Representative nuclease assays with MRE11 , MR , MRN pCtIP , as indicated , on 5 ' - end labeled dsDNA with a single streptavidin - blocked end ( S ) . substrate contained 5 phosphorothioate bonds ( PTO ) at both 3 ' - ends to prevent exonucleolytic degradation . Reactions were incubated for 2 h .

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

Representative nuclease assays with MRE11, MR, MRN and pCtIP, as indicated, on 5'-end labeled dsDNA with a single streptavidin-blocked end (S). The substrate contained 5 phosphorothioate bonds (PTO) at both 3'-ends to prevent exonucleolytic DNA degradation. Reactions were incubated for 2 h.


Representative nuclease assays as in ( A ) , but with a substrate containing a nick in top oligonucleotide ( 20 nt away from 3 ' - end , see cartoon ) .

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

Representative nuclease assays as in (A), but with a substrate containing a nick in the top oligonucleotide (20 nt away from the 3'-end, see cartoon).


Quantitation of experiments such as shown in ( A ) , with pCtIP . Averages shown ; n ≥ 4 ; error bars , .

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

Quantitation of experiments such as shown in (A), with pCtIP. Averages shown; n ≥ 4; error bars, SEM.


Quantitation of experiments such as shown in ( B ) , with without pCtIP . Averages shown ; n = 3 ; error bars , .

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

Quantitation of experiments such as shown in (B), with and without pCtIP. Averages shown; n = 3; error bars, SEM.


Population doublings ( PDs ) determined in BJ cultured with without folate for 28 days .

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

Population doublings (PDs) determined in BJ cells cultured with and without folate for 28 days.


Example of a single combed molecule labeled with IdU ( green ) CldU ( red ) , showing replication from three adjacent origins . Horizontal arrows indicate fork orientation .

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

Example of a single combed DNA molecule labeled with IdU (green) and CldU (red), showing replication from three adjacent origins. Horizontal white arrows indicate fork orientation.


Representative examples of single combed molecules from control grown for 7 days in a folate - free medium .

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

Representative examples of single combed DNA molecules from control cells and cells grown for 7 days in a folate-free medium.


Fork rate ( Kb / min ) distribution . blue bars : BJ ( n = 126 ) ; black bars : BJ that were cultured for 7 days in a folate - free medium ( n = 131 ) ; blue bars : BJ cultured for 7 days in a folate - free medium supplemented with A , G , C , T nucleosides for last 48 h of experiment ( n = 138 ) . Box plot summarizing fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 360 ) ; − folate ( n = 372 ) ; − folate + AGCT ( n = 361 ) .

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

Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 126); black bars: BJ cells that were cultured for 7 days in a folate-free medium (n = 131); blue bars: BJ cells cultured for 7 days in a folate-free medium and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 138).Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 360); −folate (n = 372); −folate + AGCT (n = 361).


Fork ( Kb ) distribution . color code is as in ( D ) . Control ( n = 72 ) ; − folate ( n = 71 ) ; − folate + AGCT ( n = 75 ) . Box plot summarizing fork distribution ( Kb ) of three independent experiments . Control ( n = 212 ) ; − folate ( n = 215 ) ; − folate + AGCT ( n = 209 ) .

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

Fork distance (Kb) distribution. The color code is as in (D). Control (n = 72); −folate (n = 71); −folate + AGCT (n = 75).Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 212); −folate (n = 215); −folate + AGCT (n = 209).


A Population doublings ( PDs ) determined in BJ cultured at indicated folate concentrations for 48 days .

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

A Population doublings (PDs) determined in BJ cells cultured at the indicated folate concentrations for 48 days.


B , C average replication rate ± ( B ) average fork ± ( C ) in indicated folate concentrations at 14 21 days . At least 115 were analyzed at each at each time point to determine average replication rate . At least 71 replication forks were analyzed at each at each time point to determine average fork .

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

B, C The average replication rate ± SEM (B) and the average fork distance ± SEM (C) in the indicated folate concentrations at 14 and 21 days. At least 115 DNA fibers were analyzed at each concentration and at each time point to determine the average replication rate. At least 71 replication forks were analyzed at each concentration and at each time point to determine the average fork distance.


