Figure 4 ( A ) BMDM from wt or Trem2 ko mice were cultured on APP / PS1micebrain cryosections incubated with or without mAb11 ( 1 µg / ml ) or an isotype control ( IC ; 1 µg / ml ) for 24 h . Sections were then probed with methoxy - X04 . Scale bar , 500 µm . ( B ) The amyloid plaque load was quantified from the entire sagittal section . Sections incubated with medium ( no cell ) were set as baseline . ( n = 6 , + / - SEM ; 2 way ANOVA , interaction P < 0 . 0001 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; Tukey ' s multiple comparisons tests ; wt vs . ko for the following conditions : no antibody P = 0 . 0304 , IC P = 0 . 0049 , mAb11 P = 0 . 0212 ; wt : IC vs . wt : mAb11 P = 0 . 0008 ; ko : IC vs . ko : mAb11 P = 0 . 0001 ) . ( C , D ) Equal numbers of wt and Trem2 ko BMDM were added and cell numbers were analyzed after termination of experiments by quantifying the CD68 positive cells on top of the sections . ( n = 4 , + / - SEM ; t - test ; n . s . , nonsignificant , P = 0 . 5004 ) . Scale bar , 200 µm . ( E ) Aβ was extracted by urea buffer from replicate slices of the experiment shown in ( A ) and total Aβ was identified by western blotting .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) BMDM from wt or Trem2 ko mice were cultured on APP/PS1micebrain cryosections incubated with or without mAb11 (1 µg/ml) or an isotype control (IC; 1 µg/ml) for 24 h. Sections were then probed with methoxy-X04. Scale bar, 500 µm.(B) The amyloid plaque load was quantified from the entire sagittal section. Sections incubated with medium (no cell) were set as baseline. (n = 6, +/- SEM; 2 way ANOVA, interaction P<0.0001, genotype P<0.0001, treatment P<0.0001; Tukey's multiple comparisons tests; wt vs. ko for the following conditions: no antibody P=0.0304, IC P=0.0049, mAb11 P=0.0212; wt : IC vs. wt : mAb11 P=0.0008; ko : IC vs. ko : mAb11 P=0.0001).(C, D) Equal numbers of wt and Trem2 ko BMDM were added and cell numbers were analyzed after termination of experiments by quantifying the CD68 positive cells on top of the sections. (n = 4, +/-SEM; t-test; n.s., nonsignificant, P=0.5004). Scale bar, 200 µm.(E) Aβ was extracted by urea buffer from replicate slices of the experiment shown in (A) and total Aβ was identified by western blotting.


Figure 5 ( A ) Cryosections from unfixed brain of 6 - months old APP / PS1mice were pre - incubated with increasing concentrations of mAb11 ( 0 . 001 , 0 . 01 , 0 . 1 , 1 , 5 µg / ml ) . BMDM from wt or Trem2 ko mice were added for 24 h . Sections were stained with methoxy - X04 . Scale bar , 500 µm . ( B ) Methoxy - X04 signals were quantified from the entire sagittal section . ( n = 5 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0082 , genotype P < 0 . 0001 , treatment P < 0 . 0001 . Fisher ' s LSD post hoc comparisons ; * show statistics between wt and ko under the same experimental condition . # in black show wt compares to no antibody stimulation ; # in grey show ko compares to no antibody stimulation ; wt vs . ko for the following conditions : no antibody P = 0 . 0053 , mAb11 0 . 001 µg / ml P = 0 . 0003 , mAb11 0 . 01 µg / ml P < 0 . 0001 , mAb11 0 . 1 µg / ml P = 0 . 0001 , mAb11 1 µg / ml P = 0 . 0007 , mAb11 5 µg / ml P = 0 . 0011 ; Following conditions compare to wt / no antibody : wt / mAb11 0 . 01 µg / ml P = 0 . 0166 , wt / mAb11 0 . 1 µg / ml P = 0 . 0002 , wt / mAb11 1 µg / ml P < 0 . 0001 , wt / mAb11 5 µg / ml P < 0 . 0001 ; Following conditions compare to ko / no antibody : ko / mAb11 0 . 1 µg / ml P = 0 . 0099 , ko / mAb11 1 µg / ml P < 0 . 0001 , ko / mAb11 5 µg / ml P < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Cryosections from unfixed brain of 6-months old APP/PS1mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher's LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.


Figure 1B . Phenotype of the patients as indicated . ( a ) Sparse woolly hair . ( b ) Protrusion of upper lip due to deformity of teeth . ( c ) Ichthyosis - like fine scale and erythema . C . Images of the heart of the patients as indicated . ( a ) M - mode echocardiography taken from the parasternal long axis view of the left ventricle ( LV ) illustrating poor LV function with severe involvement of the interventricular septum ( IVS ) and reduced motion of the left ventricular posterior wall ( LVPW ) . ( b ) bi - dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function . The SMOG is enhanced in the right atrium . ( c ) bi - dimensional apical four chamber view with free tricuspid valve regurgitation ( TR ) due to lack of cooptation of the tricuspid valve leaflets during systole ( marked by a red bar ) as a result of critical reduction of right ventricular function and right ventricular enlargement . ( d ) Right and left ventricular thrombi seen in the short axis apical parasternal view . T1 - large right ventricular thrombus , T2 & amp ; amp ; T3 - left ventricular thrombi . ( e ) The color - flow Doppler picture : TR . Free regurgitation of the tricuspid valve shown in modified four chambers apical view . RA - right atrium , RV - right ventricle , LA - left atrium , LV - left ventricle , TR - tricuspid regurgitation color - flow Doppler . D . Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10 . a c - post - mortem heart sections ( 2 . 7Y ) . ( a ) Prominent fibrosis and lymphocytic interstitial inflammation ( Hematoxylin Eosin x50 ) . ( c ) Sub - epicardial inflammation and mild interstitial " myocarditis " ( Hematoxylin Eosin x100 ) . ( b d ) - Fetal heart ( VI7 ) homozygous for p . Tyr747Ter . No inflammatory infiltrate or other histological abnormalities are seen . ( b ) Normal arrayed myocardium ( Hematoxylin Eosin x50 ) . ( d ) Sub - epicardial region with no histopathological changes . ( Hematoxylin Eosin x100 ) . F . Segregation of c . 2241C > G , p . Y747X in PPP1R13L in the studied family : The affected girl VI10 , is homozygous for the causative SV p . Y747X , as are the four fetuses that were aborted . The parents are obligate carriers as shown and , of the two healthy sisters , one carries the wild allele only , and the other is heterozygous for the causative SV .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Phenotype of the patients as indicated. (a) Sparse woolly hair. (b) Protrusion of upper lip due to deformity of teeth. (c) Ichthyosis-like fine scale and erythema.C. Images of the heart of the patients as indicated. (a) M-mode echocardiography taken from the parasternal long axis view of the left ventricle (LV) illustrating poor LV function with severe involvement of the interventricular septum (IVS) and reduced motion of the left ventricular posterior wall (LVPW). (b) bi-dimensional apical four chamber view with swirling of SMOG heart chambers as a result of poor myocardial function. The SMOG is enhanced in the right atrium. (c) bi-dimensional apical four chamber view with free tricuspid valve regurgitation (TR) due to lack of cooptation of the tricuspid valve leaflets during systole (marked by a red bar) as a result of critical reduction of right ventricular function and right ventricular enlargement. (d) Right and left ventricular thrombi seen in the short axis apical parasternal view. T1-large right ventricular thrombus, T2&amp;T3-left ventricular thrombi. (e) The color-flow Doppler picture: TR. Free regurgitation of the tricuspid valve shown in modified four chambers apical view. RA-right atrium, RV-right ventricle, LA-left atrium, LV-left ventricle, TR-tricuspid regurgitation color-flow Doppler.D. Photomicrografh demonstrating fibrosis and interstitial inflammation in the heart of patient VI10. a c- post-mortem heart sections (2.7Y). (a) Prominent fibrosis and lymphocytic interstitial inflammation (Hematoxylin Eosin x50). (c) Sub-epicardial inflammation and mild interstitial "myocarditis" (Hematoxylin Eosin x100). (b d)- Fetal heart (VI7) homozygous for p.Tyr747Ter. No inflammatory infiltrate or other histological abnormalities are seen. (b) Normal arrayed myocardium (Hematoxylin Eosin x50). (d) Sub-epicardial region with no histopathological changes. (Hematoxylin Eosin x100).F. Segregation of c.2241C> G, p.Y747X in PPP1R13L in the studied family: The affected girl VI10, is homozygous for the causative SV p.Y747X, as are the four fetuses that were aborted. The parents are obligate carriers as shown and, of the two healthy sisters, one carries the wild allele only, and the other is heterozygous for the causative SV.


