EMBO/soda-nel huggingface dataset:
The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment (2016)
Figure 5

Figure 5. Antagonism or deficiency of CXCR2 inhibits enhanced cardiac neutrophil accumulation during active phase (ZT13).

A Representative flow cytometry analysis and quantification of CXCR2high neutrophils in the blood after ZT5 and ZT13 MI. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for all time points in both groups; ZT5 vs. ZT13: *P = 0.0001 (4h), *P = 0.0001 (8h), *P = 0.0447 (12h).

B Percentage and mean fluorescence intensity (MFI) of CXCX2 expression by cardiac neutrophils 12h post-MI after ZT5 and ZT13 MI. Student`s t-test; n = 4 mice in both groups; *P = 0.0001.

C Percentage of CXCR2high neutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13).

D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13).

E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0002 (ZT13).

F Mean fluorescence intensity (MFI) of CXCX2 expression at ZT5 in the blood 24h after MI in wildtype (WT) and CXCR2 KO mice. Student`s t-test. N = 6 mice in both groups; *P = 0.0001.

G Percentage of CXCR2hi blood neutrophils 24h after ZT5 or ZT13 MI in WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0001 (ZT13).

H Flow cytometry quantification of neutrophils in hearts 24h after MI in ZT5 and ZT13 operated WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n= n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0461 (ZT13), ns = not significant.

Data information: All data are expressed as mean ± SEM.
The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment (2016)
Figure 6

Figure 6. Antagonism of CXCR2 inhibits enhanced cardiac neutrophil accumulation after ischemia reperfusion during active phase (ZT13).

A Schematic representation of transient ischemia and reperfusion protocol, performed at ZT5 and ZT13. The CXCR2 antagonist SB225002 or vehicle was injected 5 min before reopening the LAD.

B Percentage of CXCR2hi neutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0046 (ZT13); ZT5 vs. ZT13: *P = 0.0153 (DMSO).

C Flow cytometry quantification of cardiac neutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0006 (ZT13); ZT5 vs. ZT13: *P = 0.0001 (DMSO).

D Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0,0486 (ZT13).

Data information: All data are expressed as mean ± SEM.
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 1

Figure 1. Mouse genetic ablation of endothelial Tfeb leads to vascular defects.

(A,B) Tfeb expression in the vasculature (mice n=10). (A) Representative images of retinal vessels (p5) (A) (scale bar: 50 µm) and (B) glomerular, arterial (scale bars: 10 µm) and interstitial vessels (scale bar: 50 µm) of kidney (p17) of Tfeb-EGFP mice stained with anti-iB4 (A), anti-CD31 (B) and anti-GFP (A,B).

(C) Alterations in the embryonic vasculature in TfebEC-/- mice at E10.5 (mice n=25). Representative images of whole-mount embryos and yolk sacs (i, ii) (scale bars: 0.5 mm). Vessels of the head (iii), ocular (iv) and intrasomitic regions (v) were stained with anti-endomucin Ab (scale bars: 100 µm).

(D) Alteration of retinal vascular maturation in TfebiEC-/- at p5. Representative images of whole mounts of retina and immunostaining of vascular front and vascular plexus with an anti-iB4 Ab (scale bars: 50 µm). Bar graphs indicate the percent of vascular area versus total area of the retina, the percent of vascular density, the number of branches per field, vessel diameter, the number of filopodia per field and the length of filopodia (mice n=10, mean±SEM; ***p< 0.0001 versus control mice by Student’s t-test).

(E) Alteration of the glomerular ultrastructure in TfebiEC-/- mice at p17 (mice n=5). Representative transmission electron micrographs of renal tissue. Magnification: i) x6000, ii) x20000), iii-iv) x10000, v-vi) x25000; scale bars: 1µm). Asterisks indicate the accumulation of extracellular matrix in mesangium; white and black triangles indicate the fusion of podocyte foot processes and the lack of endothelial fenestrae, respectively.
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 2

Figure 2. Tfeb deletion reduces EC proliferation in vivo.