D - G BJ were grown for 14 days in 100 nM folate with without nucleoside supplementation . ( D ) Fork rate ( Kb / min ) distribution . blue bars : BJ ( n = 115 ) ; gray bars : BJ that were cultured for 14 days in 100 nM folate ( n = 117 ) ; blue bars : BJ cultured for 14 days in 100 nM folate supplemented with A , G , C , T nucleosides for last 48 h of experiment ( n = 117 ) . ( E ) Box plot summarizing fork rate distribution ( Kb / min ) of three independent experiments . Control ( n = 352 ) ; 100 nM folate ( n = 364 ) ; 100 nM folate + AGCT ( n = 355 ) . Main box represents values from lower to upper quartile ( 25th to 75th percentile ) . middle represents median . ( F ) Fork ( Kb ) distribution . color code is as in ( D ) . Control ( n = 69 ) ; 100 nM folate ( n = 74 ) ; 100 nM folate + AGCT ( n = 72 ) . ( G ) Box plot summarizing fork distribution ( Kb ) of three independent experiments . Control ( n = 201 ) ; 100 nM folate ( n = 220 ) ; 100 nM folate + AGCT ( n = 228 ) . Main box represents values from lower to upper quartile ( 25th to 75th percentile ) . middle represents median . * * P < 0 . 001 .

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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-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 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 O O O O O O O O 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 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 O O 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-G BJ cells were grown for 14 days in 100 nM folate with and without nucleoside supplementation. (D) Fork rate (Kb/min) distribution. Light blue bars: BJ cells (n = 115); gray bars: BJ cells that were cultured for 14 days in 100 nM folate (n = 117); blue bars: BJ cells cultured for 14 days in 100 nM folate and supplemented with A, G, C, and T nucleosides for the last 48 h of the experiment (n = 117). (E) Box plot summarizing the fork rate distribution (Kb/min) of three independent experiments. Control (n = 352); 100 nM folate (n = 364); 100 nM folate + AGCT (n = 355). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. (F) Fork distance (Kb) distribution. The color code is as in (D). Control (n = 69); 100 nM folate (n = 74); 100 nM folate + AGCT (n = 72). (G) Box plot summarizing the fork distance distribution (Kb) of three independent experiments. Control (n = 201); 100 nM folate (n = 220); 100 nM folate + AGCT (n = 228). Main box represents the values from the lower to upper quartile (25th to 75th percentile). The middle line represents the median. **P < 0.001.


Cyclin E - expressing BJ were grown for 7 days with without folate . Fork rate ( Kb / min ) distribution . bars : BJ expressing an empty vector ( n = 145 ) ; gray bars : BJ expressing cyclin E oncogene ( n = 147 ) ; gray bars : BJ cultured for 7 days in a folate - free medium ( n = 135 ) ; black bars : BJ expressing cyclin E oncogene cultured for 7 days in a folate - free medium ( n = 138 ) .

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 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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Fork rate (Kb/min) distribution. White bars: BJ cells expressing an empty vector (n = 145); dark gray bars: BJ cells expressing the cyclin E oncogene (n = 147); light gray bars: BJ cells cultured for 7 days in a folate-free medium (n = 135); black bars: BJ cells expressing the cyclin E oncogene cultured for 7 days in a folate-free medium (n = 138).


Cyclin E - expressing BJ were grown for 7 days with without folate . Fork distribution ( Kb ) . color code is as in ( A ) . Empty vector ( n = 78 ) ; CycE ( n = 79 ) ; empty vector − folate ( n = 71 ) ; CycE − folate ( n = 80 ) .

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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Fork distance distribution (Kb). The color code is as in (A). Empty vector (n = 78); CycE (n = 79); empty vector −folate (n = 71); CycE −folate (n = 80).


Cyclin E - expressing BJ were grown for 7 days with without folate . of origins with indicated progression between sister forks . Empty vector ( n = 158 ) ; CycE ( n = 155 ) ; empty vector − folate ( n = 160 ) ; CycE − folate ( n = 154 ) . * P < 0 . 05 .