Figure 2A . PPP1R13L mRNA expression level is lower in VI10 PDFs ( set as 1 ) than in CFs . B . and C . Western blots ( WBs ) analyses demonstrated the absence of iASPP in VI10 , VI4 and VII1 PDFs . D . WB analysis demonstrated that PPP1R13L - shRNA downregulated the putative iASPP band .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O

Figure 2A. PPP1R13L mRNA expression level is lower in VI10 PDFs (set as 1) than in CFs.B. and C. Western blots (WBs) analyses demonstrated the absence of iASPP in VI10, VI4 and VII1 PDFs.D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.


Figure 3A . Higher induction of inflammatory cytokine mRNA levels in LPS - stimulated VI10 PDFs than in CFs . The expression level in resting conditions of the PDFs was set as 1 . Differences between knockdown and control with p values ≤ 0 . 05 ( Student ' s t test ) are indicated with an asterisk . B . as in A . for patient VII1 . C . knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with asterisks in the graph .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Higher induction of inflammatory cytokine mRNA levels in LPS-stimulated VI10 PDFs than in CFs. The expression level in resting conditions of the PDFs was set as 1. Differences between knockdown and control with p values ≤ 0.05 (Student's t test) are indicated with an asterisk. B. as in A. for patient VII1.C. knockdown of PPP1R13L in CFs resulted in increased mRNA expression levels of inflammatory mediators. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks in the graph.


Figure 4A . WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs . B . ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS - stimulated PDFs than in LPS - stimulated - CFs . The binding activity at the IL1B promoter was set as 1 . Differences between knockdown and control with p value = 0 . 00436 ( Student ' s t test ) are indicated with an asterisk . C . NF - kB inhibitor BOT - 64 blocked the inducible mRNA expression of the inflammatory cytokines . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with an asterisks and exact values .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. WB analysis of nuclear and cytosolic fractions demonstrated that the duration of p65 in the nucleus is longer in the VI10 PDFs than in CFs.B. ChIP assay shows that p65 is associated more strongly with inflammatory cytokine gene promoters in LPS-stimulated PDFs than in LPS-stimulated-CFs. The binding activity at the IL1B promoter was set as 1. Differences between knockdown and control with p value= 0.00436 (Student's t test) are indicated with an asterisk.C. NF-kB inhibitor BOT-64 blocked the inducible mRNA expression of the inflammatory cytokines. Differences between knockdown and control with p values (Student's t test) are indicated with an asterisks and exact values.


Figure 5A . WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes . B . qPCR showed that ppp1r13l - knocked down murine cardiomyocytes ( using two alternative shRNA sequences ) expressed higher level of pro - inflammatory cytokine mRNAs after 4 hrs of LPS stimulation . The expression in ppp1r13l - knocked down murine cardiomyocytes was set as 1 . Genome wide expression analysis is presented in Figure 1S . Differences between knockdown and control with p values ( Student ' s t test ) are indicated with asterisks and exact values .

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes.B. qPCR showed that ppp1r13l-knocked down murine cardiomyocytes (using two alternative shRNA sequences) expressed higher level of pro-inflammatory cytokine mRNAs after 4 hrs of LPS stimulation. The expression in ppp1r13l-knocked down murine cardiomyocytes was set as 1. Genome wide expression analysis is presented in Figure 1S. Differences between knockdown and control with p values (Student's t test) are indicated with asterisks and exact values.


Figure 6A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C ( Mybpc3 ) , phospholamban ( Pln ) , ryanodine receptor 2 ( Ryr2 ) , and sarcoglycan gamma ( Sgcg ) . Hearts removed from perfused mice and mRNA was subjected for q PCR . Difference between knockdown and control with p values = 0 . 03 ( Student ' s t test ) is indicated with an asterisk . B . A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn , 7 - week - and 12 - week - hearts ( p < 0 . 05 threshold , calculated by Deseq algorithm " R " ) . The expression level of s100a9 was higher in wa3 mice than in WT by 2 . 8 times . For the full list of genes see Tables S1 and S2 , and functional analyses in Figures S3 - S6 . C . Hierarchical clustering heat map of differentially expressed genes in the hearts of 7 - and 12 - week old wa3 mice in comparison to WT .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A wa3 hearts express higher level of Il1 than littermate WT hearts without significant changes in selected structural and regulatory genes as myosin binding protein C (Mybpc3), phospholamban (Pln), ryanodine receptor 2 (Ryr2), and sarcoglycan gamma (Sgcg). Hearts removed from perfused mice and mRNA was subjected for q PCR. Difference between knockdown and control with p values = 0.03 (Student's t test) is indicated with an asterisk.B. A scatter plot showing the deviation from normal expression by highlighting the differentially expressed genes of newborn, 7-week- and 12-week- hearts (p<0.05 threshold, calculated by Deseq algorithm "R"). The expression level of s100a9 was higher in wa3 mice than in WT by 2.8 times. For the full list of genes see Tables S1 and S2, and functional analyses in Figures S3-S6.C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.


Figure 7A . LPS ( 5mg / Kg for 2 hrs ) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts . The results are presented as fold induction of untreated controls . The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts , while of Mybpc3 and Sgcg was unchanged . For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7 . Differences between knockdown and control with p values = 0 . 04998 ( Student ' s t test ) are indicated with an asterisk . B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS ( 1mg / Kg ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. LPS (5mg/Kg for 2 hrs) induced higher expression levels of cytokine genes in wa3 hearts than in littermate WT hearts. The results are presented as fold induction of untreated controls. The mRNA expression levels of Pln and Ryr2 was reduced in both wa3 and WT hearts, while of Mybpc3 and Sgcg was unchanged. For the full list see Dataset EV3 and for functional clustering in Appendix Figure S7. Differences between knockdown and control with p values =0.04998 (Student's t test) are indicated with an asterisk.B shows that LPS induced a higher mortality rate of wa3 mice than of littermate controls following two weekly subsequent injections of LPS (1mg/Kg).


