GST or H2ApT120 (E). The relative pulldown efficiency was determined (F). Means and error bars representing S.D. from three independent experiments are shown (unpaired t-test).

Data information: Scale bars, 10 μm. See also Figure EV3.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 4

Figure 4. Sgo1 and Sgo2 are not required for the enrichment of TOP2A at mitotic centromeres

A, B HeLa cells and the Sgo1-K492A mutant cells were immunostained with the indicated antibodies. Example images are shown (A). The immunofluorescence intensity ratio of centromeric INCENP/ACA was determined on around 250 chromosomes in 10 cells (B).

C, D HeLa and the Sgo1-K492A cells were exposed to nocodazole for 4 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (C). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 150 chromosomes in 10 cells (D).

E, F HeLa and the Sgo1-K492A cells were transfected with siRNA, and immunostained with the indicated antibodies. Example images are shown (E). The immunofluorescence intensity ratio of centromeric Aurora B/ACA was determined on around 250 chromosomes in 10 cells (F).

G, H HeLa and the Sgo1-K492A cells were transfected with siRNA, and treated with nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 280 chromosomes in 10 cells (H).

Data information: Means and error bars representing S.D. are shown (B, D, F and H; unpaired t-test). Scale bars, 10 μm.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 5

Figure 5. The C-gate formed by the coiled-coil domain is important for TOP2A localization at mitotic centromeres

A Schematic description of the domain organization of human TOP2A.

B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins, and then exposed to nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown.

C-F HeLa cells were transfected and treated as in (B). Example images are shown (C). The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells.

G, H HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained as in (B). Example images are shown (G). The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 250 chromosomes in 10 cells (H).

I-K U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Example images are shown (I). The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Cell lysates were immunoblotted (K).

Data information: Means and error bars representing S.D. are shown (D-F, H and J; unpaired t-test). Scale bars, 10 μm. See also Figure EV4.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 6

Figure 6. The extreme C-terminal region contributes to H2ApT120-dependent localization of TOP2A at centromeres

A HeLa cells were transfected with siRNA and plasmids encoding the indicated proteins, and then exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown.

B-D U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and the indicated EGFP-TOP2A proteins were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (B). The relative enrichment of EGFP signal at the LacO repeats was quantified in 30 cells (C). Cell lysates were immunoblotted (D). Arrows point to the transgene loci.

E, F HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained as in (A). Example images are shown (E). The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 300 chromosomes in 15 cells (F).

G, H HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were treated and immunostained as in (A). Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A-Myc-6xHis/arm TOP2A-Myc-6xHis was determined on around 200 chromosomes in 10 cells (H).

I-K U2OS cells stably expressing the indicated CENP-B fusion proteins were transfected with the plasmid encoding TOP2A-Myc-6xHis or TOP2A-ΔChT-Myc-6xHis, and then subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and H2ApT120. Example images are shown (I). The relative enrichment of anti-Myc staining signal at the centromere region versus that in the nearby nuclear region was quantified on around 200 chromosomes in 10 cells (J). Cell lysates were immunoblotted (K).

L MBP-TOP2A (429-1531)-10xHis and MBP-TOP2A (429-1531)-ΔChT-10xHis were subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP or GST.

Data information: Means and error bars representing S.D. are shown (C, F, H and J; unpaired t test). Scale bars, 10 μm.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 7

Figure 7. The H2ApT120-dependent intracellular localization of TOP2A and Sgo1 is competitive

A-D HeLa cells with or without transient expression of H2B-GFP or EGFP-TOP2A were exposed to nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI and antibodies for GFP, Sgo1 and ACA. Example images are shown (A). The immunofluorescence intensity ratio of centromeric Sgo1/ACA was determined on 400 chromosomes in 20 cells (B). The means and S.D.s from three independent experiments with the Sgo1/ACA ratio determined on around total 1200 chromosomes in 60 cells are shown (C). Lysates of asynchronous cells were immunoblotted (D). * represents non-specific bands.

E-H U2OS-LacO cells transiently expressing Myc-LacI-Bub1, with or without EGFP-TOP2A (970-1531), were subjected to immunofluorescence staining with DAPI and antibodies for H2ApT120 and Sgo1. Example images are shown (E). The relative enrichment of EGFP-TOP2A (F) and Sgo1 (G) at the LacO repeats was quantified in 120 cells. Cell lysates were immunoblotted (H).

I-L U2OS-LacO cells transiently expressing EGFP-LacI-Bub1-K together with VSV-Sgo1 or a control vector were subjected to immunofluorescence staining with DAPI and antibodies for TOP2A and VSV-tag. Example images are shown (I). The relative enrichment of TOP2A (J) and VSV-Sgo1 (K) at the LacO repeats was quantified in 50 cells. Cell lysates were immunoblotted (L). * represents non-specific bands.

