Beclin ‐ 1 depletion prolongs prometaphase by delaying chromosome congression . ( A - H ) Time ‐ lapse films of HeLa cells stably expressing YFP ‐ α ‐ tubulin and H2B ‐ Cherry and treated with the indicated siRNA . The pictures are acquired every 10 min . Time lapse starts at nuclear envelope breakdown . ( A ) Mitosis in control cells . ( B ) Western blot of whole ‐ cell lysates from Control , Beclin ‐ 1 and Beclin ‐ 1 + Mad2 siRNA ‐ treated cells . ( C - E ) Three different classes of mitotic progression were observed upon Beclin ‐ 1 depletion . Higher magnification of lagging chromatids in the anaphase of Beclin ‐ 1 ‐ depleted cells is shown on the right of ( D ) .

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Beclin‐1 depletion prolongs prometaphase by delaying chromosome congression. (A-H) Time‐lapse films of HeLa cells stably expressing YFP‐α‐tubulin and H2B‐Cherry and treated with the indicated siRNA. The pictures are acquired every 10 min. Time lapse starts at nuclear envelope breakdown. (A) Mitosis in control cells. (B) Western blot of whole‐cell lysates from Control, Beclin‐1 and Beclin‐1+Mad2 siRNA‐treated cells. (C-E) Three different classes of mitotic progression were observed upon Beclin‐1 depletion. Higher magnification of lagging chromatids in the anaphase of Beclin‐1‐depleted cells is shown on the right of (D).


( F ) The prometaphase delay is dependent on MAD2 and the spindle checkpoint . Cells treated simultaneously with siRNA targeting MAD2 and Beclin ‐ 1 rapidly exit mitosis . ( G ) Average duration of mitosis in different classes of Beclin ‐ 1 ‐ depleted cells , compared to control and MAD2 depletions . n = number of counted cells in three independent experiments . Error bars on graphs represent s . d .

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(F) The prometaphase delay is dependent on MAD2 and the spindle checkpoint. Cells treated simultaneously with siRNA targeting MAD2 and Beclin‐1 rapidly exit mitosis. (G) Average duration of mitosis in different classes of Beclin‐1‐depleted cells, compared to control and MAD2 depletions. n=number of counted cells in three independent experiments. Error bars on graphs represent s.d.


( H ) The frequency of the different Beclin ‐ 1 phenotypes depended on the efficiency of Beclin ‐ 1 knockdown . Mitotic progression in cells treated with increasing concentrations of Beclin ‐ 1 siRNA was followed by video ‐ microscopy . Left panel : western blot of Beclin ‐ 1 upon RNAi treatment ; right panel : quantification of the appearance of the three different mitotic progression patterns , that is , prolonged mitosis with normal anaphase ( as C ) , with lagging chromatids ( as D ) and mitotic exit without clear anaphase ( as E ) upon increased Beclin ‐ 1 knockdown experiments . NEB , nuclear envelop breakdown ; RNAi , RNA interference ; siRNA , small interfering RNA ; YFP , yellow fluorescent protein .

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(H) The frequency of the different Beclin‐1 phenotypes depended on the efficiency of Beclin‐1 knockdown. Mitotic progression in cells treated with increasing concentrations of Beclin‐1 siRNA was followed by video‐microscopy. Left panel: western blot of Beclin‐1 upon RNAi treatment; right panel: quantification of the appearance of the three different mitotic progression patterns, that is, prolonged mitosis with normal anaphase (as C), with lagging chromatids (as D) and mitotic exit without clear anaphase (as E) upon increased Beclin‐1 knockdown experiments. NEB, nuclear envelop breakdown; RNAi, RNA interference; siRNA, small interfering RNA; YFP, yellow fluorescent protein.


( A ) Inter ‐ kinetochore distance is reduced in unaligned chromosomes of Beclin ‐ 1 ‐ depleted cells . The distance between each kinetochore pair was quantified in siRNA ‐ treated and control cells treated with nocodazole for 3 h before fixation . Cells were stained with anti ‐ CREST antibody and DAPI . Quantification of the distance between kinetochore pairs ( indicated by coloured bars ) is reported at the bottom and done on sister kinetochores present in the same focal plane . One asterisk indicates significant result ( P = 0 . 0365 ) . Graphs represent three independent experiments ( error bars = s . d . ) .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Inter‐kinetochore distance is reduced in unaligned chromosomes of Beclin‐1‐depleted cells. The distance between each kinetochore pair was quantified in siRNA‐treated and control cells treated with nocodazole for 3 h before fixation. Cells were stained with anti‐CREST antibody and DAPI. Quantification of the distance between kinetochore pairs (indicated by coloured bars) is reported at the bottom and done on sister kinetochores present in the same focal plane. One asterisk indicates significant result (P=0.0365). Graphs represent three independent experiments (error bars=s.d.).