Figure 1Telomeric lncRNA levels in the indicated deletion strains as analyzed by dot blot ( top ) or Northern blot hybridization ( bottom ) using a double ‐ stranded telomeric probe . Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA . Bars and error bars are averages and s . d . from 4 independent experiments . U6 and 18S rRNA are shown as loading controls for Northern blotting . The asterisk indicates unspliced U6 . Molecular weights are on the left in kilobases . ARRET and αARRETNorthern blot analysis of the indicated strains using strand ‐ specific probes . Telomere length analysis of ApaI ‐ digested DNA from the indicated strains . Quantification of subtelomeric DNA from ChIP experiments using strains carrying the indicated Myc ‐ tagged proteins ( 2A9 and 8B7 : SPBC2A9 . 02 ‐ Myc and SPBP8B7 . 08c ‐ Myc , respectively ) . Immunoprecipitated DNA is expressed as fraction of input DNA . An untagged strain ( − ) served as a negative control . Bars and error bars are averages and s . d . from three independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * * P 0 . 001 relative to untagged strain .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Telomeric lncRNA levels in the indicated deletion strains as analyzed by dot blot (top) or Northern blot hybridization (bottom) using a double‐stranded telomeric probe. Telomeric dot blot signals are expressed as fold increase relative to wtA after normalization through U6 snRNA. Bars and error bars are averages and s.d. from 4 independent experiments. U6 and 18S rRNA are shown as loading controls for Northern blotting. The asterisk indicates unspliced U6. Molecular weights are on the left in kilobases.ARRET and αARRETNorthern blot analysis of the indicated strains using strand‐specific probes.Telomere length analysis of ApaI‐digested DNA from the indicated strains.Quantification of subtelomeric DNA from ChIP experiments using strains carrying the indicated Myc‐tagged proteins (2A9 and 8B7: SPBC2A9.02‐Myc and SPBP8B7.08c‐Myc, respectively). Immunoprecipitated DNA is expressed as fraction of input DNA. An untagged strain (−) served as a negative control. Bars and error bars are averages and s.d. from three independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to untagged strain.


Figure 2Chromosome maps of differentially expressed genes in cay1Δ cells shown as fold enrichment over wt . Red dots are overexpressed genes and Tf2 retrotransposons , blue dots are overexpressed LTR sequences . Northern blot analysis of tlh1 / 2 + , Tf2 retrotransposons , and 18S rRNA ( loading control ) in wt , cay1Δ , and trichostatin A ( TSA ) ‐ treated wt cells . Black arrowhead indicates tlh1 / 2 + mRNA ; red arrowhead unprocessed Tf2 transcripts ; and blue arrowhead processed Tf2 transcripts . ChIP analysis of Cay1 ‐ Myc binding to the indicated genomic loci . subtel : subtelomeres ; Tf2 : Tf2 retrotransposon genes ; LTR : Tf2 LTR sequences ; Tlh : tlh1 / 2 + genes ; cen : centromeres . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over untagged strains ( unt ) . ChIP analysis of H3K9ac , H3K9me3 , and total H3 at the indicated genomic loci . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only .

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Figure 2Chromosome maps of differentially expressed genes in cay1Δ cells shown as fold enrichment over wt. Red dots are overexpressed genes and Tf2 retrotransposons, blue dots are overexpressed LTR sequences.Northern blot analysis of tlh1/2+, Tf2 retrotransposons, and 18S rRNA (loading control) in wt, cay1Δ, and trichostatin A (TSA)‐treated wt cells. Black arrowhead indicates tlh1/2+ mRNA; red arrowhead unprocessed Tf2 transcripts; and blue arrowhead processed Tf2 transcripts.ChIP analysis of Cay1‐Myc binding to the indicated genomic loci. subtel: subtelomeres; Tf2: Tf2 retrotransposon genes; LTR: Tf2 LTR sequences; Tlh: tlh1/2+ genes; cen: centromeres. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over untagged strains (unt).ChIP analysis of H3K9ac, H3K9me3, and total H3 at the indicated genomic loci. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only.


Figure 3Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains . Act1 was used as a loading control . Asterisks indicate cross ‐ reacting bands . RT - PCR analysis of splicing efficiencies of the indicated pre ‐ mRNAs in wt and cay1 ∆ cells . The prp1 ‐ 1 strain carries a thermosensitive allele of the splicing factor Prp1 and was used as a positive control for splicing impairment . prp1 ‐ 1 cells were grown at 36 ° C for 4 h before harvesting . Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names , respectively . rap1T indicates amplification products obtained with oligonucleotides spanning rap1 + exon 3 and thereby amplifying both spliced and unspliced mRNA . Intronless act1 + was used as a loading control . Numbers at the bottom of each panel are ratios between spliced and unspliced forms ( s / u ) and spliced and act1 + ( s / act1 ) . Values are expressed as fold increase over wt . Western blot analysis of Rap1 - YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition . Membranes were probed with antibodies against GFP and Act1 ( loading control ) . Quantification of Rap1 - YFP protein levels in experiments as in ( C ) . Rap1 - YFP levels are expressed as fold increase over time 0 after normalization through Act1 . Data points and error bars are averages and s . d . from at least four independent experiments . Statistical significance was assayed using the unpaired , two - tailed Student ' s t - test . * P 0 . 05 , * * P 0 . 01 for cay1 + versus cay1 ∆ samples .

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Figure 3Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains. Act1 was used as a loading control. Asterisks indicate cross‐reacting bands.RT-PCR analysis of splicing efficiencies of the indicated pre‐mRNAs in wt and cay1∆ cells. The prp1‐1 strain carries a thermosensitive allele of the splicing factor Prp1 and was used as a positive control for splicing impairment. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1+ exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.Western blot analysis of Rap1-YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition. Membranes were probed with antibodies against GFP and Act1 (loading control).Quantification of Rap1-YFP protein levels in experiments as in (C). Rap1-YFP levels are expressed as fold increase over time 0 after normalization through Act1. Data points and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t-test. *P 0.05, **P 0.01 for cay1+ versus cay1∆ samples.


Figure 4Telomere length analysis of ApaI ‐ digested DNA from cay1 ∆ trt1 ∆ cells harvested at increasing generation doublings ( gen ) . Telomere length analysis of ApaI ‐ digested DNA from the indicated strains . dh : centromeric dh repeats shown as loading control . PFGE analysis of telomeric fusions in strains grown to logarithmic phase ( log ) or G1 ‐ arrested by nitrogen starvation ( G1 ) . Genomic DNA was digested with NotI and hybridized to C , I , L , and M probes detecting terminal fragments of chromosomes I and II . Bands corresponding to chromosome end fusions are indicated ( fused ) . Northern blot analysis of ARIA , ARRET , Tf2 retrotransposons , and 18S rRNA ( loading control ) in the indicated strains . qRT - PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1 + mRNA . Bars and error bars are averages and s . d . from 3 independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * P 0 . 01 relative to wt . ChIP analysis of H3K9ac and total H3 for the indicated strains . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars and error bars are averages and s . d . from at least four independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * * P 0 . 001 relative to wt .

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Figure 4Telomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen).Telomere length analysis of ApaI‐digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control.PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused).Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains.qRT-PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1+ mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. **P 0.01 relative to wt.ChIP analysis of H3K9ac and total H3 for the indicated strains. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to wt.


Figure 5Serial dilutions of the indicated strains were spotted on complete medium and grown at 30 ° C or 20 ° C . Otrt1 ∆ and Otrt1 ∆ cay1 ∆ are strains with circularized chromosomes lacking telomeric sequence ( Supplementary Fig S7 ) . Representative images of wt and cay1 ∆ cells grown for 24 h at 30 ° C or 20 ° C . Scale bar , 20 μm . Quantification of cell length for strains grown in complete medium for 24 h at 30 ° C or 20 ° C . Each dot represents a single cell and median cell length ( m ) is indicated for each sample . Data are from three independent experiments and at least 100 cells were scored per sample in each experiment . Top : Examples of DAPI ‐ stained wt and cay1 ∆ cells grown in complete medium for 24 h at 30 ° C or 20 ° C . The white arrowhead indicates a chromosome bridge . Scale bar , 10 μm . Bottom : Quantification of chromosome bridges . Bars and error bars are averages and s . d . from three independent experiments and at least 200 cells were scored per sample in each experiment . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * * * P 0 . 001 relative to 30 ° C .

