A ) Identification of Cdc14 phospho - targets during damage response by mass spectrometry . Wild - type cdc14 - 1 were grown overnight blocked in G2 / M by using nocodazole to avoid cycle - dependent changes in phosphorylation between both strains . After block was attained , were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h . Differential phosphorylation of phospho - peptides detected between wild - type cdc14 - 1 were grouped into broad categories depending on molecular function of . table includes damage checkpoint - related with Cdc14 - dependent hyper - phosphorylated status relative between wild - type cdc14 - 1 during damage response . Red blue indicate relative amount of residue phosphorylation between both strains ( Red , high ; Blue , low ) .

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

A) Identification of Cdc14 phospho-targets during the DNA damage response by mass spectrometry analysis. Wild-type and cdc14-1 cells were grown overnight and blocked in G2/M by using nocodazole to avoid cell cycle-dependent changes in protein phosphorylation between both strains. After the block was attained, cells were transferred to 37ºC for 45 minutes to deplete Cdc14 activity prior HO induction by galactose addition for 4h. Differential phosphorylation of phospho-peptides detected between the wild-type and cdc14-1 were grouped into broad categories depending on the molecular function of the proteins. The table includes the DNA damage and checkpoint-related proteins with Cdc14-dependent hyper-phosphorylated status and the relative ratio between the wild-type and cdc14-1 during the DNA damage response. Red and blue indicate relative amount of the residue phosphorylation between both strains (Red, high; Blue, low).


B ) Cdc14 dephosphorylates Spc110 in response to a DSB . Spc110 was tagged with 6HA epitope in wild - type , cdc14 - 1 spc110S36 - 91A backgrounds . were grown overnight in raffinose - containing media supplemented with galactose to induce HO expression . Samples were collected at each time - point indicated . were TCA extracted , separated on Phos - Tag containing gels blotted . Coomassie is shown as loading control .

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

B) Cdc14 dephosphorylates Spc110 in response to a DSB. Spc110 was tagged with the 6HA epitope in wild-type, cdc14-1 and spc110S36-91A backgrounds. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as loading control.


C ) Lambda - phosphatase treatment of extracts in absence of damage reduces Spc110 phosphorylation to same extent as samples taken during damage response . were grown overnight in raffinose containing media ( 1 ) incubated with galactose for 2 . 5 hours to induce HO expression ( 2 ) . Extracts from raffinose - containing cultures were treated with mock ( 3 ) or λ - PPase ( 4 ) . samples were separated in a Phos - Tag containing gel blotted . Coomassie is shown as loading control .

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 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 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

C) Lambda-phosphatase treatment of protein extracts in the absence of DNA damage reduces Spc110 phosphorylation levels to the same extent as samples taken during the DNA damage response. Cells were grown overnight in raffinose containing media (line 1) and incubated with galactose for 2.5 hours to induce HO expression (line 2). Extracts from raffinose-containing cultures were treated with mock (line 3) or λ-PPase (line 4). All samples were separated in a Phos-Tag containing gel and blotted. Coomassie staining is shown as loading control.


D ) canonical FEAR pathway is required for Spc110 dephosphorylation during damage response . Spc110 was labelled with 3HA tag in both wild - type esp1 - 1 background strains . were grown overnight in raffinose - containing media supplemented with galactose to induce HO expression . Samples were collected at each time - point indicated . were TCA extracted , separated on Phos - Tag containing gels blotted . Coomassie is shown as a control for loading .

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

D) The canonical FEAR pathway is required for Spc110 dephosphorylation during the DNA damage response. Spc110 was labelled with the 3HA tag in both wild-type and esp1-1 background strains. Cells were grown overnight in raffinose-containing media and supplemented with galactose to induce the HO expression. Samples were collected at each time-point indicated. Proteins were TCA extracted, separated on Phos-Tag containing gels and blotted. Coomassie staining is shown as a control for loading.


A ) Time - lapse experiments of living harbouring a non - reparable DSB were carried out after expressing HO endonuclease in wild - type cdc14 - 1 mutant . Spc110 - RFP was used as SPB reporter Tub1 - GFP was used as tubulin marker . Three z - planes every 5 - second intervals over a period of 5 minutes were captured used to quantify average SPB track velocity using Spc110 - RFP as SPB marker . At least 100 were scored . A representative maximum projection image is depicted . Scale bar : 3μm .

O 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 B-MEASURED_VAR O O 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 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

A) Time-lapse experiments of living cells harbouring a non-reparable DSB were carried out after expressing the HO endonuclease in wild-type and cdc14-1 mutant cells. Spc110-RFP was used as SPB reporter and Tub1-GFP was used as tubulin marker. Three z-planes images every 5-second intervals over a period of 5 minutes were captured and used to quantify the average SPB track length and velocity using Spc110-RFP as SPB marker. At least 100 cells were scored.A representative maximum projection image is depicted. Scale bar: 3μm.


A ) A Western blot is shown to confirm equal amount of Tub1 - GFP in both wild - type cdc14 - 1 backgrounds . Coomassie blue is shown as loading control .

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

A) A Western blot is shown to confirm the equal amount of Tub1-GFP in both wild-type and cdc14-1 backgrounds. Coomassie blue staining is shown as loading control.


B ) steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to damage . Wild - type , cdc14 - 1 spc110S36 - 91A expressing Spc110 - RFP were scored for their ability to maintain spindle polarity at axial plane of upon induction of a single DSB induced by HO endonuclease . A maximum projection of nine z - planes was used to score . At least 100 from three independent experiments were scored . Graph represents ± SD of with oriented / miss - oriented SPBs with respect to axial plane of . P value was calculated using a 2 - unpaired Student t .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The steady state phosphorylation of Spc110 is required to maintain spindle polarity in response to DNA damage. Wild-type, cdc14-1 and spc110S36-91A cells expressing Spc110-RFP were scored for their ability to maintain spindle polarity at the axial plane of the cell upon induction of a single DSB induced by the HO endonuclease. A maximum projection of nine z-planes was used to score the cells. At least 100 cells from three independent experiments were scored. Graph represents the percentage ± SD of cells with oriented/miss-oriented SPBs with respect to the axial plane of the cell. P value was calculated using a 2-tailed unpaired Student t test.


C ) asymmetric loading of dynein into astral MTs emanating from daughter SPB is affected in cdc14 - 1 spc110S36 - 91A during damage response . Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1 - GFP in strains containing Spc110 - RFP . Graph represents ± SD of grouped into three categories depending on pattern of dynein distribution on astral MTs : Type1 , Dynein is exclusively loaded on astral MTs emerging from daughter SPB ; Type 2 , Dynein is equally loaded on astral MTs emanating from both SPBs ; Type 3 , Dynein is absent from astral MTs . At least 100 from three independent experiments were scored . A representative picture showing three patterns is included . Scale bar : 3μm .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The asymmetric loading of dynein into astral MTs emanating from the daughter SPB is affected in cdc14-1 and spc110S36-91A cells during the DNA damage response. Dynein distribution on astral MTs emerging from each SPB was scored by using Dyn1-GFP in strains containing Spc110-RFP. Graph represents the percentage ± SD of cells grouped into three categories depending on the pattern of dynein distribution on astral MTs: Type1, Dynein is exclusively loaded on astral MTs emerging from the daughter SPB; Type 2, Dynein is equally loaded on astral MTs emanating from both SPBs; Type 3, Dynein is absent from astral MTs. At least 100 cells from three independent experiments were scored. A representative picture showing the three patterns is included. Scale bar: 3μm.


A ) treated with nocodazole are affected in DSB - SPB interaction . Cnm67 - CFP Ddc2 - RFP were used as SPB DSB markers respectively . Diagram depicts genomic background used . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . D1 , D2 D3 distances after inducing a DSB in presence of nocodazole or mock dimethyl sulfoxide ( DMSO ) were scored plotted . Graph represents mean ± SD of D1 , D2 D3 distances of three independent experiments . At least 100 per experiment were scored using maximum projection of three z - planes . P values were calculated using a 2 - unpaired Student t . A representative picture is shown . Scale bar : 3μm .