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-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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate. Percent of origins with the indicated progression ratio between sister forks. Empty vector (n = 158); CycE (n = 155); empty vector −folate (n = 160); CycE −folate (n = 154). *P < 0.05.


Cyclin E - expressing BJ were grown for 7 days with without folate . Examples of nuclei with γH2AX 53BP1 foci following cyclin E expression ( CycE ) ( n = 65 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( empty vector − folate ) ( n = 67 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 70 ) . Red : γH2AX ; green : 53BP1 ; blue : DAPI . of nuclei with indicated number of γH2AX - 53BP1 co - localized foci . * * P < 0 . 01 .

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 B-CONTROLLED_VAR O O 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 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

Cyclin E-expressing BJ cells were grown for 7 days with and without folate.Examples of nuclei with γH2AX and 53BP1 foci following cyclin E expression (CycE) (n = 65), empty vector (n = 65), folate-free medium for 7 days (empty vector −folate) (n = 67) or oncogene expression under folate-free conditions (CycE −folate) (n = 70). Red: γH2AX; green: 53BP1; blue: DAPI staining.Percent of nuclei with the indicated number of γH2AX-53BP1 co-localized foci. **P < 0.01.


Cyclin E - expressing BJ grown for 7 days with without folate . Immunoblotting with anti - phosphorylated ATM anti - phosphorylated CHK1 antibodies . Anti - β - catenin anti - actin antibodies were used as loading controls . level .

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

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Immunoblotting with anti-phosphorylated ATM and anti-phosphorylated CHK1 antibodies. Anti-β-catenin and anti-actin antibodies were used as loading controls.Protein level quantification.


Cyclin E - expressing BJ grown for 7 days with without folate . Examples of nuclei with RAD51 foci following cyclin E expression ( CycE ) ( n = 67 ) , empty vector ( n = 65 ) , folate - free medium for 7 days ( − folate ) ( n = 71 ) or oncogene expression under folate - free conditions ( CycE − folate ) ( n = 75 ) . Green : RAD51 , blue : DAPI . of nuclei with indicated number of RAD51 foci ( n = 66 ) . * * P < 0 . 01 .

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-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 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

Cyclin E-expressing BJ cells grown for 7 days with and without folate.Examples of nuclei with RAD51 foci following cyclin E expression (CycE) (n = 67), empty vector (n = 65), folate-free medium for 7 days (−folate) (n = 71) or oncogene expression under folate-free conditions (CycE −folate) (n = 75). Green: RAD51, blue: DAPI staining.Percent of nuclei with the indicated number of RAD51 foci (n = 66). **P < 0.01.


A , B Cyclin E - Ras ( H - RasV12 ) - expressing 3T3 grown in 100 nM folate for 4 then two additional in a normal medium . Control were grown in a normal medium for whole period . ( A ) Examples of anchorage - independent growth in soft agar of 3T3 . ( B ) Average number of colonies per soft agar plate of 3T3 . number of colonies per plate is expressed as average ± from three independent experiments .

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

A, B Cyclin E- and Ras (H-RasV12)-expressing 3T3 cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (A) Examples of anchorage-independent growth in soft agar of 3T3 cells. (B) Average number of colonies per soft agar plate of 3T3 cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments.


C - E Ras ( H - RasV12 ) - expressing MCF10A grown in 100 nM folate for 4 then two additional in a normal medium . Control were grown in a normal medium for whole period . ( C ) Examples of anchorage - independent growth in soft agar of MCF10A . ( D ) Average number of colonies per soft agar plate of MCF10A . number of colonies per plate is expressed as average ± from three independent experiments . ( E ) of - free flanks at indicated time points after injection . Ten mice were injected in both sides in each group .