( B ) Depletion of Beclin ‐ 1 leads to defects in K ‐ MT attachment . siRNA ‐ treated cells were cooled on ice before fixation to reveal stable k ‐ fibres and then stained with anti ‐ α ‐ tubulin ( red ) and anti ‐ CREST ( green ) antibodies . Numbers point to magnified areas and indicate the mode of attachment of k ‐ fibres to kinetochores ( 1 , bi ‐ oriented ; 2 , unattached ; 3 , mono ‐ oriented kinetochores ) . Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; NS , not significant ; siRNA , small interfering RNA .

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(B) Depletion of Beclin‐1 leads to defects in K‐MT attachment. siRNA‐treated cells were cooled on ice before fixation to reveal stable k‐fibres and then stained with anti‐α‐tubulin (red) and anti‐CREST (green) antibodies. Numbers point to magnified areas and indicate the mode of attachment of k‐fibres to kinetochores (1, bi‐oriented; 2, unattached; 3, mono‐oriented kinetochores). Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; NS, not significant; siRNA, small interfering RNA.


( A ) CENP ‐ E and CREST . Two asterisks indicate significant results , P = 0 . 0053 .

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(A) CENP‐E and CREST. Two asterisks indicate significant results, P=0.0053.


( B ) CENP ‐ F and CREST . Three asterisks indicate significant results , P = 0 . 0006 .

(B) CENP‐F and CREST. Three asterisks indicate significant results, P=0.0006.


( C ) ZW10 and CREST . Three asterisks indicate significant results , P = 0 . 0007 .

(C) ZW10 and CREST. Three asterisks indicate significant results, P=0.0007.


( D ) MAD2 and CREST . Two asterisks indicate significant results , P = 0 . 0032 .

(D) MAD2 and CREST. Two asterisks indicate significant results, P=0.0032.


( F ) KNL ‐ 1 and CREST . On the right , magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown . Graphs represent three independent experiments ( error bars = s . d . ) . Scale bar , 10 μm .

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(F) KNL‐1 and CREST. On the right, magnified areas and quantification of the fluorescence intensity of the protein of interest normalized to the fluorescence intensity of CREST staining are shown. Graphs represent three independent experiments (error bars=s.d.). Scale bar, 10 μm.


( G ) Western blot of whole ‐ cell lysates from control and Beclin ‐ 1 siRNA ‐ treated cells . siRNA , small interfering RNA .

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(G) Western blot of whole‐cell lysates from control and Beclin‐1 siRNA‐treated cells. siRNA, small interfering RNA.


( A ) Immunoprecipitation of endogenous Zwint ‐ 1 in HeLa cell extracts expressing HA ‐ Beclin ‐ 1 or HA ‐ GFP . Input is 3 . 5 % of the immunoprecipitated material .

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(A) Immunoprecipitation of endogenous Zwint‐1 in HeLa cell extracts expressing HA‐Beclin‐1 or HA‐GFP. Input is 3.5% of the immunoprecipitated material.


( B ) GST ‐ Zwint ‐ 1 or GST ‐ beads were used to pull down recombinant purified His6 ‐ Beclin ‐ 1 . ( Left ) Coomassie ‐ blue ‐ stained gel of the purified recombinant proteins ( 1 μg ) . ( Right ) A western blot of GST proteins and His6 ‐ Beclin ‐ 1 in the input and bound fractions . His6 ‐ Beclin ‐ 1 in the input represents 4 % of the amount used for the pull ‐ down .

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(B) GST‐Zwint‐1 or GST‐beads were used to pull down recombinant purified His6‐Beclin‐1. (Left) Coomassie‐blue‐stained gel of the purified recombinant proteins (1 μg). (Right) A western blot of GST proteins and His6‐Beclin‐1 in the input and bound fractions. His6‐Beclin‐1 in the input represents 4% of the amount used for the pull‐down.