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Figure 5Serial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C. Otrt1∆ and Otrt1∆cay1∆ are strains with circularized chromosomes lacking telomeric sequence (Supplementary Fig S7).Representative images of wt and cay1∆ cells grown for 24 h at 30°C or 20°C. Scale bar, 20 μm.Quantification of cell length for strains grown in complete medium for 24 h at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from three independent experiments and at least 100 cells were scored per sample in each experiment.Top: Examples of DAPI‐stained wt and cay1∆ cells grown in complete medium for 24 h at 30°C or 20°C. The white arrowhead indicates a chromosome bridge. Scale bar, 10 μm. Bottom: Quantification of chromosome bridges. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to 30°C.


Figure 6RT - PCR analysis of pre ‐ mRNA splicing efficiencies of rap1 + and poz1 + pre ‐ mRNAs in cay1 + and cay1Δ cells expressing Cay1 ‐ YFP or Rap1 ‐ YFP . Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names , respectively . rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA . Intronless act1 + was used as a loading control . Numbers at the bottom of each panel are ratios between spliced and unspliced forms ( s / u ) and spliced and act1 + ( s / act1 ) . Values are expressed as fold increase over wt . Western blot analysis of total proteins from early generation cay1 + and cay1Δ cells expressing Cay1 ‐ YFP , Rap1 ‐ YFP , and Taz1 ‐ YFP using antibodies against Rap1 , Taz1 , and Act1 ( loading control ) . Numbers at the bottom are ratios between Rap1 proteins ( endogenous plus ectopic ) and Act1 ( Rap1 / Act1 ) . Values are expressed as fold increase over wt . Left : Representative YFPfluorescence microscopy and DIC images of cay1 ∆ strains expressing Cay1 ‐ YFP . Scale bar , 10 μm . Right : Representative YFP and RFP fluorescence images of Cay1 ‐ YFP ‐ and Pot1 ‐ RFP ‐ expressing cells . Arrows point to YFP and RFP foci within the same nucleus demonstrating lack of co ‐ localization . Scale bar , 5 μm . Representative YFPfluorescence microscopy and DIC images of cay1 + and cay1 ∆ strains expressing Rap1 ‐ YFP . Scale bar , 5 μm .

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Figure 6RT-PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1Δ cells expressing Cay1‐YFP or Rap1‐YFP. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.Western blot analysis of total proteins from early generation cay1+ and cay1Δ cells expressing Cay1‐YFP, Rap1‐YFP, and Taz1‐YFP using antibodies against Rap1, Taz1, and Act1 (loading control). Numbers at the bottom are ratios between Rap1 proteins (endogenous plus ectopic) and Act1 (Rap1/Act1). Values are expressed as fold increase over wt.Left: Representative YFPfluorescence microscopy and DIC images of cay1∆ strains expressing Cay1‐YFP. Scale bar, 10 μm. Right: Representative YFP and RFP fluorescence images of Cay1‐YFP‐ and Pot1‐RFP‐expressing cells. Arrows point to YFP and RFP foci within the same nucleus demonstrating lack of co‐localization. Scale bar, 5 μm.Representative YFPfluorescence microscopy and DIC images of cay1+ and cay1∆ strains expressing Rap1‐YFP. Scale bar, 5 μm.


Figure 7Telomere length analysis of ApaI ‐ digested DNA from cells harvested at early ( left ) and late generations ( right ) after expression of YFP ‐ tagged proteins . Northern blot analysis of ARIA , ARRET , and 18S rRNA ( loading control ) at early generations after expression of YFP ‐ tagged proteins . Numbers at the bottom are ratios between ARRET and 18S rRNA signals , expressed as fold increase over wt . ChIP analysis of subtelomeric H3K9ac , H3K9me3 , and total H3 in the indicated strains at early generations . Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars and error bars are averages and s . d . from 3 independent experiments . Statistical significance was assayed using the unpaired , two ‐ tailed Student ' s t ‐ test . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 relative to wt . Serial dilutions of early generation cay1 + and cay1 ∆ cells expressing Cay1 ‐ YFP and Rap1 ‐ YFP were spotted on complete medium and grown at 30 ° C and 20 ° C . Quantification of cell length in strains grown for 24 h in complete medium at 30 ° C or 20 ° C . Each dot represents a single cell and median cell length ( m ) is indicated for each sample . Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment . Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30 ° C or 20 ° C . Bars and error bars are averages and s . d . from three independent experiments and at least 200 cells were scored per sample in each experiment .

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Figure 7Telomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins.Northern blot analysis of ARIA, ARRET, and 18S rRNA (loading control) at early generations after expression of YFP‐tagged proteins. Numbers at the bottom are ratios between ARRET and 18S rRNA signals, expressed as fold increase over wt.ChIP analysis of subtelomeric H3K9ac, H3K9me3, and total H3 in the indicated strains at early generations. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt.Serial dilutions of early generation cay1+ and cay1∆ cells expressing Cay1‐YFP and Rap1‐YFP were spotted on complete medium and grown at 30°C and 20°C.Quantification of cell length in strains grown for 24 h in complete medium at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment.Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30°C or 20°C. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment.


Figure 8Northern blot analysis of Tf2 retrotransposons , U6 snRNA , and act1 + mRNA ( loading control ) in wt , cay1Δ , and prp1 ‐ 1 cells . prp1 ‐ 1 cells were grown at 36 ° C for 4 h before harvesting . Black arrowhead indicates unprocessed Tf2 transcripts . The asterisk indicates unspliced U6 . Numbers at the bottom are the ratios between Tf2 and act1 + signals ( Tf2 / Act1 ) and expressed as fold increase over wt . Northern blot analysis as in ( A ) in mpn1 + and mpn1 ∆ strains carrying a stably integrated extra copy of the snu6 + gene or empty vector control plasmids ( ev ) .

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Figure 8Northern blot analysis of Tf2 retrotransposons, U6 snRNA, and act1+ mRNA (loading control) in wt, cay1Δ, and prp1‐1 cells. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Black arrowhead indicates unprocessed Tf2 transcripts. The asterisk indicates unspliced U6. Numbers at the bottom are the ratios between Tf2 and act1+ signals (Tf2/Act1) and expressed as fold increase over wt.Northern blot analysis as in (A) in mpn1+ and mpn1∆ strains carrying a stably integrated extra copy of the snu6+ gene or empty vector control plasmids (ev).


Figure 1OTULIN DNA sequence chromatograms showing the homozygous single base substitution ( c . 841G > A , p . Gly281Arg , arrowhead ) . Data are representative to two independent experiments .

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Figure 1OTULIN DNA sequence chromatograms showing the homozygous single base substitution (c.841G>A, p.Gly281Arg, arrowhead). Data are representative to two independent experiments.


Figure 2Affinity measurements by fluorescence polarisation ( FP ) with recombinant catalytically inactive OTULINC129A or OTULINC129A / G281R and FlAsH - labelled M1 - linked diUb . Data represent mean ± SD of one experiment performed in technical triplicate . Data are representative of three independent experiments . a . u . , arbitrary units . K D , dissociation constant . M1 - linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver - stained SDS - PAGE gels . Data are representative of three independent experiments . First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R ( 1 . 0 mg / mL ) measured by nano differential scanning fluorimetry ( nanoDSF ) . Apparent melting temperatures ( Tm ) are indicated ( dashed lines ) . Data are representative to two independent experiments .

