B ) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage . A strain containing Cdc14 - GFP and Net1 - HA was blocked in G1 by using the pheromone alpha - factor and released into the cell cycle . Samples were taken at different time - points to analyse Cdc14 spatial localization and Net1 phosphorylation levels . A representative picture of non - released ( T0 ) , partially released ( T60 ) and fully released ( T75 ) Cdc14 from the nucleolus is shown . Scale bar : 3μm . Graph represents the percentage ± SD from three independent experiments of cells with no released , partially released ( FEAR ) and totally released ( MEN ) Cdc14 from the nucleolus along the cell cycle .

O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O 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-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O 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-GENEPROD O O B-SUBCELLULAR O O O O O

B) Cell cycle profile of Cdc14 localization and Net1 phosphorylation in the absence of DNA damage. A strain containing Cdc14-GFP and Net1-HA was blocked in G1 by using the pheromone alpha-factor and released into the cell cycle. Samples were taken at different time-points to analyse Cdc14 spatial localization and Net1 phosphorylation levels. A representative picture of non-released (T0), partially released (T60) and fully released (T75) Cdc14 from the nucleolus is shown. Scale bar: 3μm. Graph represents the percentage ± SD from three independent experiments of cells with no released, partially released (FEAR) and totally released (MEN) Cdc14 from the nucleolus along the cell cycle.


C ) Cells expressing Cdc14 - YFP and Cnm67 - RFP were grown overnight and blocked in G2 / M by using nocodazole prior phleomycin treatment ( 1μM ) . Samples were taken every 30 minutes to follow Cdc14 re - localization . The efficiency of the arrest was determined as in Fig 3A . Scale bar : 3μm . Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion ( left graph ) and cells arrested in metaphase ( right graph ) from three independent experiments .

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O

C) Cells expressing Cdc14-YFP and Cnm67-RFP were grown overnight and blocked in G2/M by using nocodazole prior phleomycin treatment (1μM). Samples were taken every 30 minutes to follow Cdc14 re-localization. The efficiency of the arrest was determined as in Fig 3A. Scale bar: 3μm. Graphs represent the percentage ± SD of cells with Cdc14nucleolar exclusion (left graph) and cells arrested in metaphase (right graph) from three independent experiments.


D ) Live - cell imaging of Cdc14 localization in the presence of a non - reparable DSB at the MAT locus . Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media , thus producing a DSB on chromosome III . Cdc14 , the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 ( used as DSB reporter ) were labelled with the green , red and cyan fluorescent proteins respectively . Micrographs display the maximum projection of 9 planes showing Cdc14 co - localizing with the DSB and the SPBs in response to DNA damage ( arrows ) . Scale bar : 3μm . Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction ( left graph ) and the percentage of Cdc14 - SPB - DSB co - localization after 5h in galactose ( right graph ) .

O O O O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O O O B-SUBCELLULAR 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-GENEPROD O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O

D) Live-cell imaging of Cdc14 localization in the presence of a non-reparable DSB at the MAT locus. Cells were grown overnight and the HO endonuclease was expressed by adding galactose to the media, thus producing a DSB on chromosome III. Cdc14, the SPB component Cnm67 and the DNA damage checkpoint protein Ddc2 (used as DSB reporter) were labelled with the green, red and cyan fluorescent proteins respectively. Micrographs display the maximum projection of 9 planes showing Cdc14 co-localizing with the DSB and the SPBs in response to DNA damage (arrows). Scale bar: 3μm. Graphs represent the percentage ± SD from three independents experiment of cells with nucleoplasmic Cdc14 along the HO endonuclease induction (left graph) and the percentage of Cdc14-SPB-DSB co-localization after 5h in galactose (right graph).


E ) Western blot showing Net1 - 6HA phosphorylation state before and after the generation of a DSB induced by the HO expression . A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control . The phospho - bands were resolved by using Phos - tag polyacrylamide gels as in Fig 3A and Fig 3B . Coomassie staining is depicted as loading control .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O 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-EXP_ASSAY O O O O O O O

E) Western blot showing Net1-6HA phosphorylation state before and after the generation of a DSB induced by the HO expression. A strain lacking the HO endonuclease under the galactose promoter was used as undamaged control. The phospho-bands were resolved by using Phos-tag polyacrylamide gels as in Fig 3A and Fig 3B. Coomassie staining is depicted as loading control.