( C ) Zwint ‐ 1 levels do not change in Beclin ‐ 1 ‐ depleted cells . Cells were treated with nocodazole before fixation . On the right , magnified individual kinetochore pairs stained for Zwint ‐ 1 ( red ) and CREST ( green ) , and quantification of Zwint ‐ 1 levels normalized to CREST signal from three independent experiments ( error bars = s . d . ) .

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(C) Zwint‐1 levels do not change in Beclin‐1‐depleted cells. Cells were treated with nocodazole before fixation. On the right, magnified individual kinetochore pairs stained for Zwint‐1 (red) and CREST (green), and quantification of Zwint‐1 levels normalized to CREST signal from three independent experiments (error bars=s.d.).


( D ) Beclin ‐ 1 is associated with both the centrosome and the mitotic spindle during mitosis . HeLa cells were permeabilized before fixation and immunostained for Beclin ‐ 1 ( green ) , α ‐ tubulin ( red ) and DNA ( DAPI , blue ) .

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(D) Beclin‐1 is associated with both the centrosome and the mitotic spindle during mitosis. HeLa cells were permeabilized before fixation and immunostained for Beclin‐1 (green), α‐tubulin (red) and DNA (DAPI, blue).


( E ) Beclin ‐ 1 is retained on the k ‐ fibres . HeLa cells were cooled on ice before permeabilization and fixation , and stained with anti ‐ α ‐ tubulin ( red ) and anti ‐ Beclin ‐ 1 ( green ) antibodies .

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(E) Beclin‐1 is retained on the k‐fibres. HeLa cells were cooled on ice before permeabilization and fixation, and stained with anti‐α‐tubulin (red) and anti‐Beclin‐1 (green) antibodies.


( F ) Beclin ‐ 1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule ‐ dependent manner . Untreated cells ( left ) and nocodazole ‐ treated cells ( right ) were pre ‐ extracted and fixed before labelling with Beclin ‐ 1 ( green ) and Zwint ‐ 1 ( red ) antibodies .

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(F) Beclin‐1 forms discrete foci in the vicinity of the outer kinetochore in a microtubule‐dependent manner. Untreated cells (left) and nocodazole‐treated cells (right) were pre‐extracted and fixed before labelling with Beclin‐1 (green) and Zwint‐1 (red) antibodies.


( G ) The fluorescent ‐ tagged Cherry ‐ Beclin ‐ 1 forms foci in the vicinity of the outer kinetochore . HeLa cells transfected with indicated constructs were pre ‐ extracted and fixed before labelling with Zwint ‐ 1 ( green ) antibody . Magnified areas are shown on the right .

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(G) The fluorescent‐tagged Cherry‐Beclin‐1 forms foci in the vicinity of the outer kinetochore. HeLa cells transfected with indicated constructs were pre‐extracted and fixed before labelling with Zwint‐1 (green) antibody. Magnified areas are shown on the right.


( H ) Beclin ‐ 1 localization close to the kinetochore does not depend on the ZW10 protein . Cells treated with Control , Beclin ‐ 1 and ZW10 siRNA were pre ‐ extracted and fixed before labelling with Beclin ‐ 1 ( green ) , CREST ( blue ) and ZW10 ( red ) antibodies . DNA was visualized with DAPI . ( C - H ) Scale bar , 10 μm . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DNA , deoxyribonucleic acid ; GFP , green fluorescent protein ; GST , glutathione S ‐ transferase ; HA , haemagglutinin .

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(H) Beclin‐1 localization close to the kinetochore does not depend on the ZW10 protein. Cells treated with Control, Beclin‐1 and ZW10 siRNA were pre‐extracted and fixed before labelling with Beclin‐1 (green), CREST (blue) and ZW10 (red) antibodies. DNA was visualized with DAPI. (C-H) Scale bar, 10 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole; DNA, deoxyribonucleic acid; GFP, green fluorescent protein; GST, glutathione S‐transferase; HA, haemagglutinin.


Dynamic fluorescence patterns according to cell density in 8 × GTIIC - EmGFP 293T cells .

Dynamic fluorescence patterns according to cell density in 8×GTIIC-EmGFP 293T cells.


EmGFP intensity in LATS2 knockdown , or forced expression of a constitutively active form of YAP ( YAP - S127A ) . Scale bars indicate 100 μm .