O O O O O O O O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells treated with nocodazole are affected in DSB-SPB interaction. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. Diagram depicts the genomic background used. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. D1, D2 and D3 distances after inducing a DSB in the presence of nocodazole or mock dimethyl sulfoxide (DMSO) were scored and plotted. Graph represents the mean ± SD of D1, D2 and D3 distances of three independent experiments. At least 100 cells per experiment were scored using the maximum projection of three z-planes. P values were calculated using a 2-tailed unpaired Student t test. A representative picture is shown. Scale bar: 3μm.


B ) Physical of wild - type containing repair pathway choice described in Fig 2B . diagram with genomic information , restriction enzymes used location of probe are shown . were synchronized in G1 by using α - factor pheromone released into fresh media for 1 hour . Induction of HO expression was attained by adding galactose for 2 hours . After formation of DSB , glucose was added to repress HO in presence of nocodazole or mock DMSO samples were taken to analyse kinetics of repair . was extracted , digested with EcoRV , separated on agarose gels blotted . Both probes for MATa - distal HIS3 gene ( as loading control ) were used . Graphs show of MATa restoration ( NHEJ ) , mating type switching by gene conversion ( HR ) , DSB kinetics formation . data were normalized with HIS3 gene . Graphs show mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR 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 O O O O O O O O O O O O O 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) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. DNA was extracted, digested with EcoRV, separated on agarose gels and blotted. Both probes for the MATa-distal sequence and the HIS3 gene (as loading control) were used. Graphs show the quantification of MATa restoration (NHEJ), mating type switching by gene conversion (HR), and DSB kinetics formation. All data were normalized with the HIS3 gene. Graphs show the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test.


B ) Physical of wild - type containing repair pathway choice described in Fig 2B . diagram with genomic information , restriction enzymes used location of probe are shown . were synchronized in G1 by using α - factor pheromone released into fresh media for 1 hour . Induction of HO expression was attained by adding galactose for 2 hours . After formation of DSB , glucose was added to repress HO in presence of nocodazole or mock DMSO samples were taken to analyse kinetics of repair . FACS analyses are shown .

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

B) Physical analysis of wild-type cells containing the repair pathway choice assay described in Fig 2B. The diagram with the genomic information, the restriction enzymes used and the location of the probe are shown. Cells were synchronized in G1 by using the α-factor pheromone and released into fresh media for 1 hour. Induction of HO expression was attained by adding galactose for 2 hours. After formation of the DSB, glucose was added to repress the HO in the presence of nocodazole or mock DMSO and samples were taken to analyse the kinetics of the repair. FACS analyses are shown.


A ) Growth sensitivity to MMS phleomycin of a Cdk phospho - deficient mutant of Spc110 . Ten - fold serial dilutions from overnight cultures of wild - type , spc110S36 - 91A a cdc14 - 1 grown on solid rich media containing mock DMSO , MMS or Phleomycin at 30ºC .

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

A) Growth sensitivity to MMS and phleomycin of a Cdk phospho-deficient mutant of Spc110. Ten-fold serial dilutions from overnight cultures of wild-type, spc110S36-91A and a cdc14-1 cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.


B ) Diagram showing genomic background approximation used to measure DSB - SPB interaction . Spc110 spc110S36 - 91A were labelled with RFP in a background containing Ddc2 - GFP as DSB reporter . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . At least 100 from three independent experiments were scored using maximum projection of nine z - planes . Graph shows mean ± S . D . P values were calculated using a 2 - unpaired Student t .

O O O O O O O O O O O O O O O B-MEASURED_VAR B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Spc110 and spc110S36-91A were labelled with the RFP in a background containing Ddc2-GFP as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.


C ) Time - lapse experiments of living expressing HO endonuclease in wild - type spc110S36 - 91A mutants . Five z - planes every 5 - second intervals over a period of 5 minutes were captured . Quantifications were made using average SPB track velocity of at least 100 . Spc110 spc110S36 - 91A were labelled with RFP used as SPB reporter .

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

C) Time-lapse experiments of living cells expressing the HO endonuclease in wild-type and spc110S36-91A mutants cells. Five z-planes every 5-second intervals over a period of 5 minutes were captured. Quantifications were made using the average SPB track length and velocity of at least 100 cells. Spc110 and spc110S36-91A were labelled with the RFP and used as SPB reporter.


D ) Southern blot of wild - type spc110S36 - 91A mutant harbouring inter - chromosomal gene conversion portrayed . An overnight culture was induced by adding galactose at 32ºC to express HO endonuclease . Samples were collected at different time - points , genomic was extracted , digested with EcoRI analysed by Southern blot . Blots were probed with a MATa - only actin ( for loading control ) sequences . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

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 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 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 O O O O O O O O O O O O O O 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) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


D ) Southern blot of wild - type spc110S36 - 91A mutant harbouring inter - chromosomal gene conversion portrayed . An overnight culture was induced by adding galactose at 32ºC to express HO endonuclease . Samples were collected at different time - points , genomic was extracted , digested with EcoRI analysed by Southern blot . Blots were probed with a MATa - only actin ( for loading control ) sequences . FACS profiles for content are included . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

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 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

D) Southern blot of wild-type and spc110S36-91A mutant harbouring the inter-chromosomal gene conversion assay portrayed. An overnight culture was induced by adding galactose at 32ºC to express the HO endonuclease. Samples were collected at different time-points, genomic DNA was extracted, digested with EcoRI and analysed by Southern blot. Blots were probed with a MATa-only and actin (for loading control) DNA sequences. FACS profiles for DNA content are included. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


A ) damage sensitivity to MMS phleomycin of a Cdk phospho - mimic mutant of Spc110 . Ten - fold serial dilutions from mid - log phase cultures of wild - type , spc110S36 - 91A spc110S36 - 91D grown on solid rich media containing mock DMSO , MMS or Phleomycin at 30ºC .

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

A) DNA damage sensitivity to MMS and phleomycin of a Cdk phospho-mimic mutant of Spc110. Ten-fold serial dilutions from mid-log phase cultures of wild-type, spc110S36-91A and spc110S36-91D cells grown on solid rich media containing mock DMSO, MMS or Phleomycin at 30ºC.


B ) Schematic representation of genomic background approximation used to measure DSB - SPB tethering . Wild - type Spc110 spc110S36 - 91D were labelled with RFP . Ddc2 was tagged with GFP to be used as DSB reporter . D1 : inter - SPB ; D2 : DSB to proximal SPB ; D3 : DSB to distal SPB . At least 100 from three independent experiments were scored using maximum projection of nine z - planes . Graph shows mean ± S . D . P values were calculated using a 2 - unpaired Student t .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O O O 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) Schematic representation of the genomic background and the approximation used to measure DSB-SPB tethering. Wild-type Spc110 and spc110S36-91D were labelled with the RFP. Ddc2 was tagged with the GFP to be used as DSB reporter. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. At least 100 cells from three independent experiments were scored using the maximum projection of nine z-planes. Graph shows the mean ± S.D. P values were calculated using a 2-tailed unpaired Student t test.


C ) Time - lapse experiments of living after induction of HO endonuclease in wild - type spc110S36 - 91D . Three z - planes every 10 - second intervals over a period of 5 minutes were captured to measure average SPBs track velocity . Spc110 spc110S36 - 91D were labelled with RFP used to measured SPBs kinetics . At least 100 from three independent experiments were scored .

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 B-MEASURED_VAR 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

C) Time-lapse experiments of living cells after induction of the HO endonuclease in wild-type and spc110S36-91D cells. Three z-planes every 10-second intervals over a period of 5 minutes were captured to measure the average SPBs track length and velocity. Spc110 and spc110S36-91D were labelled with the RFP and used to measured SPBs kinetics. At least 100 cells from three independent experiments were scored.