A ) Diagram showing the genomic background and the approximation used to measure DSB - SPB interaction . Cnm67 - RFP and Ddc2 - GFP were used as SPB and DSB markers respectively . D1 : inter - SPB distance ; D2 : DSB distance to proximal SPB ; D3 : DSB distance to distal SPB . Graph represents the mean ± SD of D1 , D2 and D3 distances from 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 of a wild - type strain depicting DSB - SPB interaction is shown . Scale bar : 3μm .

O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O

A) Diagram showing the genomic background and the approximation used to measure DSB-SPB interaction. Cnm67-RFP and Ddc2-GFP were used as SPB and DSB markers respectively. D1: inter-SPB distance; D2: DSB distance to proximal SPB; D3: DSB distance to distal SPB. Graph represents the mean ± SD of D1, D2 and D3 distances from 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 of a wild-type strain depicting DSB-SPB interaction is shown. Scale bar: 3μm.


B ) Time - lapse experiments in living cells showing the efficiency of DSB - SPB interaction upon induction of a single DSB . Cnm67 - CFP and Ddc2 - RFP were used as SPB and DSB markers respectively . D1 , D2 and D3 distances in wild - type and cdc14 - 1 cells were tracked using the maximum projection of three z - planes images at 15 - seconds intervals over period of 7 . 5 minutes . A total of 32 time - lapses for each strain were measured . A representative picture is shown . Examples of cells defective in DSB - SPB interaction are marked with arrows . Scale bar : 3μm .

O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O

B) Time-lapse experiments in living cells showing the efficiency of DSB-SPB interaction upon induction of a single DSB. Cnm67-CFP and Ddc2-RFP were used as SPB and DSB markers respectively. D1, D2 and D3 distances in wild-type and cdc14-1 cells were tracked using the maximum projection of three z-planes images at 15-seconds intervals over period of 7.5 minutes. A total of 32 time-lapses for each strain were measured. A representative picture is shown. Examples of cells defective in DSB-SPB interaction are marked with arrows. Scale bar: 3μm.


C ) An RFP - marked I - SceI recognition site is re - localized to the SPBs after expressing the endonuclease . Cells harbouring a TetO array adjacent to the I - SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC - Ade prior galactose induction . To check reproducibility with previous results a Rad52 - CFP was used . Cnm67 was labelled with the GFP to be used as SPB reporter . D1 , D2 and D3 distances were measured in wild - type and cdc14 - 1 mutants before and after endonuclease expression by using both TetI - RFP and Rad52 - CFP foci using the maximum projection of nine z - planes images . Graphs represent the average of three independent experiments . P values were calculated using a 2 - tailed unpaired Student t test . A representative picture of a wild - type strain depicting TetI - Rad52 - SPB interaction is shown . Scale bar : 3μm .

O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O

C) An RFP-marked I-SceI recognition site is re-localized to the SPBs after expressing the endonuclease. Cells harbouring a TetO array adjacent to the I-SceI site and a plasmid containing the endonuclease under the control of the galactose promoter were grown overnight in SC-Ade prior galactose induction. To check reproducibility with previous results a Rad52-CFP was used. Cnm67 was labelled with the GFP to be used as SPB reporter. D1, D2 and D3 distances were measured in wild-type and cdc14-1 mutants before and after endonuclease expression by using both TetI-RFP and Rad52-CFP foci using the maximum projection of nine z-planes images. Graphs represent the average of three independent experiments. P values were calculated using a 2-tailed unpaired Student t test. A representative picture of a wild-type strain depicting TetI-Rad52-SPB interaction is shown. Scale bar: 3μm.


D ) DSB - SPB tethering requires the N - terminus domain of Mps3 . Cells expressing a wild - type Mps3 , a mps3Δ2 - 64 and a mps3Δ75 - 150 fused to the RFP as the sole source of the protein were scored for their ability to enhance DSB - SPB tethering in both wild - type and cdc14 - 1 backgrounds . Ddc2 - YFP was used as DSB reporter . Measurements were carried out using a maximum projection of nine z - planes images . Graphs represent the average distribution of D1 , D2 and D3 of three independent experiments . At least 100 cells for each experiment were scored . P values were calculated using a 2 - tailed unpaired Student t test .