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EmGFP intensity in LATS2 knockdown, or forced expression of a constitutively active form of YAP (YAP-S127A). Scale bars indicate 100 μm.


Scatter plots of mean frequency of each shRNA per gene . Average of v HOXA4 is indicated as a red dot .

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Scatter plots of mean frequency of each shRNA per gene. Average of v HOXA4 is indicated as a red dot.


Augmented 8 × GTIIC - luciferase reporter activity by loss of HOXA4 . ShSAV1 was used as an experimental control . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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Augmented 8×GTIIC-luciferase reporter activity by loss of HOXA4. ShSAV1 was used as an experimental control. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Quantitative real - time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with

Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4


Quantitative real - time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression

Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression vector


Quantitative real - time PCR analysis of CTGF , CYR61 , and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4 . ShLATS1 / 2 and shYAP were used as an experimental control . * P < 0 . 05 , * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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Quantitative real-time PCR analysis of CTGF, CYR61, and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4. ShLATS1/2 and shYAP were used as an experimental control. *P<0.05, **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


8 × GTIIC - luciferase reporter assays in HEK 293T cells transfected HOXA4 and / or shLATS1 / 2 . * * P < 0 . 01 vs shCtrl , by two - way ANOVA with Sidak ' s correction .

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8×GTIIC-luciferase reporter assays in HEK 293T cells transfected HOXA4 and/or shLATS1/2. **P<0.01 vs shCtrl, by two-way ANOVA with Sidak's correction.


Quantitative real - time PCR analysis of HOXA4 , YAP , CTGF , and CYR61 in HEK 293T cells with overexpression of HOXA4 and / or constitutive active form of YAP ( YAP - 5SA ) . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with LacZ , by ANOVA with Sidak ' s correction .

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Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with overexpression of HOXA4 and/or constitutive active form of YAP (YAP -5SA). *P<0.05, **P<0.01 vs YAP-5SA with LacZ, by ANOVA with Sidak's correction.


Quantitative real - time PCR analysis of HOXA4 , YAP , CTGF , and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and / or overexpression of YAP - 5SA . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with shCtrl , by ANOVA with Sidak ' s correction .

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Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and/or overexpression of YAP-5SA. *P<0.05, **P<0.01 vs YAP-5SA with shCtrl, by ANOVA with Sidak's correction.


Association of HOXA4 with TEAD1 indicated by co - IP experiments . HEK 293T cells were co - transfected with FLAG - HOXA4 and Myc -

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Association of HOXA4 with TEAD1 indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-TEAD1


Association of HOXA4 but not YAP indicated by co - IP experiments . HEK 293T cells were co - transfected with FLAG - HOXA4 and Myc - YAP .

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Association of HOXA4 but not YAP indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-YAP.


Interaction between endogenous TEAD1 and HOXA4 by co - IP experiments in HEK 293T cells . The interaction between endogenous TEAD1 and YAP was used as a positive control .

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Interaction between endogenous TEAD1 and HOXA4 by co-IP experiments in HEK 293T cells. The interaction between endogenous TEAD1 and YAP was used as a positive control.


Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1 .

Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1.


Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4 . Arrowheads indicate IgG heavy chain . Arrows indicate TEAD1 ( a . a . 1 - 426 ) .

Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).


Requirement of TEAD1 ( a . a . 30 - 135 ) for its interaction with HOXA4 via its homeodomain ( a . a . 216 - 274 ) .

Requirement of TEAD1 (a.a. 30-135) for its interaction with HOXA4 via its homeodomain (a.a. 216-274).


Failure of TEAD1 ( a . a . 30 - 102 ) , TEA domain alone , to interact with HOXA4 .

Failure of TEAD1 (a.a. 30-102), TEA domain alone, to interact with HOXA4.


ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4 . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Data are expressed as the means ± SEM of three independent experiments .

ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Competitive co - IP assay and densitometry showing significantly reduced TEAD - YAP interaction by overexpression of HOXA4 in a dose - dependent manner in HEK 293T cells . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments .

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Competitive co-IP assay and densitometry showing significantly reduced TEAD-YAP interaction by overexpression of HOXA4 in a dose-dependent manner in HEK 293T cells. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Expression of Hoxa4 in various mouse tissues using qRT - PCR . Expression of glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) mRNA was used as an internal control . WAT ; white adipose tissue , BAT ; brown adipose tissue , Skm ; skeletal muscle .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Expression of Hoxa4 in various mouse tissues using qRT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control. WAT; white adipose tissue, BAT; brown adipose tissue, Skm; skeletal muscle.