D ) Southern blot of wild - type spc110S36 - 91D mutant bearing inter - chromosomal gene conversion depicted . Mid - log phase were HO - induced at 32ºC by adding galactose to media . Samples were collected at different time - points genomic was extracted . Preps were digested with EcoRI analysed by Southern blot . Blots were hybridized with MATa - only actin probes . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

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 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-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

D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


D ) Southern blot of wild - type spc110S36 - 91D mutant bearing inter - chromosomal gene conversion depicted . Mid - log phase were HO - induced at 32ºC by adding galactose to media . Samples were collected at different time - points genomic was extracted . Preps were digested with EcoRI analysed by Southern blot . Blots were hybridized with MATa - only actin probes . FACS profiles for content are displayed . Graphs show of gene conversion , DSB induction crossover vs non - crossover . data were normalized using actin signal . Graphs represent mean ± S . D . from three independent experiments . P values were calculated using a 2 - unpaired Student t . Asterisk denotes an overexposed film to visualize formation of crossover products .

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 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 O O

D) Southern blot of wild-type and spc110S36-91D mutant bearing the inter-chromosomal gene conversion assay depicted. Mid-log phase cells were HO-induced at 32ºC by adding galactose to the media. Samples were collected at different time-points and the genomic DNA was extracted. Preps were digested with EcoRI and analysed by Southern blot. Blots were hybridized with MATa-only and actin probes. FACS profiles for DNA content are displayed. Graphs show quantification of gene conversion, DSB induction and crossover vs non-crossover ratio. All data were normalized using the actin signal. Graphs represent the mean ± S.D. from three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. Asterisk denotes an overexposed film to visualize the formation of crossover products.


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) RNAP2 ( data from ( Larochelle et al , 2018 ) ) across ( A ) a 25 kb region of chromosome 3 containing several tRNA 5S rRNA

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

Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genes


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) RNAP2 ( data from ( Larochelle et al , 2018 ) ) across U6 snRNA

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

Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the U6 snRNA snu6


Snapshots of ChIP - seq signals of Sen1 ( this study ) , RNAP3 ( this study ) RNAP2 ( data from ( Larochelle et al , 2018 ) ) across srp7 loci .

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

Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across the srp7 loci.


Genome - wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp .

Genome-wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp.


ChIP - qPCR of Flag - tagged Sen1 at indicated loci in a population of cycling ( mean ± standard deviation from 4 biological replicates ) .

O O 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

ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates).


ChIP - qPCR of Flag - tagged RNAP3 subunit Rpc37 Flag - tagged Sen1 across SPCTRNAARG . 10 tRNA locus , whose TATA box was either mutated ( TATA - ) or not ( TATA + ) . mutations introduced to disrupt putative TATA boxes are indicated in red above graphs . enrichment values were normalized to SPCTRNATHR . 10 ( mean ± standard deviation from 4 biological replicates ; p - value obtained using Wilcoxon - Mann Whitney statistical ) .

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

ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA-) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


( left ) ChIP - qPCR of Rpc37 in presence or absence of Sen1 at indicated loci in a population of cycling ( mean ± standard deviation from 4 biological replicates ) . ( right ) Western blot of Rpc37 in presence or absence of Sen1 . Tubulin was used as a loading control .

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

(left) ChIP-qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates). (right) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1. Tubulin was used as a loading control.


( left ) Total from indicated strains were separated on a 2 , 8 % agarose gel . ( right ) of overall tRNA ( mean ± standard deviation from 3 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 O O O

(left) Total RNA from the indicated strains were separated on a 2,8% agarose gel. (right) Quantification of overall tRNA levels (mean ± standard deviation from 3 biological replicates).


Strand - specific RT - qPCR was used to quantify indicated RNAP3 transcripts . Transcript were normalized to act1 ( mean ± standard deviation from 3 biological replicates ) .

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 O O O O

Strand-specific RT-qPCR was used to quantify the indicated RNAP3 transcripts. Transcript levels were normalized to act1 (mean ± standard deviation from 3 biological replicates).


were grown in minimal medium during 18 hours to induce strong expression of RnhA . R - ChIP using a catalytically - inactive RNase H1 ( Rnh1 - D129N ) was used to quantify R - loop formation at RNAP3 - transcribed genes ( mean ± standard deviation from 4 biological replicates ) .

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 B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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

Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA. R-ChIP using a catalytically-inactive RNase H1 (Rnh1-D129N) was used to quantify R-loop formation at RNAP3-transcribed genes (mean ± standard deviation from 4 biological replicates).


( left ) ChIP - qPCR of 13myc - tagged RNAP3 subunit Rpc25 in indicated strains at indicated loci ( mean ± standard deviation of 4 biological replicates ) . ( right ) Western blot of Myc - tagged Rpc25 Flag - tagged RnhA in indicated strains . Tubulin was used as a loading control .

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

(left) ChIP-qPCR of the 13myc-tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci (mean ± standard deviation of 4 biological replicates). (right) Western blot analysis of Myc-tagged Rpc25 and Flag-tagged RnhA protein levels in the indicated strains. Tubulin was used as a loading control.


Snapshots of ChIP - seq signals of RNAP3 subunits Rpc1 Rpc2 in presence or absence of Sen1 across a representative ( A ) tRNA gene , ( B ) 5S rRNA gene ( C ) srp7 . Boxed regions highlight increased density of reads in downstream region of genes in absence of Sen1 .

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

Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1.


Average ChIP - seq profile of Rpc1 Rpc2 across isolated tRNA 5S rRNA genes in presence absence of Sen1 .

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

Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1.


Scanning of Rpc37 - 3flag occupancy at three different tDNA loci in absence of Sen1 by ChIP - qPCR ( mean ± standard deviation from 6 biological replicates ) .

O O B-MEASURED_VAR 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

Scanning of Rpc37-3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP-qPCR (mean ± standard deviation from 6 biological replicates).


of indicated genotypes that carried or not DRT5T dimeric tRNA construct ( schematized on top ) were grown either in presence of optimum of adenine ( left ) or in presence of a limiting of adenine ( right ) . Two independent clones of same genotype ( # 1 # 2 ) were used . See text for details . 3F refers to 3 Flag epitope tag at C - terminus of Rpc37 .

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

Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.


Strand - specific RT - qPCR was used to quantify of read - through transcripts ( see Methods ) . mean ± standard deviation from 4 biological replicates is represented here . P - values were obtained using Wilcoxon - Mann Whitney statistical .

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

Strand-specific RT-qPCR was used to quantify the levels of read-through transcripts (see Methods). The mean ± standard deviation from 4 biological replicates is represented here. P-values were obtained using the Wilcoxon-Mann Whitney statistical test.


Northern blot of tRNA SPATRNAPRO . 02 using an intron - specific probe ( TCTAAACTCAGCATACAAGTGGGG ) . U5 snRNA was used as a loading control .

O O O O B-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

Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.


ChIP - qPCR of Rpc37 around SPCTRNAARG . 10 gene in strong terminator mutant ( mean ± standard deviation from 4 biological replicates ) .

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

ChIP-qPCR analysis of Rpc37 around SPCTRNAARG.10 gene in the strong terminator mutant (mean ± standard deviation from 4 biological replicates).


Strand specific RT - qPCR was used to quantify read - through transcripts at SPCTRNAARG . 10 ( arg10 ) SPCTRNASER . 09 ( ser09 ) in strong terminator mutant ( mean ± standard deviation from 4 biological replicates ; p - value obtained using Wilcoxon - Mann Whitney statistical ) .

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

Strand specific RT-qPCR was used to quantify read-through transcripts at SPCTRNAARG.10 (arg10) and SPCTRNASER.09 (ser09) in the strong terminator mutant (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).


C . MA plot highlighting genes differentially expressed between state independent of ( glm state term , false discovery rate - adjusted P value ( FDR ) < 0 . 05 log2FC > 1 ) . pluripotent TFs highlighted for each state ( maroon = naive ESCs , teal = formative EpiLCs ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MA plot highlighting genes differentially expressed between state independent of strain (glm state term, false discovery rate-adjusted P value (FDR) < 0.05 and log2FC >1). Core pluripotent TFs highlighted for each state (maroon = naive ESCs, teal = formative EpiLCs).