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Figure 2Affinity measurements by fluorescence polarisation (FP) with recombinant catalytically inactive OTULINC129A or OTULINC129A/G281R and FlAsH-labelled M1-linked diUb. Data represent mean ± SD of one experiment performed in technical triplicate. Data are representative of three independent experiments. a.u., arbitrary units. KD, dissociation constant.M1-linked tetraUb hydrolysis by recombinant OTULINWT and OTULING281R using the indicated OTULIN concentrations and visualised on silver-stained SDS-PAGE gels. Data are representative of three independent experiments.First derivative of tryptophan fluorescence upon thermal unfolding of recombinant OTULINcatWT and OTULINcatG281R (1.0 mg/mL) measured by nano differential scanning fluorimetry (nanoDSF). Apparent melting temperatures (Tm) are indicated (dashed lines). Data are representative to two independent experiments.


Figure 3Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts . Data are representative of three independent experiments . Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 ( 10 μM ) for 24 h . Data are representative of two independent experiments . Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts . Data are representative of three independent experiments . Immunoblot ( left ) and densitometry ( right ) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments .

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Figure 3Immunoblot analysis of whole cell lysates from untreated primary healthy control and patient fibroblasts. Data are representative of three independent experiments.Immunoblot analysis of whole cell lysates from primary healthy control and patient fibroblasts either left untreated or treated with the proteasome inhibitor MG132 (10 μM) for 24 h. Data are representative of two independent experiments.Immunoblot analysis of whole cell lysates from untreated primary healthy control or patient fibroblasts. Data are representative of three independent experiments.Immunoblot (left) and densitometry (right) analysis of IκBα levels in primary healthy control and patient fibroblasts treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.


Figure 4Immunoblot analysis of IκBα phosphorylation and degradation as well as p65 / RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Densitometry analysis of p65 / RelA phosphorylation as presented in ( A ) from three independent experiments . ELISA analysis of IL - 8 secretion in response to TNF stimulation ( 10 ng / mL ) in healthy control or OTULING281R primary fibroblasts . Bars represent mean ± SEM ( n = 4 ) . Immunoblot analysis of the native TNF - RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts . Data are representative of three independent experiments .

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Figure 4Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in healthy control or OTULING281R fibroblasts primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments. Immunoblot analysis of phosphorylation of the MAP kinases p38 and JNK in healthy control or OTULING281R primary fibroblasts in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.Densitometry analysis of p65/RelA phosphorylation as presented in (A) from three independent experiments.ELISA analysis of IL-8 secretion in response to TNF stimulation (10 ng/mL) in healthy control or OTULING281R primary fibroblasts. Bars represent mean ± SEM (n=4).Immunoblot analysis of the native TNF-RSC purified by immunoprecipitation from healthy control or OTULING281R primary fibroblasts. Data are representative of three independent experiments.


Figure 5Immunoblot analysis of whole cell lysate from untreated human THP - 1 monocytes with stable expression of a non - targeting control shRNA or and shRNA targeting OTULIN . Data are representative of two independent experiments . Immunoblot ( left ) and densitometry ( right ) analysis of IκBα levels in shControl and shOTULIN THP - 1 cells treated with cycloheximide ( CHX ) ( 50 μg / mL ) as indicated . Data are representative of three independent experiments . ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP - 1 cells . Data represents mean ± SEM ( n = 4 ) and were analysed using the two - way ANOVA test of statistical significance with Sidak ' s correction for multiple comparisons . Immunoblot analysis of IκBα phosphorylation and degradation as well as p65 / RelA phosphorylation in shControl and shOTULIN THP - 1 cells in response to stimulation with TNF ( 10 ng / mL ) . Data are representative of three independent experiments . Densitometry analysis of p65 / RelA phosphorylation as presented in ( D ) from three independent experiments .

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Figure 5Immunoblot analysis of whole cell lysate from untreated human THP-1 monocytes with stable expression of a non-targeting control shRNA or and shRNA targeting OTULIN. Data are representative of two independent experiments.Immunoblot (left) and densitometry (right) analysis of IκBα levels in shControl and shOTULIN THP-1 cells treated with cycloheximide (CHX) (50 μg/mL) as indicated. Data are representative of three independent experiments.ELISA analysis of spontaneous TNF secretion over 96 h in shControl and shOTULIN THP-1 cells. Data represents mean ± SEM (n=4) and were analysed using the two-way ANOVA test of statistical significance with Sidak's correction for multiple comparisons.Immunoblot analysis of IκBα phosphorylation and degradation as well as p65/RelA phosphorylation in shControl and shOTULIN THP-1 cells in response to stimulation with TNF (10 ng/mL). Data are representative of three independent experiments.Densitometry analysis of p65/RelA phosphorylation as presented in (D) from three independent experiments.


Figure 6Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , as indicated was analysed using MTT reduction assays . Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , and Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays . Viability of shControl and shOTULIN THP - 1 cells ( C - D ) after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , shControl and shOTULIN THP - 1 cells ( C - D ) after 24 h and 6 h , respectively , of treatment with TNF ( 100 ng / mL ) , CHX ( 50 μg / mL ) , Q - VD - OPh ( 10 μM ) , and Nec - 1 ( 10 μM ) as indicated was analysed using MTT reduction assays . Increased apoptotic cell death in the skin of ORAS patient III . 2 . Serial sections of normal skin ( top panels ) and a skin biopsy from patient III . 2 taken at an inflammatory flare ( bottom panels ) were immunostained for cleaved caspase - 3 ( centre panels ) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) assay ( right panels ) . Arrowheads indicate cleaved caspase - 3 - positive mesenchymal cells . None of these markers were present in the healthy control skin . H & E , haematoxylin and eosin . Boxes in H & E panels indicate the areas magnified in the cleaved caspase - 3 and TUNEL panels . Scale bars , 100 μm . Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III . 2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), as indicated was analysed using MTT reduction assays.Viability of healthy control or OTULING281R primary fibroblasts after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.Viability of shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL),shControl and shOTULIN THP-1 cells (C-D) after 24 h and 6 h, respectively, of treatment with TNF (100 ng/mL), CHX (50 μg/mL), Q-VD-OPh (10 μM), and Nec-1 (10 μM) as indicated was analysed using MTT reduction assays.Increased apoptotic cell death in the skin of ORAS patient III.2. Serial sections of normal skin (top panels) and a skin biopsy from patient III.2 taken at an inflammatory flare (bottom panels) were immunostained for cleaved caspase-3 (centre panels) or analysed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (right panels). Arrowheads indicate cleaved caspase-3-positive mesenchymal cells. None
of these markers were present in the healthy control skin. H&E, haematoxylin and eosin. Boxes in H&E panels indicate the areas magnified in the cleaved caspase-3 and TUNEL panels. Scale bars, 100 μm.
Data are representative of stained sections from three healthy controls and one biopsy from ORAS patient III.2.


Figure 7Analysis of the blood cell chimerism in patient III . 2 after HSCT . Short terminal repeat ( STR ) analysis was used to determine the percentage of donor cells in peripheral blood . Arrow indicates point of relapse . Photograph of patient III . 2 at age ~ 10 years at an episode of inflammation caused by delayed etanercept administration . Arrows indicate the erythematous subcutaneous nodules ( panniculitis ) .

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Figure 7Analysis of the blood cell chimerism in patient III.2 after HSCT. Short terminal repeat (STR) analysis was used to determine the percentage of donor cells in peripheral blood. Arrow indicates point of relapse.Photograph of patient III.2 at age ~10 years at an episode of inflammation caused by delayed etanercept administration. Arrows indicate the erythematous subcutaneous nodules (panniculitis).