(A, B) Reduced EC proliferation in the retina (p5) and kidney (p17) of in TfebiEC-/- mice. (A) Representative images of vessels of the vascular front and vascular plexus in the retina of control and TfebiEC-/- mice stained with anti-iB4 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percentage of EC Ki-67+ nuclei versus total nuclei co-localized with iB4+ vessels (mice n=6, mean±SEM; **p<0.001 and *p< 0.01 versus control mice by Student’s t-test). (B) Representative images of vessels of the kidney in control and TfebiEC-/- mice stained with anti-CD31 and Ki-67 antibodies (scale bars: 50 µm). Bar graph indicates the percent of EC Ki-67+ nuclei versus total nuclei co-localized with CD31+ vessels (mice n=6, mean±SEM; **p<0.001 versus control mice by Student’s t-test). Podocin staining is shown to glomerular localization.

(C, D) TFEB silencing reduced EC proliferation. Representative graph of scr-shRNA and sh-TFEB ECs treated for 24 hours with FCS (20%) or VEGF-A (30 ng/ml). (C) DNA incorporation of EdU was detected by flow cytometry. The percentage of proliferating cells is indicated (n=4, mean±SEM; ***p<0.0001 versus scr-shRNA ECs by Student’s t-test). (D) DNA content was determined by propidium iodide staining and assessed by fluorescences-activated cell sorter analysis (representative experiment out of 4 with similar results).

(E) TFEB silencing reduced human EC morphogenesis. Representative images of tube-like-structure of scr-shRNA and sh-TFEB human ECs stained with phalloidin-555. Bar graph indicates the percentage of phalloidin+ area in sh-TFEB and scr-shRNA ECs (scale bars: 0.25 mm; n=6, mean±SEM; ***p<0.0001 versus scr-shRNA by Student’s t-test).
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 3

Figure 3. TFEB gene regulation in human ECs.

(A) Heatmap showing unsupervised hierarchical clustering of human differentially expressed genes between human scr-shRNA and sh-TFEB ECs. Red: up-regulated genes; green: down-regulated genes.

(B) Selection of enriched functional GO categories by DAVID analysis in differentially expressed genes between human sh-TFEB and scr-shRNA ECs. GO analyses were performed individually on down‐ or up‐regulated genes using DAVID tool (biological process). GO terms are ranked by P‐value corrected by Benjamini Hockberg method, and the number of genes is indicated.

(C) Selection enriched of Molecular Pathways by GSEA analysis of microarray data comparing human sh-TFEB and scr-shRNA ECs. Normalized enrichment scores (NESs) and p-values are reported.

(D) Selection of enriched functional GO categories by DAVID analysis on ChiP-seq data set performed in human ECs overexpressing TFEBS142A. GO analyses were performed using DAVID tool (biological process). GO terms are ranked by P‐value corrected by BH method, and the number of genes is indicated.
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 4

Figure 4. TFEB regulates cell cycle genes.

(A-C) (A) Heatmap, (B) qPCR and (C) immunoblots showing the differentially expressed cell cycle related genes between scr-shRNA and sh-TFEB ECs. (A) Red: up-regulated genes; green: down-regulated genes. (B) Data are expressed as relative fold-change compared with the expression in scr-shRNA cells after normalization to the housekeeping gene TBP (n=3, mean±SEM; **p<0.001 and ***p<0.0001 by Student’s t-test). (C) Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with specific Abs. The bar graph shows the densitometric analysis expressed as the ratio between the cell cycle genes and α-tubulin (n=3, mean±SEM; *p<0.01, **p<0.001 and ***p0.0001 versus scr-shRNA by Student’s t-test).

(D) Modulation of cell cycle genes expression in the lung ECs derived from control and TfebiEC-/- mice. Data are expressed as relative fold-change compared with the expression in ECs of control mice after normalization to the housekeeping gene TBP (n=3, mean±SEM; **p<0.001 and ***p<0.0001, by Student’s t-test).

(E-G) Analysis of TFEB binding and modulation of the CDK4 promoter in human ECs. (E) Representative snapshot of TFEB binding on CDK4 in human ECs. (F) ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti-TFEB (indicated in the bar graph as “+Ab anti-TFEB”), followed by qPCR for CDK4. Bar graph shows the percent enrichment (n=3, mean±SD). (G) Analysis of TFEB modulation of CDK4 promoter in human ECs. Bar graph shows the relative luciferase activity % evaluated in control and TFEBS142A human ECs after transfection of CDK4 full promoter and CDK4 promoter deleted of TFEB binding site (n=3, mean±SEM; *p<0.01 and **p<0.001 by Student’s t-test).