H . of motif deviations from 3 biological replicates of B6 D2 ESCs for TFs highlighted in G .

H. Heatmap of motif deviations from 3 biological replicates of B6 and D2 ESCs for TFs highlighted in G.


A - D . Example expression patterns for select genes identified by applying a general linear model ( glm ) including state , genotype , interaction terms . Dots represent biological replicates ( N = 3 ) ; lines highlight changes in mean values for replicates between each state ( log2FC > 1 FDR < 0 . 05 ) . A . State dependent expression is exemplified by Nanog which showed no difference between strains . B . Expression of Zfp277 exemplifies - dependence being consistently higher in B6 . C & D . A significant genotype x state ( GXS ) interaction was identified for Nr5a2 ( C , a marker for pluripotency ) Sox1 ( D , a marker for neuronal differentiation ) .

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

A-D. Example expression patterns for select genes identified by applying a general linear model (glm) including state, genotype, and interaction terms. Dots represent individual biological replicates (N = 3); lines highlight changes in mean values for replicates between each state (log2FC >1 and FDR < 0.05). A. State dependent expression is exemplified by Nanog which showed no difference between strains. B. Expression of Zfp277 exemplifies strain-dependence being consistently higher in B6. C & D. A significant genotype x state (GXS) interaction was identified for Nr5a2 (C, a marker for pluripotency) and Sox1 (D, a marker for neuronal differentiation).


D . Feature plot of expression gradients for indicated gene overlaid on UMAP from a . Three lineage markers that help to distinguish identity are shown for five clusters .

O O O O O O 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. Feature plot of expression gradients for indicated gene overlaid on UMAP from a. Three lineage markers that help to distinguish cellular identity are shown for five cell clusters.


E . Pseudocolor plots of FACS on from EBs for both B6 D2 strains . were labeled with anti - EpCAM ( top ) or biological replicates with anti - SOX1 antibody ( bottom ) . Gated population indicates percentages of EpCAM + ( Q3 ) or SOX1 + .

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

E. Pseudocolor plots of FACS analysis on cells from EBs for both B6 and D2 strains. Cells were labeled with anti-EpCAM (top) or biological replicates with anti-SOX1 antibody (bottom). Gated population indicates percentages of EpCAM+ (Q3) or SOX1+ cells.


B . Scatterplot of genomic locations of accessibility ( ca ) QTL ( x - axis , LOD > 5 ) versus location of ATAC peak being regulated ( y - axis ) for ESCs . Genetic effects that act locally ( n = 3 , 973 ) , i . e . in cis , fall on diagonal , while distal acting QTL ( n = 13 , 767 ) lie off diagonal . Several prominent distal QTL hotspots appear as vertical lines . C . Similar to B , plotting expression ( e ) QTL position versus gene location ( LOD > 5 , n = 701 local - eQTL , n = 357 distal - eQTL ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatterplot of genomic locations of individual chromatin accessibility (ca)QTL (x-axis, LOD > 5) versus location of ATAC peak being regulated (y-axis) for ESCs. Genetic effects that act locally (n = 3,973), i.e. in cis, fall on the diagonal line, while distal acting QTL (n = 13,767) lie off the diagonal. Several prominent distal QTL hotspots appear as vertical lines. C. Similar to B, plotting expression (e)QTL position versus gene location (LOD > 5, n = 701 local-eQTL, n = 357 distal-eQTL). D


G . Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3 , ATAC , read depth from merged replicates . Locations of Chr 7 trans caQTL targets are indicated with black bars LOD scores listed below .

O O O O O B-MEASURED_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

G. Coverage profile for Gstp2 locus on Chr 19 showing H3K4me3, ATAC, and RNA read depth from merged replicates. Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below.


F . Left - Coverage profiles ( averaged biological replicates , N = 3 ) for ChIP factor occupancy at Cd59a on Chr 2 under trans - regulation by Chr 13d QTL . Distal ca - QTL target region used for is indicated by a black box with LOD score below . Right - Scatterplots for ChIP factors comparing quantitative level of modification / binding for independent replicates between B6 D2 ESCs .

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

F. Left - Coverage profiles (averaged biological replicates, N=3) for ChIP factor occupancy at Cd59a on Chr 2 under trans-regulation by the Chr 13d QTL. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below. Right - Scatterplots for ChIP factors comparing quantitative level of modification/binding for independent replicates between B6 and D2 ESCs.


G . difference in occupancy of P300 in parental strains is correlated to difference in accessibility in 33 BXDs on genotype of BXD at QTL for Chr 13 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 61 ) . H . TRIM28 occupancy at same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal ( Pearson ' s r = - 0 . 64 ) .

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

G. The difference in occupancy of P300 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 13 caQTL targets (LOD > 7, Pearson's r = -0.61). H. TRIM28 occupancy at the same targets of Chr 13d caQTL in F were negatively correlated with ATAC signal (Pearson's r = -0.64). I


I . Similar to F , coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932 , targeted by Chr 12 trans caQTL , showed greater occupancy of POU5F1 increased open compared to D2 . Distal ca - QTL target region used for is indicated by a black box with LOD score below .

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 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

I. Similar to F, coverage profiles for ChIP factor occupancy for a putative regulatory element on Chr 5 near Zfp932, targeted by Chr 12 trans caQTL, showed greater occupancy of POU5F1 and increased open chromatin compared to D2. Distal ca-QTL target region used for quantification is indicated by a black box with LOD score below.


J . difference in occupancy of POU5F1 in parental strains is correlated to difference in accessibility in 33 BXDs on genotype of BXD at QTL for Chr 12 caQTL targets ( LOD > 7 , Pearson ' s r = - 0 . 66 ) . K . Similar to J showing negative correlation between TRIM28 occupancy accessibility ( Pearson ' s r = - 0 . 51 ) .

O 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 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

J. The difference in occupancy of POU5F1 in the parental strains is correlated to difference in chromatin accessibility in the 33 BXDs based on the genotype of the BXD at the QTL for Chr 12 caQTL targets (LOD > 7, Pearson's r = -0.66). K. Similar to J showing negative correlation between TRIM28 occupancy and chromatin accessibility (Pearson's r = -0.51). D


A . Coverage profile for H3K4me3 , ATAC , P300 , TRIM28 ChIP at a target locus for QTL on Chr 4 for both accessibility ( black boxes ) gene expression ( Riok3 Rmc1 ) . LOD scores are listed under each target locus .

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

A. Coverage profile for H3K4me3, ATAC, P300, and TRIM28 ChIP at a target locus for the QTL on Chr 4 for both chromatin accessibility (black boxes) and gene expression (Riok3 and Rmc1). LOD scores are listed under each target locus.


E . Box whisker plot indicating change in H3K9me3 between WT Chr 4 KO mESCs grouped by whether accessibility is higher when QTL is B6 ( left ) or D2 ( right ) . Dots represent mean values from biological duplicate experiments lines connect means . Orange indicates loci for which H3K9me3 signal decreases in KO ( p - value indicated on top , two - sided paired Wilcoxon ) .

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 O O O O 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 O O O O O

E. Box and whisker plot indicating change in H3K9me3 levels between WT and Chr 4 KO mESCs grouped by whether chromatin accessibility is higher when the QTL is B6 (left) or D2 (right). Dots represent mean values from biological duplicate experiments and lines connect means. Orange indicates loci for which H3K9me3 signal decreases in the KO (p-value indicated on top, two-sided paired Wilcoxon test).


H . MA - plot similar to D showing change in gene expression between WT Chr 4 KO mESCs ( n = 2 ) . Chr 4 QTL targets are indicated in magenta increase expression in KO .

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

H. MA-plot similar to D showing change in gene expression between WT and Chr 4 KO mESCs (n = 2). Chr 4 QTL targets are indicated in magenta and increase expression in the KO.


Flag - INVS interacted with HA - Akt ( lanes 1 - 3 ) , but not with HA - PDK1 ( lanes 4 - 6 ) or HA - PrKA ( lanes 7 - 9 ) in HEK293T . Expression of HA - Akt , PDK1 , PrKA , Flag - INVS were similar , as determined by immunoblotting ( HA : anti - HA antibody ; F : anti - Flag antibody ; C : control antibody ) . results presented ( A - E ) are representative of at least two independent experiments .