Figure 1A , B Representative time - lapse images ( A ) and kymograph ( B ) of mouse zygotes expressing H2B - RFP ( magenta ) and labelled with SiR - Tubulin ( green ) . Shown is an example of marked chromosomal displacement to the cortex . C Changes in chromosome - to - cell centre distances ( d ) over time relative to anaphase - onset ( n = 50 blastomeres ) . Data information : In ( A , time is shown as h post - metaphase and scale bar = 20 µm . In ( C , data are shown as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex. C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time is shown as h post-metaphase and scale bar = 20 µm. In (C, data are shown as mean ± SEM.


Figure 2B , C Representative time - lapse images of embryos in which DMSO ( B ) or nocodazole ( C ) was added after anaphase completion . D Changes in nucleus - to - cell centre distances ( d ) over time relative to time of drug addition following DMSO - ( n = 22 blastomeres ) or nocodazole - treatment ( n = 26 blastomeres ) at anaphase completion . Data information : In ( B , C , time is shown as h post - drug addition and scale bar = 20 µm . In ( D , data are shown as mean ± SEM . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) . E - G Representative time - lapse images of embryos in which DMSO ( E ) , Nocodazole ( F ) or Cytochalasin D ( G ) was added when nuclei had travelled to the cortex . H Changes in nucleus - to - cell centre distances ( d ) over time relative to time of drug addition DMSO - ( n = 20 blastomeres ) , nocodazole - ( n = 12 blastomeres ) or cytochalasin D - treatment ( n = 14 blastomeres ) after nuclei reached the cortex . Data information : In E - G ) , time is shown as h post - drug addition and scale bar = 20 µm . In H ) , data are shown as mean ± SEM . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B, C Representative time-lapse images of embryos in which DMSO (B) or nocodazole (C) was added after anaphase completion. D Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase completion. Data information: In (B,C, time is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).E-G Representative time-lapse images of embryos in which DMSO (E), Nocodazole (F) or Cytochalasin D (G) was added when nuclei had travelled to the cortex. H Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei reached the cortex. Data information: In E - G), time is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


Figure 3A , B Representative time - lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase ( A , n = 15 embryos ) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin ( B , n = 22 embryos ) . C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not . Each data point represents the mean maximal d value of the two blastomeres from the same embryo ( n = 45 embryos ) . D Graph of time taken to develop to the blastocyst stage in relation to maximal d values ( n = 27 embryos ) . E Graph comparing the distribution of times taken for nuclei to re - centre in embryos that developed to the blastocyst stage or not ( n = 45 embryos ) . F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re - centre ( n = 27 embryos ) . Data information : In ( A , B ) , time is shown as h post - drug washout and scale bar = 20 µm . In ( C - F ) , data are shown as mean ± SEM . In ( C , E ) , statistical significance was assessed by Welch ' s t - test , ns : not significant . In ( D , F ) , the strength of correlation ( r ) and statistical significance were assessed using the Pearson correlation test , ns : not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B Representative time-lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase (A, n = 15 embryos) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not. Each data point represents the mean maximal d value of the two blastomeres from the same embryo (n = 45 embryos). D Graph of time taken to develop to the blastocyst stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times taken for nuclei to re-centre in embryos that developed to the blastocyst stage or not (n = 45 embryos). F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re-centre (n = 27 embryos). Data information: In (A, B), time is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.


Figure 4A Representative time - lapse image of DNA and SiR - tubulin in DMSO - and BFA - treated embryos during the first mitotic division and subsequent interphase . Time is shown as h relative to the first mitotic anaphase . B Changes in nucleus - to - cell centre distances ( d ) over time in BFA - treated embryos , relative to maximal nuclear displacement to the cortex ( n = 14 blastomeres ) . Data information : In ( A , scale bar = 20 µm . D Representative time - lapse images of the second mitotic division in DMSO - and BFA - treated embryos . Dashed white lines highlight position of nuclei shortly before NEBD . Dashed yellow lines outline daughter - cells . Time is shown as h relative to the second mitotic NEBD . E Dot plot of the duration between first mitotic division anaphase - onset and second mitotic division NEBD for DMSO - ( n = 47 blastomeres ) and BFA - treated ( n = 47 blastomeres ) embryos . F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO - ( n = 44 blastomeres ) and BFA - treated ( n = 43 blastomeres ) embryos . G Plot of the spindle - to - cell centre distances against pre - NEBD nucleus - to - cell centre distance ( n = 34 blastomeres ) . Line indicates mathematically regressed fit . Data information : In D ) , scale bar = 20 µm . In E - G , data are shown as mean ± SEM . In ( E , F , statistical significance was assessed by Mann - Whitney test , * * * * P < 0 . 0001 , ns : not significant . In ( G , the strength of correlation ( r ) and statistical significance were assessed using the Pearson correlation test . Two - tailed p values are reported ( * * P < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Representative time-lapse image of DNA and SiR-tubulin in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre distances (d) over time in BFA-treated embryos, relative to maximal nuclear displacement to the cortex (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.D Representative time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei shortly before NEBD. Dashed yellow lines outline daughter-cells. Time is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).


Figure 5A Representative time - lapse images of 2 - cell embryos cultured with SiR - DNA to label nuclei ( white circles ) and microinjected with oil droplets ( yellow circles ) . Time is shown as h : min relative to maximal nuclear displacement . B Plot of the distances between oil droplets and nuclei to the cell - centre over time ( n = 14 oil - nucleus pairs ) . Data information : Scale bar = 20 µm in A , In ( B ) , data are shown as mean ± SEM . E Top panel : an example of fluorescent bead distribution in a 2 - cell embryo . Lower panel : an example of region imaged for bead tracking at high temporal resolution . F , G Trajectories of bead movement over a 20 s - duration in oocytes ( F , n = 217 beads from 23 oocytes ) and 2 - cell embryos ( n = 277 beads from 26 embryos ) . Each coloured line represents a single bead track . Data information : Scale bar = 20 µm in E , except 2 µm in the lower panel of E .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Representative time-lapse images of 2-cell embryos cultured with SiR-DNA to label nuclei (white circles) and microinjected with oil droplets (yellow circles). Time is shown as h:min relative to maximal nuclear displacement. B Plot of the distances between oil droplets and nuclei to the cell-centre over time (n = 14 oil-nucleus pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.E Top panel: an example of fluorescent bead distribution in a 2-cell embryo. Lower panel: an example of region imaged for bead tracking at high temporal resolution. F, G Trajectories of bead movement over a 20 s-duration in oocytes (F, n = 217 beads from 23 oocytes) and 2-cell embryos (n = 277 beads from 26 embryos). Each coloured line represents a single bead track. Data information: Scale bar = 20 µm in E, except 2 µm in the lower panel of E.