(H) Modulation of Rb protein in sh-TFEB ECs. Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with phospho-Rb and total Rb Abs. The bar graph shows the densitometric analysis of the immunoblotting expressed as % of sh-TFEB vs scr-shRNA and the ratio between phospho-Rb and total Rb in the different conditions (n=3, mean±SEM; **p<0.001, ***p<0.0001 versus scr-shRNA by Student’s t-test).
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 5

Figure 5. Regulation of VEGFR2 by TFEB in ECs.

(A-C) Increase of Vegfr2 expression in the vasculature of TfebEC-/- and TfebiEC-/- mice. (A) Representative immunostaining images of TfebEC-/- embryonic vessels (E10.5) stained with anti-endomucin and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in endomucin+ vessel areas (embryos n=6, mean±SEM; ***p< 0.0001 versus control embryos by Student’s t-test). (B) Representative immunostaining images (i) and detail (ii) of the vascular front and vascular plexus of the retina (p5) of control and TfebiEC-/- mice with anti-iB4 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graph indicates the Vegfr2 mean intensity only in iB4+ vessel areas (mice n=6, mean±SEM; **p<0.001 and ***p<0.0001 versus control mice by Student’s t-test). (C) Representative immunostaining images of the glomerulus (p17) of control and TfebiEC-/- mice with anti-podocin, anti-CD31 and anti-Vegfr2 Abs (scale bars: 50 µm). Bar graphs indicate the Vegfr2 mean intensity in CD31+ vessel areas (mice n=6, mean±SEM; **p<0.001 versus control mice by Student’s t-test).

(D-F) VEGFR2 expression is regulated by TFEB. (D, E) qPCR of VEGFR2 in lung ECs obtained from control and TfebiEC-/- mice (D) and in human sh-TFEB (E). Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP (n=3, mean±SEM; **p<0.001 and ***p<0.0001, by Student’s t-test). (F) Immunoblots of total lysates from scr-shRNA and sh-TFEB ECs probed with anti-VEGFR2 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin (n=3, mean±SEM; ***p<0.0001 versus scr-shRNA by Student’s t-test).

(G) Analysis of TFEB binding to the VEGFR2 promoter in human ECs. ChIP was performed using digested chromatin from human control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti-TFEB (indicated in the bar graph as “+Ab anti-TFEB”), followed by qPCR for VEGFR2. Bar graph shows the percent enrichment (n=3, mean±SD).
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 6

Figure 6. Role of TFEB in regulating VEGFR2 localization and activity in human ECs.

(A, B) Silenced TFEB alters the localization of VEGFR2. (A) FACS analysis of surface VEGFR2 expression on human scr-shRNA and sh-TFEB ECs. Bar graph shows the ratio between total and PM VEGFR2 (n=6, mean±SEM; **p<0.001 versus scr-shRNA by Student’s t-test). (B) Representative TIRF and epifluorescence images of human scr-shRNA and sh-TFEB ECs after staining with anti-VEGFR2 Ab (scale bars: 10 µm). Bar graphs show the ratio of VEGFR2 analyzed in epifluorescence and TIRF mode analyzed by TIRF (n=40, mean±SEM; ***p<0.0001 versus scr-shRNA by Student’s t-test).

(C) Silenced TFEB alters the localization and the phosphorylation state of VEGFR2 and its signal transduction. Representative immunoblot of PM biotinylated portion and total cell lysates of scr-shRNA and sh-TFEB ECs after VEGF-A stimulation (30 ng/ml). Blots of total or PM cell lysates were probed with anti-VEGFR2. Blots of total cell lysates were probed with anti-p-Y1175-VEGFR2, anti-PLCγ, p-PLCγ, anti-ERK-1/2, anti-pERK1/2, anti-p-Src, anti-Src, anti CD31 and α-tubulin Abs. The bar graphs (i,ii) show densitometric analysis of stimulated versus unstimulated scr-shRNA and sh-TFEB ECs expressed as: (i) % of VEGFR2 on PM fraction (n=3, mean±SEM; ANOVA p<0.02; **p<0.001 versus scr-shRNA by Bonferroni post-test), (ii) % of VEGFR2 total (n=3, mean±SEM; ANOVA p>0.05; *p<0.05 and **p<0.001 versus scr-shRNA by Bonferroni post-test).