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

Flag-INVS interacted with HA-Akt (lanes 1-3), but not with HA-PDK1 (lanes 4-6) or HA-PrKA (lanes 7-9) in HEK293T cells. Expression levels of HA-Akt, PDK1, PrKA, and Flag-INVS were similar, as determined by immunoblotting (HA: anti-HA antibody; F: anti-Flag antibody; C: control antibody). The results presented (A-E) are representative of at least two independent experiments.


B . Flag - tagged INVS interacted with HA - tagged Akt1 ( lane 1 - 3 ) , Akt2 ( lane 4 - 6 ) , Akt3 ( lane 7 - 9 ) in co - immunoprecipitationassays . Expression of each Akt isoform INVS were similar .

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

B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar.


C . INVS was co - immunoprecipitated with endogenous Akt from HEK293 , in which INVS is also endogenously expressed ( lane 2 ) .

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

C. INVS was co-immunoprecipitated with endogenous Akt from HEK293 cells, in which INVS is also endogenously expressed (lane 2).


D . To determine Akt domain responsible for interacting with INVS , Akt subfragments were generated . C - terminus of Akt is domain for INVS interaction ( lanes 7 - 9 ) . Similar of each Akt fragment were immunoprecipitated using anti - HA antibody .

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

D. To determine the Akt domain responsible for interacting with INVS, Akt subfragments were generated. The C-terminus of Akt is the primary domain for INVS interaction (lanes 7-9). Similar levels of each Akt fragment were immunoprecipitated using anti-HA antibody.


E . Schematic showing structure functional domains of Flag - tagged full - fragmented INVS in mammalian expression vectors ( N - Term : 1 - 421 , Intermediate domain : 421 - 678 , C - Term : 678 - 1065 ) used in current study . Intermediate ( lanes 6 - 9 ) C - terminal ( lanes 10 - 12 ) fragments of INVS were important for interaction with Akt .

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

E. Schematic showing the structure and the functional domains of Flag-tagged full-length and fragmented INVS in mammalian expression vectors (N-Term: 1-421, Intermediate domain: 421-678, and C-Term: 678-1065) used in the current study. Intermediate (lanes 6-9) and C-terminal (lanes 10-12) fragments of INVS were important for interaction with Akt.


A . indicated recombinant INVS ( lanes 1 - 4 ) were used for IVK . Akt phosphorylates WT ( lane5 ) , 1 - 970 ( lane 6 ) , 1 - 898 ( lane 7 ) INVS , but failed to phosphorylate 1 - 670 ( lane 8 ) INVS .

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

A. The indicated recombinant INVS proteins (lanes 1-4) were used for IVK. Akt phosphorylates WT (lane5), 1-970 (lane 6), and 1-898 (lane 7) INVS, but failed to phosphorylate 1-670 (lane 8) INVS.


indicated recombinant INVS ( lanes 1 - 10 ) were used for IVK . Akt phosphorylates WT ( lane 6 ) 1 - 898 ( lane7 ) INVS , but failed to phosphorylate other subfragments of INVS ( 1 - 822 , 1 - 746 , 1 - 670 , in lane 8 , 9 , 10 , respectively ) , as detected using a phospho - Akt substrate antibody . .

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

The indicated recombinant INVS proteins (lanes 1-10) were used for IVK. Akt phosphorylates WT (lane 6) and 1-898 (lane7) INVS, but failed to phosphorylate other subfragments of INVS (1-822, 1-746, and 1-670, in lane 8, 9, and 10, respectively), as detected using a phospho-Akt substrate antibody..


C . Using indicated subfragments of INVS for IVK , we confirmed that Akt phosphorylates 675 - 1065 ( lane 4 ) , but not 675 - 822 ( lane 5 ) , or 675 - 746 ( lane 6 ) INVS .

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

C. Using the indicated subfragments of INVS for IVK, we confirmed that Akt phosphorylates 675-1065 (lane 4), but not 675-822 (lane 5), or 675-746 (lane 6) INVS.


E . Purified recombinant WT or 3A INVS were generated ( top panel , lanes 1 - 4 ) . In IVK , Akt phosphorylated WT fragment ( 824 - 898 , middle panel , lane 3 ) , but failed to phosphorylate 3A INVS ( middle panel , lane 4 ) , as detected by using a phospho - Akt substrate antibody .

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

E. Purified recombinant WT or 3A INVS were generated (top panel, lanes 1-4). In IVK, Akt phosphorylated the WT fragment (824-898, middle panel, lane 3), but failed to phosphorylate 3A INVS (middle panel, lane 4), as detected by using a phospho-Akt substrate antibody.


F . Akt phosphorylated WT ( lane 10 ) , 1 - 898 ( lane 12 ) 675 - 1065 ( lane 15 ) INVS , but failed to phosphorylate both full - 675 - 1065 , ∆ 727 - 896 , 1 - 857 , or 1 - 822 , 3A INVS ( lanes 16 , 11 , 14 , 15 , respectively ) , of which lacked T864 / S865 / T866 target acid .

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 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 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

F. Akt phosphorylated WT (lane 10), 1-898 (lane 12) and 675-1065 (lane 15) INVS, but failed to phosphorylate both full-length and 675-1065, ∆727-896, 1-857, or 1-822, 3A INVS (lanes 16, 11, 14, and 15, respectively), all of which lacked the T864/S865/T866 target amino acid.


A . Confocal microscopy shows that active Akt ( phospho - S473Akt ) is localized in basal of cilia ( right side panels show higher magnification ) . B . INVS is localized in proximal region of INVS compartment in cilia ( INVS compartment , right side panels show higher magnification ) . C . INVS co - localizes with γ - tubulin , a centrosome marker . D . Anti - phospho - Akt substrates stain positively at basal region of cilia ( arrows ) , as determined by anti - acetylated tubulin ( right side panels show higher magnification ) .

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

A. Confocal microscopy shows that active Akt (phospho-S473Akt) is localized in the basal body of primary cilia (right side panels show higher magnification).B. INVS is localized in the proximal region of the INVS compartment in primary cilia (INVS compartment, right side panels show higher magnification).C. INVS co-localizes with γ-tubulin, a centrosome marker.D. Anti-phospho-Akt substrates stain positively at the basal region of the primary cilia (arrows), as determined by anti-acetylated tubulin (right side panels show higher magnification).


E . Silver - intensified immunogold electron microscopy shows that both phospho - Akt ( left side panels ) INVS ( right side panels ) were localized to ciliary pocket of cilia in presence of PDGF - AA ( arrow ) .

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

E. Silver-intensified immunogold electron microscopy shows that both phospho-Akt (left side panels) and INVS (right side panels) were localized to the ciliary pocket of primary cilia in the presence of PDGF-AA (white arrow).


PDGF - AA stimulation enhances localization of active Akt ( phospho - S473Akt ) at basal of cilia ( right side panels show higher magnification ) .

B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

PDGF-AA stimulation enhances localization of active Akt (phospho-S473Akt) at the basal body of primary cilia (right side panels show higher magnification).


of co - localization of phosphorylated Akt ( green ) INVS ( red ) at indicated time points after PDGF - AA stimulation revealed a time - dependent increase . Colocalization was measured by counting yellow pixels ( colocalized ) using Imaris software ( Bitplane AG ) . Results presented are means ± SE ( n = 31 at time 0 , n = 35 at 1 min . , n = 33 at 3 min , respectively ) . Three independent experiments were analyzed with similar results . Statistical significance was determined by student ' s t .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 co-localization of phosphorylated Akt (green) and INVS (red) at the indicated time points after PDGF-AA stimulation revealed a time-dependent increase. Colocalization was measured by counting yellow pixels (colocalized area) using Imaris software (Bitplane AG). Results presented are means ±SE (n=31 at time 0, n=35 at 1 min., and n=33 at 3 min, respectively). Three independent experiments were analyzed with similar results. Statistical significance was determined by student's t test.