Figure 7B Shown is a representative 2 - cell embryo immunostained for β - tubulin ( green ) and DNA ( magenta ) at anaphase completion ( n = 8 embryos ) . C Dot plot of mean β - tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus ( cortex - facing aspect , dashed white triangle in B ) or behind the nucleus ( cleavage plane - facing aspect , dashed yellow triangle in B ) ( n = 14 embryos ) . Data information : In ( B scale bar = 20 µm . In ( C , data are shown as mean ± SEM . In ( C , statistical significance was assessed by Welch ' s t - test , * * * * P < 0 . 0001 , ns = not significant . D Representative time - lapse images of embryos expressing H2B - RFP and labelled with SiR - tubulin following the addition of dynarrestin at anaphase completion ( n = 14 embryos ) . Data information : In ( D , time is shown as h post - drug addition . In D , scale bar = 20 µm . E Dot plot of maximal d values between DMSO - and dynarrestin - treated embryos ( n = 28 blastomeres per group ) . Data information : In E , data are shown as mean ± SEM . In E ) , statistical significance was assessed by Welch ' s t - test , * * * * P < 0 . 0001 , ns = not significant . G Representative time - lapse images of embryos expressing H2B - RFP and labelled with SiR - tubulin following the addition of taxol at maximal nuclear displacement . Data information : In G , scale bar = 20 µm . H Changes in nucleus - to - cell centre distances ( d ) over time relative to the time of drug addition in DMSO - ( n = 19 ) and taxol - ( n = 19 ) treated 2 - cell blastomeres ( mean ± SEM ) . Data information : In H ) , data are shown as mean ± SEM . In ( H ) , statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unremarked datapoints = not significant ) . I Representative images of embryos immunostained for β - tubulin and Hoechst following the completion of live imaging ( shown in E ) in DMSO - and taxol - treated embryos ( n = 9 embryos per group ) . Time is shown as h post - drug addition . Scale bar = 20 µm . Data information : In I ) , scale bar = 20 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos). C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos). Data information: In (B scale bar = 20 µm. In (C, data are shown as mean ± SEM. In (C, statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos). Data information: In (D, time is shown as h post-drug addition. In D, scale bar = 20 µm.E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement. Data information: In G, scale bar = 20 µm.H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.


Figure 1B Pim1 , 2 , or 3 and GFP - EDC3 expression vectors were transfected into HEK - 293T . GFP - Trap Nano - body beads ( GFP - Trap ) were used to immunoprecipitate ( IP ) GFP - EDC3 , and the associated proteins were analyzed by Western blotting with the indicated Abs .

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Figure 1B Pim1, 2, or 3 and GFP-EDC3 expression vectors were transfected into HEK-293T. GFP-Trap Nano-body beads (GFP-Trap) were used to immunoprecipitate (IP) GFP-EDC3, and the associated proteins were analyzed by Western blotting with the indicated Abs.


Figure 2B HEK - 293T cells were transfected with Pim1 and each EDC3 domain ( Fig . 2A ) fused to the C - terminus of the GFP - tagged vector . The individual fragments were immunoprecipitated with GFP - Trap beads and analyzed by Western blotting with the indicated Abs . EDC3 wild type ( W ) , F1 , F2 , F3 , F4 were EDC3 fragments vector fused to GFP . The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control ( pcDNA3 ) .

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Figure 2B HEK-293T cells were transfected with Pim1 and each EDC3 domain (Fig. 2A) fused to the C-terminus of the GFP-tagged vector. The individual fragments were immunoprecipitated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs. EDC3 wild type (W), F1, F2, F3, F4 were EDC3 fragments vector fused to GFP. The C1 lane was only transfected with the GFP Flag vector while C2 contained EDC3 wild type fused to GFP and no Pim1 but empty vector control (pcDNA3).


Figure 3F PC3 - LN4 CRISPR - Ctr and Pim1 KO cells were treated with PIMi ( PIM447 , 3μM ) , AKTi ( GSK690693 , 5μM ) , or the combination for 24 h . Extracts were subjected to Western blotting with the indicated Abs .

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Figure 3F PC3-LN4 CRISPR-Ctr and Pim1 KO cells were treated with PIMi (PIM447, 3μM), AKTi (GSK690693, 5μM), or the combination for 24 h. Extracts were subjected to Western blotting with the indicated Abs.


Figure 4Quantification of EDC3 / DCP1a foci number from Control ( Ctrl ) , PIMi , AKTi and the AKTi + PIMi combination in ( A ) . Data information : The quantification data of ( B are presented as mean + / - SEM ( n = 30 cells / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - test , are considered significant . p values > 0 . 05 not significant ( n . s . ) . The analyses done included multiple independent fields . D Quantification of DCP1a foci number for Control ( Ctrl ) , PIMi , AKTi , and AKTi + PIMi combination in ( C ) . Data information : The quantification data of D ) are presented as mean + / - SEM ( n = 30 cells / treatment group ) . p values < 0 . 05 , calculated using unpaired student t - test , are considered significant . p values > 0 . 05 not significant ( n . s . ) . The analyses done included multiple independent fields .

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Figure 4Quantification of EDC3/DCP1a foci number from Control (Ctrl), PIMi, AKTi and the AKTi + PIMi combination in (A). Data information: The quantification data of (B are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.D Quantification of DCP1a foci number for Control (Ctrl), PIMi, AKTi, and AKTi + PIMi combination in (C). Data information: The quantification data of D) are presented as mean +/- SEM (n = 30 cells/treatment group). p values<0.05, calculated using unpaired student t-test, are considered significant. p values>0.05 not significant (n.s.). The analyses done included multiple independent fields.


Figure 5A , B Western blot of whole - cell ( WCL ) and P - body ( PB ) purified from PC3 - LN4 cells stably expressing GFP - DCP1a used as a P - body tag . The P - bodies ( PB ) were isolated with GFP - Trap beads and analyzed by Western blotting with the indicated Abs .

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Figure 5A, B Western blot of whole-cell (WCL) and P-body (PB) purified from PC3-LN4 cells stably expressing GFP-DCP1a used as a P-body tag. The P-bodies (PB) were isolated with GFP-Trap beads and analyzed by Western blotting with the indicated Abs.


Figure 7A PC3 - LN4 Naïve and CRISPR / Cas9 genome - edited EDC3 S161A ( SA ) , S161D ( Fredriksson et al . ) , and EDC3 knock - out ( KO ) cell lines were plated ( 4000 cells / well in 24 well plates ) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform . The graph shown was generated with IncuCyte Basic Software following 5 days of growth . The experiments were done in triplicates . Values are mean + / - S . E . M . p values < 0 . 05 , calculated using unpaired student t - test . Data information : All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts . The data are presented as the mean + / - S . D . p values < 0 . 05 , calculated using unpaired student ' s t - test . H Relative mRNA expression of ITGA6 , ITGB1 , and KLF4 in PC3 - LN4 EDC3 KO - C60 cells , containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information : Real - time PCR dataJ Western blot analysis of proteins extracted from PC3 - LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies .

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Figure 7A PC3-LN4 Naïve and CRISPR/Cas9 genome-edited EDC3 S161A (SA), S161D (Fredriksson et al.), and EDC3 knock-out (KO) cell lines were plated (4000 cells/well in 24 well plates) and imaged every 12 h using phase contrast in the IncuCyte ZOOM platform. The graph shown was generated with IncuCyte Basic Software following 5 days of growth. The experiments were done in triplicates. Values are mean +/- S.E.M. p values<0.05, calculated using unpaired student t-test. Data information: All invasion and migration assays were performed in triplicates and 6 different fields at 10x magnification were counted in 3 separate inserts. The data are presented as the mean +/- S.D. p values<0.05, calculated using unpaired student's t-test.H Relative mRNA expression of ITGA6, ITGB1, and KLF4 in PC3-LN4 EDC3 KO-C60 cells, containing reexpressed EDC3 WT or EDC3 S161A mutant cDNAs Data information: Real-time PCR dataJ Western blot analysis of proteins extracted from PC3-LN4 cells treated with DMSO or the combination of Pim447 and AZD5363 for 96 h using the specified antibodies.