Quantitative real - time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF - BB stimulation for 24 hours . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test .

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Quantitative real-time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF-BB stimulation for 24 hours. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Quantitative real - time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP . Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test .

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Quantitative real-time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP / TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 .

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Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP/TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.


Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 .

Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.


Quantitative real - time PCR analysis of YAP / TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP . The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test .

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Quantitative real-time PCR analysis of YAP/TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Proliferation assay in VSMCs transduced with the indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cells were collected and counted at each time point as indicated . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction .

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Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.


BrdU incorporation assays in VSMCs transduced with the indicated genes . The percentage of BrdU positive cells in each group is shown . Nine to twelve images were randomly acquired and analyzed . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction .

BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.


Proliferation assay in VSMCs transduced with the indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cells were collected and counted at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction .

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Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction.


BrdU incorporation assays in VSMCs transduced with the indicated genes . The percentage of BrdU positive cells in each group is shown . Nine to twelve images were randomly acquired and analyzed . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1 / 3 / 4 . Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction.


Proliferation assay in VSMCs transduced with indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cell viability was analyzed by Cell Counting Kit - 8 at indicated days at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co - IP experiments in human VSMCs with knockdown of HOXA4 . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


ChIP assays of CTGF , CYR61 and α - SMA promoters using anti - FLAG - tag , anti - Myc - tag , anti - TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc - tagged YAP - 5SA and / or FLAG - tagged HOXA4 . * P < 0 . 05 , * * P < 0 . 01 vs FLAG - GFP , by unpaired two - tailed Student ' s t - test . Data are expressed as the means ± SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ChIP assays of CTGF, CYR61 and α-SMA promoters using anti-FLAG-tag, anti-Myc-tag, anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P<0.05, **P<0.01 vs FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Quantitative real - time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8 - week - old WT and Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) .

Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).


Quantitative real - time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at the baseline was defined as 1 . 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.


Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation ( n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O

Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation (n=6).


Quantitative real - time PCR analysis of Yap / Tead target genes in the left carotid artery of 8 - week - old WT and Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) .

Quantitative real-time PCR analysis of Yap/Tead target genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).


Quantitative real - time PCR analysis of Yap / Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at the baseline was defined as 1 . 0 .

Quantitative real-time PCR analysis of Yap/Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.


Representative western blotting analysis and densitometry of non - ligated ( right ; R ) and ligated ( left ; L ) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation ( WT : n = 6 , Hoxa4 KO : n = 7 ) . * P < 0 . 05 , * * P < 0 . 01 , by two - way ANOVA with Sidak ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative western blotting analysis and densitometry of non-ligated (right; R) and ligated (left; L) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation (WT: n=6, Hoxa4 KO: n=7). *P<0.05, **P<0.01, by two-way ANOVA with Sidak's correction.


Representative images showing Elastica van Gieson staining ( G ) , and vessel , neointima and media area ( H ) , and neointima to media layer ratio ( I ) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation ( WT : n = 14 , Hoxa4 KO : n = 11 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images showing Elastica van Gieson staining (G), and vessel, neointima and media area (H), and neointima to media layer ratio (I) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation (WT: n=14, Hoxa4 KO: n=11).


Balb / C mice were aerosolically challenged with Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR strains . Each data point represents log10 CFU obtained from the infected lung of mice and e rror bar depicts the median with interquartile range for each group . Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Balb/C mice were aerosolically challenged with Rv, Rv∆aosR, and Rv∆aosR::aosR strains. Each data point represents log10 CFU obtained from the infected lung of mice and error bar depicts the median with interquartile range for each group. Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group.


Each data point represents Log10 CFU / lung of an infected animal and the error bar depicts the median with interquartile range for each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O

Each data point represents Log10 CFU/lung of an infected animal and the error bar depicts the median with interquartile range for each group.


Relative gene expression of selected DEGs measured by qRT - PCR in Rv compared with Rv treated with CHP ( g ) and Rv as compared with Rv ∆ aosR under oxidative conditions ( h ) . Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates .

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Relative gene expression of selected DEGs measured by qRT-PCR in Rv compared with Rv treated with CHP (g) and Rv as compared with Rv∆aosR under oxidative conditions (h). Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates.