Co - immunoprecipitation assays using HEK293T show that INVS preferentially binds to WT - Akt ( second panel , lane1 ) or phospho - mimetic Akt ( T308D / S473D , lane2 ) , but fails to interact with phosphorylation - defective forms of Akt ( T308A / S473A , lane 3 ) . PDGF - AA stimulation of HEK293T results in augmented interaction of endogenous INVS with Akt ( top panel , comparison between lane 1 lane 2 , untreated PDGF - AA - stimulated conditions , respectively ) .

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

Co-immunoprecipitation assays using HEK293T cells show that INVS preferentially binds to WT-Akt (second panel, lane1) or phospho-mimetic Akt (T308D/S473D, lane2), but fails to interact with phosphorylation-defective forms of Akt (T308A/S473A, lane 3).PDGF-AA stimulation of HEK293T cells results in augmented interaction of endogenous INVS with Akt (top panel, comparison between lane 1 and lane 2, untreated and PDGF-AA-stimulated conditions, respectively).


Both WT 3A - INVS , which are localized at INVS compartment of cilia under serum - starved condition ( left side panels , lower panels show higher magnification ) , translocate to basal of cilium after PDGF - AA stimulation ( right side panels , lower panels show higher magnification ) . Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP - INVS ( green ) with γ - tubulin ( red ) in hTERT - RPE1 ( n = 28 ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t . intensities of INVS ( green ) , acetylated - α - tubulin ( blue ) , γ - tubulin ( red ) along ( a - b ) are plotted underneath .

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

Both WT and 3A-INVS, which are localized at the INVS compartment of primary cilia under serum-starved condition (left side panels, lower panels show higher magnification), translocate to the basal body of the cilium after PDGF-AA stimulation (right side panels, lower panels show higher magnification). Results presented are means ± SE of yellow pixels demonstrating colocalization of EGFP-INVS (green) with γ-tubulin (red) in hTERT-RPE1 cells (n=28). Three independent experiments showed similar results. Statistical significance was determined by student's t test. Fluorescence intensities of INVS (green), acetylated-α-tubulin (blue), and γ-tubulin (red) along the line (a-b) are plotted underneath.


A . PDGF - AA stimulation resulted in phosphorylation of WT INVS ( top panel , lane 2 ) , but not 3A INVS ( top panel , lane 6 ) . PDGF - AA - stimulated INVS phosphorylation is inhibited by Akt inhibitors ( top panel , LY294002 , MK2206 , GSK690693 , lanes 3 , 4 , 5 , respectively ) .

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

A. PDGF-AA stimulation resulted in the phosphorylation of WT INVS (top panel, lane 2), but not 3A INVS (top panel, lane 6). PDGF-AA-stimulated INVS phosphorylation is inhibited by Akt inhibitors (top panel, LY294002, MK2206, and GSK690693, lanes 3, 4, and 5, respectively).


B . Myr - Akt , a constitutively active form of Akt , was expressed together with WT or 3A INVS in HEK293 INVS phosphorylation was quantified . WT , but not 3A INVS , can be phosphorylated in presence of Myr - Akt .

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

B. Myr-Akt, a constitutively active form of Akt, was expressed together with WT or 3A INVS in HEK293 cells and INVS phosphorylation was quantified. WT, but not 3A INVS, can be phosphorylated in the presence of Myr-Akt.


C . 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS ( top panel , compare lanes 1 2 , WT phospho - defective mutant , respectively ) . Expression of Akt INVS are shown underneath . D . 3A INVS exhibited weaker dimerization as compared to WT INVS ( top panel , compare lanes 1 2 , WT 3A , respectively ) . Expression of Akt INVS are shown below each .

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

C. 3A INVS exhibited a weaker interaction with Akt as compared to WT INVS (top panel, compare lanes 1 and 2, WT and phospho-defective mutant, respectively). Expression of Akt and INVS are shown underneath.D. 3A INVS exhibited weaker dimerization as compared to WT INVS (top panel, compare lanes 1 and 2, WT and 3A, respectively). Expression of Akt and INVS are shown below each.


E . of cilia in confluent hTERT - RPE was analyzed after siRNA - mediated knock down of Akt1 / 2 . Ciliary decreased in expressing siRNA . Results presented are means ± SE of longitudinal of acetylated - tubulin ( red ) , a marker of cilia , in siRNA - transfected ( green ) hTERT - RPE1 ( n = 175 for control siRNA Akt siRNA , n = 195 for Akt siRNA plus siRNA resistant Akt , respectively ) . Three independent experiments showed similar results . Statistical significance was analyzed by student ' s t . Note that re - introduction of siRNA - resistant Akt ( Matsuda - Lennikov et al . , 2014 ) in Akt knockdown rescued effect on ciliary .

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

E. Length of primary cilia in confluent hTERT-RPE cells was analyzed after siRNA-mediated knock down of Akt1/2. Ciliary length decreased in cells expressing siRNA. Results presented are means ±SE of the longitudinal length of acetylated-tubulin (red), a marker of primary cilia, in siRNA-transfected (green) hTERT-RPE1 cells (n=175 for control siRNA and Akt siRNA, and n=195 for Akt siRNA plus siRNA resistant Akt, respectively). Three independent experiments showed similar results. Statistical significance was analyzed by student's t test. Note that re-introduction of siRNA-resistant Akt (Matsuda-Lennikov et al., 2014) in Akt knockdown cells rescued the effect on ciliary length.


F . Expression of 3A INVS ( bottom panels ) but not WT INVS ( middle panels ) inhibited development of cilia as compared to control ( top panels ) .

O O O O O B-MEASURED_VAR O O 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

F. Expression of 3A INVS (bottom panels) but not WT INVS (middle panels) inhibited the development of primary cilia as compared to control cells (top panels).


of ciliary . Results are mean ± SE of longitudinal of acetylated - tubulin ( blue ) , a marker of cilia , in EGFP - INVS ( green ) transfected in hTERT - RPE1 ( n = 58 for EGFP vector for EGFP - INVS WT , n = 52 for EGFP - INVS 3A mutant , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t .

O O 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 B-CONTROLLED_VAR 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

Quantification of ciliary length. Results are mean ± SE of longitudinal length of acetylated-tubulin (blue), a marker of primary cilia, in EGFP-INVS (green) transfected in hTERT-RPE1 cells (n=58 for EGFP vector and for EGFP-INVS WT, and n=52 for EGFP-INVS 3A mutant, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


Schematic describing measurement of spindle angle ( θ ) ( Kikuchi et al . , 2010 ) . Z - stack were obtained from 0 . 45 mm - thick sections of metaphase angle between axis of metaphase spindle that of substrate surface , which is termed spindle angle , was calculated . Results presented are scatter plots of angle ( θ ) of mitotic spindle ( n = 28 for samples of vector , HA - WT , - 3D , - 3A , - R899X , - Q671X , - R603X INVS stably transfected MDCK ) . Three independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median .

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

Schematic describing the measurement of spindle angle (θ) (Kikuchi et al., 2010). Z-stack images were obtained from 0.45 mm-thick sections of metaphase cells and the angle between the axis of metaphase spindle and that of the substrate surface, which is termed the spindle angle, was calculated. Results presented are scatter plots of the angle (θ) of the mitotic spindle (n=28 for all samples of vector, HA-WT, -3D, -3A, -R899X, -Q671X, and -R603X INVS stably transfected MDCK cells). Three independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


Spindle angles were measured in MDCK transfected with indicated INVS plasmids ( + ) . spindle axis was misaligned in expressing mutant forms of INVS as compared to expressing WT INVS or sham - transduced . Expression of 3A INVS of clinically relevant truncated forms of INVS but not WT INVS , resulted in disorientation of spindle axis during metaphase . Similar expression of INVS constructs was verified by immunoblotting ( Appendix figure S4A ) .