Data information: Means and error bars representing S.D. are shown (B, C, F, G, J and K; unpaired t test). Scale bars, 10 μm. See also Figure EV5.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 8

Figure 8. Bub1 kinase promotes the timely decatenation of sister chromatids independent of Sgo1 and Sgo2

A-C HeLa cells were transfected with siRNA and subjected to immunofluorescence staining with DAPI, ACA and the anti-PICH antibody. Example images of anaphase cells are shown (A). The percentage of PICH-positive cells was determined in at least 339 anaphase cells in each condition. Means and S.D.s from three independent experiments are shown (B). Lysates of asynchronously growing cells were immunoblotted (C). Arrows point to the UFBs.

D, E HeLa cells were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (A). Example images of anaphase cells are shown (D). The percentage of anaphases with PICH was determined (E). Arrows point to the UFBs.

F, G HeLa cells were arrested in a prometaphase-like state with nocodazole treatment for 5hr, and then BAY 1816032 or DMSO was added for 1 hr in the continued presence of nocodazole. Then, nocodazole was removed by washing into fresh medium containing BAY 1816032 or DMSO. After 1 hr, cells were fixed and subjected to immunofluorescence staining. Example images are shown (F). The percentage of anaphases with PICH was determined (G). Arrows point to the UFBs.

H-K HeLa and the Sgo1-K492A cells transfected with the indicated siRNA were synchronized and treated with BAY 1816032 or DMSO as in (D), and then immunostained with the indicated antibodies. Example images of anaphase cells are shown (H). The percentage of anaphases with PICH was determined in at least 256 cells in each condition. Means and S.D.s from three independent experiments are shown (I). Example images of prometaphase cells immunostained with the indicated antibodies are shown (J). Lysates of nocodazole-arrested mitotic cells were immunoblotted (K). Arrows point to the UFBs.

Data information: Means and error bars representing S.D. are shown (B and I; unpaired t test). Scale bars, 10 μm.
Histone H2A phosphorylation recruits topoisomerase IIα to centromeres to safeguard genomic stability (2019)
Figure 9

Figure 9. Centromeric localization of TOP2A facilitates the timely decatenation of sister chromatids

A HeLa cells were transfected with plasmids encoding CB-GFP or CB-TOP2A-GFP. Lysates of asynchronously growing cells were immunoblotted with the indicated antibodies.

B-D HeLa cells transfected with siRNA and plasmids encoding the indicated proteins were released from single thymidine block. At 12 hr post-release, cells were fixed and subjected to immunofluorescence staining with DAPI and the antibodies for PICH, GFP and CENP-C. Example images of anaphase cells are shown (B). The percentage of anaphases with PICH was determined in at least 152 cells in each condition. Means and S.D.s from three independent experiments are shown (C). Lysates of nocodazole-arrested mitotic cells were immunoblotted (D). * represents non-specific bands. Arrows point to the UFBs.

E, F HeLa cells transiently expressing CB-GFP or CB-TOP2A-GFP were released from single thymidine block, and after 7 hr, BAY 1816032 or DMSO was added for 5 hr. Cells were then fixed and subjected to immunofluorescence staining as in (B). Example images of anaphase cells are shown (E). The percentage of anaphases with PICH was determined in at least 142 cells in each condition. Means and S.D.s from three independent experiments are shown (F). Arrows point to the UFBs.

G-I HeLa cells transfected with siRNA and plasmids encoding EGFP-TOP2A (WT or ΔChT) were synchronized as in (B), and then subjected to immunofluorescence staining with DAPI, ACA and the antibodies for PICH and GFP. Example images of anaphase cells are shown (G). The percentage of anaphases with PICH was determined in at least 174 cells in each condition. Means and S.D.s from three independent experiments are shown (H). Lysates of asynchronous cells were immunoblotted (I). Arrows point to the UFBs.

Data information: Means and error bars representing S.D. are shown (C, F and H; unpaired t test). Scale bars, 10 μm.
Glomerular endothelial cell senescence drives age-related kidney disease through PAI-1 (2021)
Figure 1

Figure 1: Physiological renal aging is characterized by glomerulosclerosis, podocyte loss and glomerular endothelial senescence.

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively.

B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively.

C: TUNEL assay (upper panels) and quantification of TUNEL-positive tubular cells (upper panel) in glomeruli from young and aged mice. Panels are representative images of 6 young and old mice, respectively. Scale bar = 20 μm.

D: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. Panels are representative images of 4 young and old mice, respectively.

E: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively.

F: 53BP1/CD34 co-immunostaining in kidneys from young and aged mice (left panels) and quantification (right panels) of 53BP1 foci per glomerular endothelial cell. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively.

G: p16 mRNA expression in whole kidney from young and old mice. n = 6 for young and old mice, respectively.

H: mRNA expression of SASP components in kidneys from young and aged mice. n = 6 for young and old mice, respectively.

I: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 5 for young and old mice, respectively.

Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.