Figure 8A Two independently derived CRISPR / Cas9 PC3 - LN4 EDC3 S161A mutants ( EDC3 SA # 1 , # 2 ) and wild type cells were injected subcutaneously in the flank ( 5X106 ) of SCID mice ( n = 8 / group ) , and tumor growth was monitored until the control group reached 1000 mm3 . The subcutaneous tumor volume measurement ( mm3 ) was done as outlined in Materials and Methods . Data are presented as mean ± SEM , ( n = 8 / group ) . B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines , MCF10A and MCF12A , human breast cancer cell lines HCC38 , HCC1954 , MDA - MB - 231 , MDA - MD - 468 , BT - 549 , MRKNU1 , and mouse 4T1 by Western blotting with the indicated Abs . C Dual immunohistochemistry staining of human breast tumor with p - EDC3 Ab ( Fredriksson et al . ) and total EDC3 ( brown ) . Scale bar : 50µm . D Dual immunohistochemistry staining of human normal resected breast tissue sections with p - EDC3 Ab ( Fredriksson et al . ) and total EDC3 ( brown ) . Scale bar : 50µm .

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Figure 8A Two independently derived CRISPR/Cas9 PC3-LN4 EDC3 S161A mutants (EDC3 SA#1, #2) and wild type cells were injected subcutaneously in the flank (5X106) of SCID mice (n=8/group), and tumor growth was monitored until the control group reached 1000 mm3. The subcutaneous tumor volume measurement (mm3) was done as outlined in Materials and Methods. Data are presented as mean ± SEM, (n = 8/group).B Phosphorylated EDC3 levels were examined in normal immortalized breast cell lines, MCF10A and MCF12A, human breast cancer cell lines HCC38, HCC1954, MDA-MB-231, MDA-MD-468, BT-549, MRKNU1, and mouse 4T1 by Western blotting with the indicated Abs.C Dual immunohistochemistry staining of human breast tumor with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm. D Dual immunohistochemistry staining of human normal resected breast tissue sections with p-EDC3 Ab (Fredriksson et al.) and total EDC3 (brown). Scale bar: 50µm.


Figure 1A Montage of class averages and 3D volumes of humanTFG ( amino acids 1 ‐ 193 ; EMDataBank accession code EMD ‐ 6076 ) . Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference ‐ free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left . Scale bars , 50 Å . B Montage of class averages and 3D volumes of C . elegans TFG ( amino acids 1 ‐ 195 ; EMDataBank accession code EMD ‐ 6075 ) , generated as described in ( A ) . Scale bars , 50 Å . C Superposition of 3D volumes of full ‐ length human TFG and truncated TFG ( amino acids 1 ‐ 193 ) . The truncated form is depicted in yellow , while the full ‐ length form is depicted as gray mesh . The top view ( top ) exhibits limited differences in the structures , while the side view ( bottom ) shows extra density in the full ‐ length TFG isoform . Scale bar , 25 Å .

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Figure 1A Montage of class averages and 3D volumes of humanTFG (amino acids 1‐193; EMDataBank accession code EMD‐6076). Two sets of 2D class averages and 3D volumes are shown with the left columns showing the class averages generated by reference‐free alignment and classification and the right columns showing 3D RCT volumes corresponding to the class average to the left. Scale bars, 50 Å.B Montage of class averages and 3D volumes of C. elegans TFG (amino acids 1‐195; EMDataBank accession code EMD‐6075), generated as described in (A). Scale bars, 50 Å.C Superposition of 3D volumes of full‐length human TFG and truncated TFG (amino acids 1‐193). The truncated form is depicted in yellow, while the full‐length form is depicted as gray mesh. The top view (top) exhibits limited differences in the structures, while the side view (bottom) shows extra density in the full‐length TFG isoform. Scale bar, 25 Å.


Figure 2A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations . Error bars represent mean ± SEM ; n = 10 replicates . B Confocal microscopy images of recombinant , BODIPY ‐ labeled TFG in the presence of varying potassium acetate concentrations . Scale bar , 2 μm . C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain ‐ EM . Large arrowheads highlight TFG polymers ( only found in the presence of elevated potassium acetate ) . Small arrowheads highlight individual 11 nm cup ‐ like TFG octamers . Scale bar , 30 nm . D Circular dichroism spectroscopy was used to characterize the carboxyl ‐ terminus of C . elegans TFG ( amino acids 195 ‐ 486 ) . Samples were analyzed at different concentrations , and the data were normalized relative to one another . CD spectra were collected at 25 ° C in 25 mM sodium phosphate ( pH 7 . 2 ) using a 1 mm path length quartz cell . The spectra are characteristic of an intrinsically disordered protein .

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Figure 2A TFG particle size was determined using dynamic light scattering under increasing potassium acetate concentrations. Error bars represent mean ± SEM; n = 10 replicates.B Confocal microscopy images of recombinant, BODIPY‐labeled TFG in the presence of varying potassium acetate concentrations. Scale bar, 2 μm.C Recombinant TFG in the presence of varying potassium acetate concentrations was imaged by negative stain‐EM. Large arrowheads highlight TFG polymers (only found in the presence of elevated potassium acetate). Small arrowheads highlight individual 11 nm cup‐like TFG octamers. Scale bar, 30 nm.D Circular dichroism spectroscopy was used to characterize the carboxyl‐terminus of C. elegans TFG (amino acids 195‐486). Samples were analyzed at different concentrations, and the data were normalized relative to one another. CD spectra were collected at 25°C in 25 mM sodium phosphate (pH 7.2) using a 1 mm path length quartz cell. The spectra are characteristic of an intrinsically disordered protein.


Figure 3A The distributions of endogenous TFG and ERGIC ‐ 53 were examined in hTERT ‐ RPE ‐ 1 cells stably expressing low levels of mApple ‐ Sec16B using SR ‐ SIM ( more than 15 cells analyzed on 3 separate occasions ) . Scale bar , 5 μm . Inset scale bar , 1 μm . B Cells stably expressing low levels of GFP ‐ Sec16B and mRuby ‐ Sec23A ( Sec23A ) were fixed and imaged using SR ‐ SIM ( n = 12 ) . Scale bar , 5 μm . Inset scale bar , 1 μm . C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby ‐ Sec23A ( Sec23A ) using SR ‐ SIM ( n = 10 ) . Scale bar , 5 μm . Inset scale bar , 1 μm . D The distributions of endogenous TFG and Sec31A were examined using g ‐ STED ( n = 8 ) . Scale bar , 1 μm . E The distributions of endogenous TFG and Sec31A were examined using immunogold EM . Large arrowheads highlight 15 nm gold particles bound to α ‐ TFG antibodies . The small arrowhead highlights 5 nm gold particles bound to α ‐ Sec31A antibodies . At least 15 different cells were examined , and representative images are shown . Scale bar , 100 nm .

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Figure 3A The distributions of endogenous TFG and ERGIC‐53 were examined in hTERT‐RPE‐1 cells stably expressing low levels of mApple‐Sec16B using SR‐SIM (more than 15 cells analyzed on 3 separate occasions). Scale bar, 5 μm. Inset scale bar, 1 μm.B Cells stably expressing low levels of GFP‐Sec16B and mRuby‐Sec23A (Sec23A) were fixed and imaged using SR‐SIM (n = 12). Scale bar, 5 μm. Inset scale bar, 1 μm.C The distribution of endogenous TFG was examined in cells stably expressing low levels of mRuby‐Sec23A (Sec23A) using SR‐SIM (n = 10). Scale bar, 5 μm. Inset scale bar, 1 μm.D The distributions of endogenous TFG and Sec31A were examined using g‐STED (n = 8). Scale bar, 1 μm.E The distributions of endogenous TFG and Sec31A were examined using immunogold EM. Large arrowheads highlight 15 nm gold particles bound to α‐TFG antibodies. The small arrowhead highlights 5 nm gold particles bound to α‐Sec31A antibodies. At least 15 different cells were examined, and representative images are shown. Scale bar, 100 nm.