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

Spindle angles were measured in MDCK cells transfected with the indicated INVS plasmids (+). The spindle axis was misaligned in cells expressing mutant forms of INVS as compared to cells expressing WT INVS or sham-transduced cells. Expression of 3A INVS and of clinically relevant truncated forms of INVS but not WT INVS, resulted in disorientation of the spindle axis during metaphase. Similar expression of all INVS constructs was verified by immunoblotting (Appendix figure S4A).


WT INVS robustly inhibited transactivation of INVS in a luciferase reporter , which could be reverted by C - terminal truncated INVS ( R899X , Q671X , or Q603X ) , but not by a phospho - mimetic mutant or 3A INVS . Results presented are mean ± SE of of triplicates between sequentially measured firefly TCF / LEF signal Renilla luciferase signal in HA - INVS transfected 293T . Dual - Luciferase reporter ® System ( Promega ) was used experiments were performed in triplicates . Two independent experiments showed similar results . Statistical significance was determined by student ' s t .

O B-MEASURED_VAR O O O O B-MEASURED_VAR 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 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 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

WT INVS robustly inhibited the transactivation of INVS in a luciferase reporter assay, which could be reverted by C-terminal truncated INVS (R899X, Q671X, or Q603X), but not by a phospho-mimetic mutant or 3A INVS. Results presented are mean ± SE of the ratio of triplicates between sequentially measured firefly TCF/LEF signal and Renilla luciferase signal in HA-INVS transfected 293T cells. Dual-Luciferase reporter® Assay System (Promega) was used and experiments were performed in triplicates. Two independent experiments showed similar results. Statistical significance was determined by student's t test.


expression of HA - Dvl ( upper panels ) indicated forms of Flag - INVS ( lower panels ) are shown by immunoblots ; α - tubulin was used as an internal control .

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

The expression of HA-Dvl (upper panels) and the indicated forms of Flag-INVS (lower panels) are shown by immunoblots; α-tubulin was used as an internal control.


WT INVS - transduced MDCK in 3D cultures form uniform round tubules . However , expression of 3A or NPHP2 - related truncated INVS mutants , which lack Akt phosphorylation site ( s ) , disrupts this uniformity . Results presented are representative of three independent experiments show mean ± SE of normal acini in vector - or WT INVS - transduced MDCK ( n = 422 for Vector , n = 398 for HA - WT , n = 401 for - 3D , n = 365 for - 3A , n = 344 for - R899X , n = 331 for - Q671X , n = 325 for - R603X INVS stably transfected MDCK , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t .

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 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 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 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

WT INVS- transduced MDCK cells in 3D cultures form uniform round tubules. However, expression of 3A or NPHP2-related truncated INVS mutants, which lack Akt phosphorylation site (s), disrupts this uniformity. Results presented are representative of three independent experiments and show mean ± SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=422 for Vector, n=398 for HA-WT, n=401 for -3D, n=365 for -3A, n= 344 for -R899X, n=331 for -Q671X, and n=325 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


WT transduced MDCK form a relatively smaller lumen ; however , expression of 3A INVS or indicated NPHP2 - related truncation mutants result in relatively larger acini . Both truncation mutants ( Q671X or R603X ) lack Akt phosphorylation site exhibit a larger lumen . Three independent experiments showed similar results .

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

WT transduced MDCK cells form a relatively smaller lumen; however, expression of 3A INVS or the indicated NPHP2-related truncation mutants result in relatively larger acini. Both truncation mutants (Q671X or R603X) lack the Akt phosphorylation site and exhibit a larger lumen size. Three independent experiments showed similar results.


C . of normal lumen in transduced with WT or mutant INVS . Results presented are mean ± SE of normal acini in vector - or WT INVS - transduced MDCK ( n = 166 for Vector , n = 204 for HA - WT , n = 233 for - 3D , n = 150 for - 3A , n = 164 for - R899X , n = 128 for - Q671X , n = 137 for - R603X INVS stably transfected MDCK , respectively ) . Three independent experiments showed similar results . Statistical significance was determined by student ' s t .

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 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 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

C. Quantification of normal lumen in cells transduced with WT or mutant INVS. Results presented are mean ±SE of percent normal acini in vector- or WT INVS-transduced MDCK cells (n=166 for Vector, n=204 for HA-WT, n=233 for -3D, n=150 for -3A, n=164 for -R899X, n=128 for -Q671X, and n=137 for -R603X INVS stably transfected MDCK cells, respectively). Three independent experiments showed similar results. Statistical significance was determined by student's t test.


D . of lumen in transduced with WT or mutant INVS is shown . Results presented are scatter plots of acini ( n = 205 for Vector , n = 143 for HA - WT , n = 131 for - 3D , n = 167 for - 3A , n = 144 for - R899X , n = 128 for - Q671X , n = 144 for - R603X INVS stably transfected MDCK , respectively ) . Two independent experiments showed similar results . Statistical significance was determined by Mann - Whitney ' s non - parametric median .

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 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

D. Size of lumen in cells transduced with WT or mutant INVS is shown. Results presented are scatter plots of acini size (n=205 for Vector, n=143 for HA-WT, n=131 for -3D, n=167 for -3A, n=144 for -R899X, n=128 for -Q671X, and n=144 for -R603X INVS stably transfected MDCK cells, respectively). Two independent experiments showed similar results. Statistical significance was determined by Mann-Whitney's non-parametric median test.


B ) Electrophoretic mobility shift ( EMSA ) examination of binding affinity of CENPC - CT CM peptide to a CENP - A nucleosome . Binding to a canonical H3 nucleosome was also examined using EMSA .

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 O B-MEASURED_VAR O O O O O O O

B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA.


C ) EMSA results of binding affinities of CENPC - CT WT , CENPC - CT 3A679 - 681 , CENPC - CT ∆ 678 - 683 to CENP - A nucleosome .

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

C) EMSA results of the binding affinities of CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 to the CENP-A nucleosome.


D ) Stable expression of GFP - fused CENPC - CT its mutants ( shown in ( B ) ) in CENP - C knockout chicken DT40 . α - Tubulin ( Tub ) was probed as a loading control . Parental CENP - C knockout ( parental ) were also examined .

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

D) Stable expression of GFP-fused CENPC-CT and its mutants (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. Parental CENP-C knockout cells (parental) were also examined.


E ) Localization of GFP - fused CENPC - CT WT , CENPC - CT 3A679 - 681 , CENPC - CT ∆ 678 - 683 ( green ) on mitotic chromosomes in chicken DT40 . CENP - T ( red ) was used as a centromere marker . was stained using 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar indicates 10 μm .

O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR 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 O O O O O O O O O O O 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) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm.


F ) Localization of CENP - C in CENP - A knockout DT40 stably expressing GFP - fused gCENP - A WT , RG - AA , or ∆ RG . Scale bar indicates 10 μm . CENP - C was stained by an anti - CENP - C antibody ( red ) was stained by DAPI ( blue ) . CENP - C signals on kinetochores in mitotic were quantified in each . Bar graph indicates mean ± SD ( n = 7 ; * * * * , P < 0 . 0001 , unpaired t , two ) .

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

F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).


C ) EMSA results of binding affinities of CENP - C fragments indicated in ( B ) to CENP - A nucleosome . fragments were phosphorylated by CDK1 .

O O O O O O O O B-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

C) EMSA results of the binding affinities of CENP-C fragments indicated in (B) to the CENP-A nucleosome. All fragments were phosphorylated by CDK1.


D ) Stable expression of GFP - fused CENPC - CT CENPC643 - 864 ( shown in ( B ) ) in CENP - C knockout chicken DT40 . α - Tubulin ( Tub ) was probed as a loading control .

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

D) Stable expression of GFP-fused CENPC-CT and CENPC643-864 (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control.


E ) Localization of GFP - fused CENPC - CT CENPC643 - 864 lacking CM upstream region ( green ) on mitotic chromosomes in CENP - C knockout DT40 . CENP - T ( red ) was used as a centromere marker . was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

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

E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) position of a CDK1 phosphorylation site , T651CENP - C , is indicated in diagram of chicken CENPC - CT . EMSA was performed to examine binding affinities of phosphorylated or non - phosphorylated CENPC - CT ( left panel ) CM peptide ( right panel ) to CENP - A nucleosome .