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

C-E Ras (H-RasV12)-expressing MCF10A cells grown in 100 nM folate for 4 weeks and then two additional weeks in a normal medium. Control cells were grown in a normal medium for the whole period. (C) Examples of anchorage-independent growth in soft agar of MCF10A cells. (D) Average number of colonies per soft agar plate of MCF10A cells. The number of colonies per plate is expressed as the average ± SEM from three independent experiments. (E) Percentage of tumor-free flanks at the indicated time points after cell injection. Ten mice were injected in both sides in each group.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( A ) Representative maximum projection of 10 Z - stacks per image . Scale bar : 10 µm .

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

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (A) Representative maximum intensity projection images of 10 Z-stacks per image. Scale bar: 10 µm.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( B ) of GFP foci per 0 . 0025 mm2 . Values are presented as mean ± standard deviation of mean were calculated from at least three independent experiments with 3 individuals per replicate . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to T - .

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

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (B) Quantification of GFP foci per 0.0025 mm2. Values are presented as mean ± standard deviation of the mean and were calculated from at least three independent experiments with 3 individuals per replicate. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( C ) GFP - ATG8a cleavage immunoblot for exposed to same treatments as in ( A ) . Numbers below blots represent for given sample normalized to input relative to non - treated control . Experiments were repeated minimum 3 times with similar results .

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

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (C) GFP-ATG8a cleavage immunoblot for plants exposed to the same treatments as in (A). Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.


GFP - ATG8a expressing seedlings in Murashige & Skoog ( MS ) growth medium or 30 min after treatment with MS containing ACC , ABA , ATP , BL , 6 - BA , Flg22 , NAA or PEP1 . ( D ) NBR1 immunoblot for atg2 - 2 samples for given treatments . Numbers below blots represent for given sample normalized to input relative to non - treated control . Experiments were repeated minimum 3 times with similar results .

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

GFP-ATG8a expressing seedlings in Murashige & Skoog (MS) growth medium or 30 min after treatment with MS containing ACC, ABA, ATP, BL, 6-BA, Flg22, NAA or PEP1. (D) NBR1 immunoblot for atg2-2 samples for given treatments. Numbers below the blots represent ratio for given sample normalized to input and relative to non-treated control. Experiments were repeated minimum 3 times with similar results.


( A ) Seedlings were acclimated for 16 h in MS containing ABA then imaged 30 min after being swapped to MS containing ABA / NAA ( control ) , MS or MS containing flg22 . are representative maximum projection of 10 Z - stacks per condition . Experiments were repeated 3 times independently with similar results . Scale bar : 10 µm . ( B ) of GFP foci per 0 . 0025 mm2 , for samples treated as described in A . Values are presented as mean ± standard deviation of mean are on 3 independent experiments , with 3 individuals per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Seedlings were acclimated for 16 h in MS containing ABA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing flg22. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (B) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in A. Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( C ) GFP - ATG8a cleavage immunoblot for given treatments . of free GFP to loading control , normalized to 16h pre - treated sample ( set to 1 ) is provided below each band .

O O O O O O O O O O O O O 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) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.


( D ) Seedlings were acclimated for 16 h in MS containing NAA then imaged 30 min after being swapped to MS containing ABA / NAA ( control ) , MS or MS containing 6 - BA . are representative maximum projection of 10 Z - stacks per condition . Experiments were repeated 3 times independently with similar results . Scale bar : 10 µm . ( E ) of GFP foci per 0 . 0025 mm2 , for samples treated as described in ( D ) . Values are presented as mean ± standard deviation of mean are on 3 independent experiments , with 3 individuals per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to 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 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 O O O O O O O O O O O O O O O O O O O

(D) Seedlings were acclimated for 16 h in MS containing NAA and then imaged 30 min after being swapped to MS containing ABA/NAA (control), MS or MS containing 6-BA. Images are representative maximum intensity projection of 10 Z-stacks per condition. Experiments were repeated 3 times independently with similar results. Scale bar: 10 µm. (E) Quantification of GFP foci per 0.0025 mm2, for samples treated as described in (D). Values are presented as mean ± standard deviation of the mean and are based on 3 independent experiments, with 3 individuals per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( F ) GFP - ATG8a cleavage immunoblot for given treatments . of free GFP to loading control , normalized to 16h pre - treated sample ( set to 1 ) is provided below each band .