(D, E) Regulaton of autophagy and lysosome pathway by TFEB silencing. (D) qPCR and (E) immunoblots showing the differentially expressed autophagy and lysosome related genes between human scr-shRNA and sh-TFEB ECs. (D) Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP (n=3, mean±SEM; ***p<0.0001 by Student’s t-test). (E) Immunoblots of total lysates from human scr-shRNA and sh-TFEB ECs probed with anti-ULK-1, anti-ATG9, anti-LC3-I/II, anti-LAMP-1 and α-tubulin Abs. The bar graph shows the densitometric analysis expressed as the ratio between scr-shRNA and sh-TFEB ECs (n=3, mean±SEM; **p<0.001, ***p<0.0001 versus scr-shRNA by Student’s t-test).

(F) TFEB silencing inhibits VEGFR2 internalization. Bar graphs of VEGFR2 internalization expressed as the percent of internalized VEGFR2 versus PM VEGFR2 after VEGF-A stimulation (n=6, mean±SEM, ANOVA p<0.0001; ***p<0.0001 versus scr-shRNA by Bonferroni post-test).
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 7

Figure 7. Role of MYO1C in the localization of VEGFR2 in TFEB-silenced ECs.

(A-C) Analysis of TFEB binding and modulation of the MYO1C promoter in human ECs. (A) Representative snapshot of TFEB binding on MYO1C in human ECs. (B) ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti-TFEB (indicated in the bar graph as “+Ab anti-TFEB”), followed by qPCR for MYO1C. Bar graph shows the percent enrichment (n=3, mean±SD). (C) qPCR of MYO1C expression in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C ECs. Data are expressed as relative fold change compared with the expression in scr-shRNA ECs after normalization to the housekeeping gene TBP (n=3, mean±SEM; **p<0.001 and ***p <0.0001 by Student’s t-test).

(D) MYO1C silencing reverses the effect of TFEB silencing on the up-regulation of PM VEGFR2. Analyses were performed on human ECs carrying appropriate scr-shRNA or sh-TFEB in the presence or absence of sh-MYO1C. Representative western blot of MYOC1, total and PM biotinylated VEGFR2 (representative experiment out of 4 with similar results).

(E, F) Representative immunostaining images of the vascular plexus of the retina (p5) (E) and glomerulus (p17)(F) of control and TfebiEC-/- mice with anti-CD31 and anti-MYO1C Abs (scale bars: 50 µm). Bar graphs indicate the Myo1C mean intensity only in vessel areas CD31+ (n=6, mean±SEM; ***p<0.0001 versus control mice by Student’s t-test).

(G) Representative TIRF and epifluorescence images of VEGFR2 in scr-shRNA, sh-TFEB, sh-MYO1C and sh-TFEB+sh-MYO1C human ECs (scale bars: 10 µm). Bar graph shows the ratio between PM and total VEGFR2 (n=40, mean±SEM; **p<0.001 and ***p<0.0001 versus scr-shRNA by Student’s t-test).
TFEB CONTROLS VASCULAR DEVELOPMENT BY REGULATING THE PROLIFERATION OF ENDOTHELIAL CELLS (2018)
Figure 8

Figure 8. Indirect regulation of VEGFR2 via miR-15a/16 by TFEB in ECs.

(A) Analysis of TFEB binding to DLEU2 and SMC4 promoters in human ECs. ChIP was performed using digested chromatin from control ECs and TFEBS142A ECs incubated with IgG (indicated in the bar graph as “+IgG”) or with Ab anti-TFEB (indicated in the bar graph as “+Ab anti-TFEB”), followed by qPCR for DLEU2 and SMC4. Bar graph shows the percent enrichment (n=3, mean±SD).

(B) DLEU2 expression is regulated by TFEB. qPCR of DLEU2 in human scr-shRNA, sh-TFEB or control and TFEBS142A ECs (left panel) and lung ECs obtained from control and TfebiEC-/- mice (right panel). Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene TBP (n=3, mean±SEM; *p<0.01, and ***p<0.0001 by Student’s t-test).

(C) Human miR-15a-5p and miR-16-5p are regulated by TFEB. qPCR of miR-15a-5p (left panel) and miR-16-5p (right panel) in sh-TFEB or TFEBS142A ECs. Data are expressed as relative fold-change compared with the expression in scr-shRNA and control cells after normalization to the housekeeping gene RNU44 (n=3, mean±SEM; ***p<0.0001 by Student’s t-test).