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

A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome.


D ) constructs analyzed in ( E ) were stably expressed in CENP - C knockout .

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

D) All constructs analyzed in (E) were stably expressed in CENP-C knockout cells.


E ) Localization of GFP - fused CENPC - CT , CENPC - CTR656E , CENPC - CTR655E _ R656E ( green ) on mitotic chromosomes in chicken DT40 . was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

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

E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) Competitive pull - down assays for CENP - A nucleosome binding of CENPC - CT CENPN - NT . left panel displays a of MBP - CENPN - NT ( MBP - CN - NT ) with CENP - A nucleosome ( CA - nuc ) incubated in absence or presence of phosphorylated CENPC - CT ( CC - CT ) pulled down by MBP affinity . right panel displays a of phosphorylated MBP - CENPC - CT ( MBP - CC - CT ) with CENP - A nucleosome ( CA - nuc ) incubated in absence or presence of CENPN - NT ( CN - NT ) pulled down by MBP affinity . signal intensities of histones in CENP - A nucleosomes , which were precipitated with MBP in indicated lanes ( * ) , were quantified . signal intensities were normalized to MBP - CN - NT or MBP - CC - CT signals . Bar graph indicates mean with SD ( n = 3 ) .

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

A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3).


Immunoprecipitation of SATB2 from mouse neonatal cortical lysates . LEMD2 was detected by Western blotting in SATB2 - immunoprecipitate from control but not SATB2 - deficient cortical lysate . equal input of total from control SATB2 - deficient cortical lysates was controlled by ERK2 detection . Representative of immunoblots are shown ; I ( Input ) , F ( Flow - through ) , B ( Beads ) .

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

Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2-immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads).


Reverse immunoprecipitation with LEMD2 antibody control IgG antibody , followed by WB detection of LEMD2 SATB2 . Lysates from cortical culture lysates were used .

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

Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used.


GST pull - down assays . Representative of n = 3 independent experiments are shown . GST pull - down of transiently overexpressed V5 - tagged LEMD2 ( i ) , SATB2 ( ii ) , LEMD2ΔLEM ( iii ) from HeLa lysates using GST , GST - SATB2 , GST - LEMD2 ( 413 - 503 ) hybrid .

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

GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins.


Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa transfected with expression plasmids encoding V5 - tagged full - SATB2 ( Satb21 - 733 ) EGFP as control . equal input of total was controlled by GAPDH detection . Representative of immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular marker ) .

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 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

Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21-733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


CUT - like domain of SATB2 is required for interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from lysates of HeLa transfected with V5 - tagged Satb21 - 247 Satb21 - 157 deletion mutants . LEMD2 BAF were detected only in immunoprecipitates from HeLa transfected with deletion mutant containing CUT - like domain ( Satb21 - 247 ) . equal input of total was controlled by GAPDH detection . Representative of immunoblots are shown ; I ( Input ) , F ( Flow ‐ through ) , B ( Beads ) , M ( Molecular marker ) .

O O O O O B-MEASURED_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 B-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

The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21-247 and Satb21-157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21-247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).


CUT1 CUT2 domains are required for interaction with LEMD2 . Immunoprecipitations using anti - V5 - tag antibody from HeLa transfected with V5 - tagged Satb2346 - 733 Satb2616 - 733 deletion mutants . LEMD2 BAF were detected only in immunoprecipitates from HeLa expressing CUT1 CUT2 containing deletion mutant ( Satb2346 - 733 ) .

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

CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).


Representative of z - axis projected stacks of confocal of hippocampal neuronal nuclei immunostained for Lamin B2 . Examples of spherical infolded nuclei are shown . Scale bars : 3 μm .

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

Representative images of z-axis projected stacks of confocal images of hippocampal neuronal nuclei immunostained for Lamin B2. Examples of spherical and infolded nuclei are shown. Scale bars: 3 μm.


Activity - induced formation of infoldings is abolished in SATB2 - defficient hippocampal neurons . Bic - induced AP bursting for 1 h caused a significant increase in of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx / flx mice but not from Satb2flx / flx : : Nes - Cre mice ( Satb2NesCre ) ( n = 6 independent cultures , two - way ANOVA , F1 , 20 = 9 . 51 , significant interaction p = 0 . 0058 , simple main effects , Satb2flx / flx cultures , Bic - treated vs untreated p = 0 . 0000043 , Satb2NesCre cultures , Bic - treated vs untreated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , untreated - 680 , Satb2flx / flx cultures , Bic - treated - 711 , Satb2NesCre , untreated - 755 , Satb2NesCre , Bic - treated - 720 ) . Data are presented as mean ± , * * * p < 0 . 001 .

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

Activity-induced formation of nuclear infoldings is abolished in SATB2-defficient hippocampal neurons. Bic-induced AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in DIV10 hippocampal cultures derived from Satb2flx/flx mice but not from Satb2flx/flx::Nes-Cre mice (Satb2NesCre) (n = 6 independent primary cultures, two-way ANOVA, F1,20 = 9.51, significant interaction p = 0.0058, simple main effects analysis, Satb2flx/flx cultures, Bic-treated vs untreated p = 0.0000043, Satb2NesCre cultures, Bic-treated vs untreated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, untreated - 680, Satb2flx/flx cultures, Bic-treated - 711, Satb2NesCre, untreated - 755, Satb2NesCre, Bic-treated - 720). Data are presented as mean ± SEM, ***p < 0.001.


Activity - induced formation of infoldings is impaired in SATB2 - deficient cortical neurons . DIV14 cortical cultures form Satb2flx / flx Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h . of infolded nuclei following AP bursting was increased in control Satb2flx / flx cultures but not in SATB2 - deficient cultures ( n = 3 - 4 independent cultures , two - way ANOVA , F1 , 10 = 39 . 124 , significant interaction p = 0 . 000094 , simple main effects , Satb2flx / flx cultures : Bic - treated vs NBQX - treated p = 7 . 66E - 07 , Satb2NesCre cultures : Bic - treated vs NBQX - treated p > 0 . 05 , adjustment for multiple comparisons : Bonferroni , number of analyzed nuclei : Satb2flx / flx cultures , NBQX - 606 , Satb2flx / flx cultures , Bic - 910 ; Satb2NesCre cultures , NBQX - 604 , Satb2NesCre cultures , Bic - 835 ) . Data are presented as mean ± , * * * p < 0 . 001 .

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

Activity-induced formation of nuclear infoldings is impaired in SATB2-deficient cortical neurons. DIV14 cortical cultures form Satb2flx/flx and Satb2NesCre mice were silenced with NBQX for 1 h followed by stimulation with Bic for 1 h. The percentage of infolded nuclei following AP bursting was increased in control Satb2flx/flx cultures but not in SATB2-deficient cultures (n = 3-4 independent primary cultures, two-way ANOVA, F1,10 = 39.124, significant interaction p = 0.000094, simple main effects analysis, Satb2flx/flx cultures: Bic-treated vs NBQX-treated p = 7.66E-07, Satb2NesCre cultures: Bic-treated vs NBQX-treated p > 0.05, adjustment for multiple comparisons: Bonferroni, number of analyzed nuclei: Satb2flx/flx cultures, NBQX - 606, Satb2flx/flx cultures, Bic - 910; Satb2NesCre cultures, NBQX -604, Satb2NesCre cultures, Bic - 835). Data are presented as mean ± SEM, ***p < 0.001.


Confocal ( z - axis - projected stacks ) of Lamin B2 stained neuronal nuclei from CA1 pyramidal layer of adult Satb2flx / flx Satb2flx / flx : : CamK2a - Cre ( Satb2CamkCre ) mice . Scale bars : 10 μm .

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

Confocal images (z-axis-projected stacks) of Lamin B2 stained neuronal nuclei from the CA1 pyramidal cell layer of adult Satb2flx/flx and Satb2flx/flx::CamK2a-Cre (Satb2CamkCre) mice. Scale bars: 10 μm.