WCLs of Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15 % SDS - PAGE , transferred to nitrocellulose membrane , and probed with indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

WCLs of Rv, Rv∆aosR, and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, and probed with indicated antibodies.


pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p . i ( mean ± SEM ; n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p.i (mean ± SEM; n=3).


The promoter of sigA and mec was fused to luciferase , and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured ( mean ± SD ; n = 6 )

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

The promoter of sigA and mec was fused to luciferase, and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=6)


Rv : : pN and Rv : : pN - F - sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross - linking . 1 mg WCL was used to immunoprecipitate FLAG - SigH and 1 / 10th IP was probed with α - FLAG and 9 / 10th IP with α - AosR . 50 µg WCLs were probed with α - FLAG , α - AosR , and α - SigA as controls .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Rv::pN and Rv::pN-F-sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-AosR. 50 µg WCLs were probed with α-FLAG,α-AosR, and α-SigA as controls.


Expression of CysO , CysM , AosR and GroEL1 ( control ) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h .

O O O 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-CONTROLLED_VAR O O O O

Expression of CysO, CysM, AosR and GroEL1 (control) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h.


5 - 7 . 5 x 104 end labelled 100 bp double - stranded oligonucleotide was incubated with no protein ( lanes 1 and 6 ) or 4 μΜ BSA ( lanes 2 and 7 ) or 2 μΜ His - AosR + 2 μΜ BSA ( lanes 3 and 8 ) or , 2 μΜ His - SigH + 2 μΜ BSA ( lanes 4 and 9 ) or 2 μΜ His - AosR + 2 μΜ His - SigH ( lanes 5 and 10 ) . The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols , respectively , and resolved on 8 % PAGE gel . Protein - DNA complexes ( bound ) and free probe are indicated by arrows . To confirm the specificity of DNA - protein interaction , EMSA reaction was performed in the presence of 5 , 10 , 25 , 50 , and 100 molar excess of unlabelled 100 bp double - stranded oligonucleotide .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.


Msm : : FLAG - SigH , Msm : : FLAG - SigH - AosR - HA and Msm : : FLAG - SigH - AosRAxxxA - HA were either subjected to none or 50 µM of CHP for 3 h followed by cross - linking . 1 mg WCL was used to immunoprecipitate FLAG - SigH and 1 / 10th IP was probed with α - FLAG and 9 / 10th IP with α - HA . 50 µg WCLs were probed with α - FLAG , α - HA and α - SigA as controls .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Msm::FLAG-SigH, Msm::FLAG-SigH-AosR-HA and Msm::FLAG-SigH-AosRAxxxA-HA were either subjected to none or 50 µM of CHP for 3 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-HA. 50 µg WCLs were probed with α-FLAG, α-HA and α-SigA as controls.


Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified . Data presented is representative of one of two biological replicates , each performed in triplicates ( n = 3 ) . Y - axis represents µM thiol / µg of WCL ( mean ± SD ) . Metabolite levels of reduced mycothiol , MSH ergothioneine , ERG were measured in Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR in normal and CHP stressed cells using LC - MRM MS / MS analysis . The absolute values obtained under normal conditions were normalized to 100 % for each strain and the relative values were calculated for samples processed from CHP treated cells . The data indicates relative levels of metabolites ( mean percent ± SD ) of four replicates ( n = 4 ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified. Data presented is representative of one of two biological replicates, each performed in triplicates (n=3). Y-axis represents µM thiol /µg of WCL (mean ± SD). Metabolite levels of reduced mycothiol, MSH ergothioneine, ERG were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4).


qRT - PCR measurements for the log2 fold induction ( mean ± SD , n = 3 ) for indicated genes in Rv ∆ aosR and Rv ∆ aosR treated with 1mM L - cysteine as compared with Rv . All strains were treated with 50 μM CHP . Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates .

O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

qRT-PCR measurements for the log2 fold induction (mean ±SD, n=3) for indicated genes in Rv∆aosR and Rv∆aosR treated with 1mM L-cysteine as compared with Rv. All strains were treated with 50 μM CHP. Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates.


( B ) Western blot analysis of lysates and media from wt and mutant N9 cells ( N9 wt / mu ) using antibody anti - murine TREM2 ( clone 5F4 ) , which is raised against the murine TREM2 extracellular domain . sTREM2 , soluble TREM2 * Indicate unspecific bands . Anti - calnexin antibody was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using antibody anti-murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2* Indicate unspecific bands. Anti-calnexin antibody was used as loading control.