(D-G) VEGFR2 expression is regulated by TFEB through a miR-dependent mechanism. (D, F) qPCR of VEGFR2 in human scr-shRNA and sh-TFEB ECs (D, E) and in control and TFEBS142A ECs (F, G) treated with a specific miR-control, miR-15a-5p and miR-16-5p mimics or inhibitors. (D, F) Data are expressed as relative fold-change compared with the expression in control cells after normalization to the housekeeping gene TBP (n=3, mean±SEM; **p<0.001 and ***p<0.0001 versus control or scr-shRNA plus miR-control and ##p<0.001 and ##p<0.0001 versus sh-TFEB and TFEBS142A plus miR-control by Student’s t-test). (E, G) Representative western blot of VEGFR2 expression under the same experimental conditions previously reported. The bar graph shows the densitometric analysis expressed as the ratio between VEGFR2 and α-tubulin (n=3, mean±SEM; **p<0.001 and ***p<0.0001 versus control or scr-shRNA plus miR-control and #p<0.01 and ##p<0.001 versus sh-TFEB and TFEBS142A plus miR-control by Student’s t-test).
TIAR marks nuclear G2/M transition granules and restricts CDK1 activity under replication stress (2018)
Figure 1

Figure 1. TIAR controls mitotic entry.

A HeLa-H2B/tub cells were transfected with control or TIAR siRNAs for 48 hours and synchronized by double thymidine (TT) block. After release from the block, mitotic cells were counted by fluorescence microscopy (mean ± SD; n = 3).

B Time from S- to M-phase was measured in si-RNA transfected HeLa-H2B/tub cells by time-lapse microscopy following release from TT block (4 repeat experiments; all measurements depicted).

C Western blot analysis of unsynchronized HeLa-H2B/tub cells was carried out to monitor expression of p(S10)-H3, total H3 and TIAR in control and TIAR-depleted cells (mean ± SD; n = 3).

D HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA. 72 hours after transfection, p(S10)-H3-positive cells were quantified by flow cytometry (mean ± SD; n = 3).

E HeLadox-YFP-TIARr cells were transfected with control or TIAR siRNAs, and 24h hours later cell were cultured in the abscence or presence of 1 μg/ml doxocycline for 48 hours. The expression of p(S10)-H3, total H3 and TIAR was measured by Westen blot analysis. The graph shows the quantification of p-H3 levels normalized to total H3 (mean ± SD, n = 4).

F HeLadox-YFP-TIARr-RRM123m cells were analyzed as in panel (E) (mean ± SD, n = 4).

G HeLadox-YFP-TIARr-dQRD cells were analyzed as in panel (E) (mean ± SD, n = 4).

Data information: Statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
TIAR marks nuclear G2/M transition granules and restricts CDK1 activity under replication stress (2018)
Figure 2

Figure 2. TIAR deficiency induces chromosomal instability.

A Following transfection of HeLa cells with control or TIAR siRNAs for 72 hours, mitotic cells were analyzed by IF microscopy and scored for chromatin (mean ± SE, n = 3).

B HeLa cells were analyzed as in (A) and scored for spindle defects (mean ± SE, n = 3).

C HeLa cells were transfected with control or TIAR siRNAs for 4 days, and subjected to colcemid for 2 hours prior to preparation of metaphase spreads. Chromosomes were stained with Hoechst (red) and anti-CENP-A antibody (green); the yellow arrowhead marks a chromosome break.

D Metaphase spreads were prepared as in (C), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition).

E Metaphase spreads were prepared as in (C), and the frequency of cells with scattered chromosomes was quantified (mean ± SD, n = 3).

F HeLa cells were transfected with control or TIAR si-RNAs, alone or together with Cdc25B siRNA, and 4 days later subjected to colcemid for 2 hours prior to preparation of metaphase spreads. The frequency of cells with scattered chromatids was quantified (mean ± SD, n = 3 independent experiments, approximately 30 metaphase spreads were assessed per experiment and condition).

G Metaphase spreads were prepared as in (F), and the frequency of cells with chromosomal breaks was quantified (mean ± SD, n = 3).

Data information: In (A, B and D-G), statistical significance was determined by unpaired Student's t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.