(F) GFP-ATG8a cleavage immunoblot for given treatments. The ratio of free GFP to loading control, normalized to the 16h pre-treated sample (set to 1) is provided below each band.


( B ) Pattern correlation used to find that accumulate upon ABA / NAA treatment are removed in WT but not in atg2 after swapping to flg22 / 6 - BA .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O

(B) Pattern correlation used to find proteins that accumulate upon ABA/NAA treatment and are removed in WT but not in atg2 after swapping to flg22/6-BA.


clusters obtained after quantitative proteomics of WT ( green ) atg2 - 2 ( magenta ) samples treated as described ( C ) cluster fitting pattern displayed ( D ) cluster for that accumulate to higher in atg2 than WT upon treatment with flg22 .

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

Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (C) Protein cluster fitting the pattern displayed (D) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with flg22.


clusters obtained after quantitative proteomics of WT ( green ) atg2 - 2 ( magenta ) samples treated as described ( E ) cluster of which accumulate upon NAA treatment are removed in WT but not in atg2 after swapping to 6 - BA ( F ) cluster for that accumulate to higher in atg2 than WT upon treatment with 6 - BA .

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

Protein clusters obtained after quantitative proteomics of WT (green) and atg2-2 (magenta) samples treated as described (E) Protein cluster of proteins which accumulate upon NAA treatment and are removed in WT but not in atg2 after swapping to 6-BA (F) Protein cluster for proteins that accumulate to higher levels in atg2 than WT upon treatment with 6-BA.


( B ) Dry of WT , atg5 - 1 or atg2 - 2 grown under stable ( 21 / 16 ºC 16 / 8 - h photoperiod ) or following Swedish Spring of 2013 . Box plots : centerlines show medians ; box limits indicate 25th 75th percentiles ; whiskers extend to minimum maximum . Significance calculated by Kruskal - Wallis or Feltz Miller for equality of coefficients of variation as explained in materials methods . Asterisks represent significant pairwise differences ( ∗ ∗ ∗ ∗ P < 0 . 0001 ) ( C ) Box plots displaying heterogeneity of samples in ( B ) . Box plots : centerlines show medians ; box limits indicate 25th 75th percentiles ; whiskers extend to minimum maximum . Significance calculated by Kruskal - Wallis or Feltz Miller for equality of coefficients of variation as explained in materials methods . Asterisks represent significant pairwise differences ( ∗ ∗ P < 0 . 01 , ∗ ∗ ∗ ∗ P < 0 . 0001 )

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

(B) Dry weight of WT, atg5-1 or atg2-2 plants grown under stable (21/16 ºC 16/8-h photoperiod) or following the Swedish Spring of 2013. Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences ( ∗∗∗∗P< 0.0001) (C) Box plots displaying the heterogeneity of samples in (B). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Significance calculated by Kruskal-Wallis or Feltz and Miller test for the equality of coefficients of variation as explained in materials and methods. Asterisks represent significant pairwise differences (∗∗P < 0.01, ∗∗∗∗P< 0.0001)


( A ) Representative of root explants from WT , atg2 - 2 atg5 - 1 in CIM ( 6 days ) or CIM + SIM ( 6 + 21 days ) . Scale bar : 2

(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (6 days) or CIM + SIM (6 + 21 days). Scale bar: 2 cm


( B ) ( % ) of explants presenting shoots after 21 days after incubation on SIM . Results were obtained from 3 independent experiments with at least 20 calli per condition . Bars marked with an asterisk ( * ) are statistically significant ( P < 0 . 05 ) according to 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 O O O O O O

(B) Quantification (%) of explants presenting shoots after 21 days after incubation on SIM. Results were obtained from 3 independent experiments with at least 20 calli per condition. Bars marked with an asterisk (*) are statistically significant (P<0.05) according to the T -test.