( C ) Phagocytosis of 1 µM HiLyte ™ Fluor 488 Aβ1 - 42 ( fAβ42 ) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non - binding antibody 6687 . Cytochalasin D ( CytoD , 10 mM ) was used as control to verify phagocytic uptake . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 61 , genotype P < 0 . 0001 , treatment P = 0 . 0001 ; post hoc tests wt vs . mu for the following conditions : fAβ42 P = 0 . 0043 , fAβ42 - 2D8 P = 0 . 0436 ) .

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(C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436).


( D ) Western blot of BMDM derived from wt and Trem2 knockout ( ko ) animals using antibody 5F4 .

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(D) Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4.


( E ) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8 , or the non - binding control antibody 6687 . ( n = 3 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0005 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 P = 0 . 0021 , fAβ42 - 2D8 1 µg / ml P < 0 . 0001 , fAβ42 - 2D8 5 µg / ml P < 0 . 0001 , fAβ42 - 2D8 10 µg / ml P < 0 . 0001 , fAβ42 / 6687 10 µg / ml P = 0 . 0007 ) ( F ) Quantification of relative fAβ42uptake to lowest antibody concentration used .

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(E) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non-binding control antibody 6687. (n = 3, +/- SEM; 2 way ANOVA, interaction P=0.0005, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42 P=0.0021, fAβ42-2D8 1 µg/ml P<0.0001, fAβ42-2D8 5 µg/ml P<0.0001, fAβ42-2D8 10 µg/ml P<0.0001, fAβ42/6687 10 µg/ml P=0.0007)(F) Quantification of relative fAβ42uptake to lowest antibody concentration used.


( G ) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0223 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 - mAb11 1 µg / ml P = 0 . 0391 , fAβ42 - mAb11 5 µg / ml P = 0 . 0069 , fAβ42 - mAb11 10 µg / ml P < 0 . 0001 , fAβ42 - mAb11 20 µg / ml P = 0 . 0001 , fAβ42 - mAb11 50 µg / ml P < 0 . 0001 ) . ( H ) Quantification of relative fAβ42 uptake to lowest antibody concentration used .

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(G) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0223, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 1 µg/ml P=0.0391, fAβ42-mAb11 5 µg/ml P=0.0069, fAβ42-mAb11 10 µg/ml P<0.0001, fAβ42-mAb11 20 µg/ml P=0.0001, fAβ42-mAb11 50 µg/ml P<0.0001).(H) Quantification of relative fAβ42 uptake to lowest antibody concentration used.


( I ) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2 - deficient BMDM . Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 ( 10 µg / ml ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2-deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 µg/ml).


( K ) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 5 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 4797 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 - mAb11 5 µg / ml P = 0 . 0449 , fAβ42 - mAb11 10 µg / ml P = 0 . 0370 , fAβ42 - mAb11 20 µg / ml P = 0 . 0299 , fAβ42 - mAb11 50 µg / ml P = 0 . 0120 ) . ( L ) Quantification of relative fAβ42uptake to lowest antibody concentration used .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.4797, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 5 µg/ml P=0.0449, fAβ42-mAb11 10 µg/ml P=0.0370, fAβ42-mAb11 20 µg/ml P=0.0299, fAβ42-mAb11 50 µg/ml P=0.0120).(L) Quantification of relative fAβ42uptake to lowest antibody concentration used.


( A ) Representative histograms for Fcγ - receptors - PE ( FcγR - PE ) expression levels as used for quantification . Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR ( I , IIB / III and IV ) . ( B ) Relative quantification of cell surface levels of FcγR molecules . Absolute number of cell surface FcγR - PE molecules was determined by the BD QuantiBRITE ® method ( see methods section for details ) and normalized to expression levels of the respective wt control . ( n = 4 , + / - SEM , t test , two - tailed ; wt vs . ko : FcγRI - PE P = 0 . 0008 , FcγRIIB / III - PE P = 0 . 0033 , FcγRIV - PE P = 0 . 7001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative histograms for Fcγ-receptors-PE (FcγR-PE) expression levels as used for quantification. Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR (I, IIB/III and IV).(B) Relative quantification of cell surface levels of FcγR molecules. Absolute number of cell surface FcγR-PE molecules was determined by the BD QuantiBRITE® method (see methods section for details) and normalized to expression levels of the respective wt control. (n=4, +/- SEM, t test, two-tailed; wt vs. ko: FcγRI-PE P=0.0008, FcγRIIB/III-PE P=0.0033, FcγRIV-PE P=0.7001).