O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DSB-SPB tethering requires the N-terminus domain of Mps3. Cells expressing a wild-type Mps3, a mps3Δ2-64 and a mps3Δ75-150 fused to the RFP as the sole source of the protein were scored for their ability to enhance DSB-SPB tethering in both wild-type and cdc14-1 backgrounds. Ddc2-YFP was used as DSB reporter. Measurements were carried out using a maximum projection of nine z-planes images. Graphs represent the average distribution of D1, D2 and D3 of three independent experiments. At least 100 cells for each experiment were scored. P values were calculated using a 2-tailed unpaired Student t test.


E ) DSB - SPB interaction involves a proficient DNA damage checkpoint activation . D1 , D2 and D3 distances were scored in wild - type , sml1Δ and sml1Δ rad53Δ backgrounds using Ddc2 - GFP and Spc110 - RFP as DSB and SPB markers respectively . Measurements were performed by using a maximum projection of nine z - planes images . Graphs denote the average distribution of D1 , D2 and D3 from three independent experiments where at least 100 cells for each sample were scored . P values were calculated using a 2 - tailed unpaired Student t test .

O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DSB-SPB interaction involves a proficient DNA damage checkpoint activation. D1, D2 and D3 distances were scored in wild-type, sml1Δ and sml1Δ rad53Δ backgrounds using Ddc2-GFP and Spc110-RFP as DSB and SPB markers respectively. Measurements were performed by using a maximum projection of nine z-planes images. Graphs denote the average distribution of D1, D2 and D3 from three independent experiments where at least 100 cells for each sample were scored. P values were calculated using a 2-tailed unpaired Student t test.


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 ) .

O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O 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 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 .

O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O 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-EXP_ASSAY 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 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 .

O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 ) 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 .

O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O 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-EXP_ASSAY 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 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 .

O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O 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-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR 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 the equal amount of Tub1 - GFP in both wild - type and cdc14 - 1 backgrounds . Coomassie blue staining is shown as loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY 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 ) 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 .

O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR 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 ) 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 .

O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O 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 ) 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 .

O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY 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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O 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-SUBCELLULAR O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-SUBCELLULAR 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 .

O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O 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-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SUBCELLULAR 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 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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across ( A ) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD 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-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR

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 ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the U6 snRNA

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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 ) and RNAP2 ( data from ( Larochelle et al , 2018 ) ) across the srp7 loci .

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

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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 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 ) .

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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 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 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD 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-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD 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 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 ) .

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(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 the indicated RNAP3 transcripts . Transcript levels were normalized to act1 ( mean ± standard deviation from 3 biological replicates ) .

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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).


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 ) .

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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 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 .

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(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 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 .

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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 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1 .

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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 the absence of Sen1 by ChIP - qPCR ( mean ± standard deviation from 6 biological replicates ) .

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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).


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 .

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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 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 .

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

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


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

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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 ) 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 ) .

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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 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 ) .

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

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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 , 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 ) .

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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 cellular identity are shown for five cell clusters .

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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 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 .

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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 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 ) .

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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 , and RNA read depth from merged replicates . Locations of Chr 7 trans caQTL targets are indicated with black bars and LOD scores listed below .

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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 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 .

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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 . 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 ) .

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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 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 .

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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 . 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 ) .

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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 , 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 .

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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 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 ) .

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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 and Chr 4 KO mESCs ( n = 2 ) . Chr 4 QTL targets are indicated in magenta and increase expression in the KO .

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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 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 .

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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 ) , and Akt3 ( lane 7 - 9 ) in co - immunoprecipitationassays . Expression of each Akt isoform and INVS were similar .

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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 cells , in which INVS is also endogenously expressed ( lane 2 ) .

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


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 .

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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 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 .

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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 . 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 .

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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.


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 . .

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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 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 .

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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 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 .

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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 ) 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 .

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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 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 ) .

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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 ) and INVS ( right side panels ) were localized to the ciliary pocket of primary cilia in the presence of PDGF - AA ( white arrow ) .

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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 the basal body of primary cilia ( right side panels show higher magnification ) .

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


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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 cells and INVS phosphorylation was quantified . WT , but not 3A INVS , can be phosphorylated in the presence of Myr - Akt .

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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 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 .

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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 . 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 .

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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 the development of primary cilia as compared to control cells ( top panels ) .

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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).


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 .

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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 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 .

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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 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 ) .

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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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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.