( C ) mRNA levels of FcγR are increased in Trem2 ko BMDM . Fold changes of the respective FcγR ( I , IIB , III , IV ) mRNA levels in Trem2 ko BMDM were determined by quantitative real - time PCR . ( n = 6 , + / - SEM ; one - sample t test , two - tailed ; wt vs . ko : FcγRI P = 0 . 0118 , FcγRIIB P = 0 . 0006 , FcγRIII P = 0 . 0004 , FcγRIV P = 0 . 0284 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) mRNA levels of FcγR are increased in Trem2 ko BMDM. Fold changes of the respective FcγR (I, IIB, III, IV) mRNA levels in Trem2 ko BMDM were determined by quantitative real-time PCR. (n=6, +/- SEM; one-sample t test, two-tailed; wt vs. ko: FcγRI P=0.0118, FcγRIIB P=0.0006, FcγRIII P=0.0004, FcγRIV P=0.0284)


( D ) Phosphorylated Syk ( P - Syk ) and total Syk ( T - Syk ) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone , together with antibody 2D8 ( Aβ - 2D8 ) , or an isotype control ( Aβ - IC ) . Actin was used as a loading control . ( E ) Quantification of P - Syk normalized to T - Syk . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0490 , genotype P = 0 . 0898 , treatment P < 0 . 0001 . Bonferroni - corrected pair - wise post hoc tests , * * P = 0 . 0075 vs . wt ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Phosphorylated Syk (P-Syk) and total Syk (T-Syk) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone, together with antibody 2D8 (Aβ-2D8), or an isotype control (Aβ-IC). Actin was used as a loading control.(E) Quantification of P-Syk normalized to T-Syk. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0490, genotype P=0.0898, treatment P<0.0001. Bonferroni-corrected pair-wise post hoc tests, ** P =0.0075 vs. wt).


( B ) mAb11 but not the IC co - localized with methoxy - X04 .

(B) mAb11 but not the IC co-localized with methoxy-X04.


BMDM ( C ) or primary microglia ( D ) were cultured on cryosections pre - incubated with mAb11 ( 1 µg / ml ) . After 24 h , sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy - X04 staining to visualize Aβ . Note that both cell types internalize Aβ into intracellular vesicles ( right panels show enlargement of insets ) . Scale bar : 10µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Aβ was extracted by urea buffer from replicate slices of the experiment shown in (A) and total Aβ was identified by western blotting.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


B . 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 .

O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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.


A . PPP1R13L mRNA expression level is lower in VI10 PDFs ( set as 1 ) than in CFs .

A. 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 .

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. 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 .

D. WB analysis demonstrated that PPP1R13L-shRNA downregulated the putative iASPP band.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 .

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.


A . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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. 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 O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


A . WB analysis confirmed the reduced expression of iASPP in ppp1r13l knocked down cardiomyocytes .

A. 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 O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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.


A 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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 .

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 .

C. Hierarchical clustering heat map of differentially expressed genes in the hearts of 7- and 12- week old wa3 mice in comparison to WT.


A . 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 .

O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O

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).


Telomeric 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Telomeric 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 .

ARRET and αARRETNorthern blot analysis of the indicated strains using strand‐specific probes.


Telomere length analysis of ApaI ‐ digested DNA from the indicated strains .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Chromosome 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chromosome 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 .

O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Western blot analysis of endogenous Rap1 and Taz1 in the indicated strains . Act1 was used as a loading control . Asterisks indicate cross ‐ reacting bands .

Western 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Telomere length analysis of ApaI ‐ digested DNA from cay1 ∆ trt1 ∆ cells harvested at increasing generation doublings ( gen ) .

O O O O O O O O O O O O O O O O O O O O O O O

Telomere 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 .

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 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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.


Serial 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Serial 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


RT - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O

Representative YFPfluorescence microscopy and DIC images of cay1+ and cay1∆ strains expressing Rap1‐YFP. Scale bar, 5 μm.


Telomere length analysis of ApaI ‐ digested DNA from cells harvested at early ( left ) and late generations ( right ) after expression of YFP ‐ tagged proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Telomere 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.