( A ) Representative of root explants from WT , atg2 - 2 atg5 - 1 in CIM ( 21 days ) or CIM + SIM ( 21 + 21 days or 21 + 35 days ) . Scale bar : 2

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

(A) Representative images of root explants from WT, atg2-2 and atg5-1 in CIM (21 days) or CIM + SIM (21+ 21 days or 21+ 35 days). Scale bar: 2 cm


( B C ) Fresh weights of calli after incubation on CIM ( B ) or CIM + SIM ( 21 + 21 ) ( C ) . Box plots : centerlines show medians ; box limits indicate 25th 75th percentiles ; whiskers extend to minimum maximum . Results were obtained from 3 independent experiments with at least 20 calli per condition , asteriscs mark statistical significance to WT according to T - ( * * P < 0 . 001 ; * * * P < 0 . 0001 ) .

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

(B and C) Fresh weights of calli after incubation on CIM (B) or CIM + SIM (21+ 21) (C). Box plots: centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend to the minimum and maximum. Results were obtained from 3 independent experiments with at least 20 calli per condition, asteriscs mark statistical significance to WT according to the T -test (** P<0.001; *** P<0.0001).


A . of P4 ( top ) P21 ( bottom ) Mpdz + / + Mpdz - / - mice . Arrowheads point to domed foreheads of latter .

O O O O O 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. Images of P4 (top) and P21 (bottom) Mpdz+/+ and Mpdz-/- mice. Arrowheads point to the domed foreheads of the latter.


B . Mean weights of P18 - P21 Mpdz + / + Mpdz - / - mice ( n = 8 ÷ 12 , mean ± SD ) .

B. Mean weights of P18-P21 Mpdz+/+ and Mpdz-/- mice (n=8÷12, mean ± SD).


C . Coronal , axial , sagittal ( top to bottom ) PET of P18 - P21 Mpdz + / + Mpdz - / - mice . emission is shown as a 7 - point scale from black ( 0 ) to ( 7 ) .

O O O O O O O O O O O O O O O O O O O O O O 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. Coronal, axial, and sagittal (top to bottom) PET images of P18-P21 Mpdz+/+ and Mpdz-/- mice. The emission intensity is shown as a 7-point temperature scale from black (0) to white (7).


D . Mean PET emission intensities in indicated brain sections ( n = 4 , mean ± SD ) .

D. Mean PET emission intensities in the indicated brain sections (n=4, mean ± SD).


A . Coronal , sagittal , axial ( clockwise from top left corner image of each genotype ) MR of Mpdz + / + Mpdz - / - P18 - P21 mice . Ventricles are pseudo - colored in red 3D - reconstructed in lower left panels .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Coronal, sagittal, and axial (clockwise from top left corner image of each genotype) MR images of Mpdz+/+ and Mpdz-/- P18-P21 mice. Ventricles are pseudo-colored in red and 3D-reconstructed in the lower left panels.


B . Means of total ventricle volumes total brain volumes of Mpdz + / + Mpdz - / - mice ( n = 8 , mean ± SD ) .

O O O O O O 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. Means of total ventricle volumes and total brain volumes of Mpdz+/+ and Mpdz-/- mice (n=8, mean ± SD).


C . A coronal HE - stained section ( center ) flanked by anatomically - corresponding coronal T2 - weighted MR of Mpdz - / - P18 - P21 mice . Arrows or arrowheads show match between lateral ventricle CP in HE section in MR .

C. A coronal HE-stained section (center) flanked by anatomically-corresponding coronal T2-weighted MR images of Mpdz-/- P18-P21 mice. Arrows or arrowheads show the match between the lateral ventricle CP in the HE section and in the MR images.


D . Duplicate rows of T2 - weighted coronal anatomically - corresponding T1 - weighted coronal at 1 min at peak - signal time point after contrast - medium injection ; bottom row shows a similar set of axial . Areas surrounded by squares are magnified in insets ; arrows mark contrast - medium signal in T1 - weighted . Each row corresponds to one mouse aged 18 - 21 - days . E . Time courses of normalized T1 - weighted image intensities corresponding to areas surrounded by numbered squares on T2 - weighted . Scale bars , 1 mm ; insets , 0 . 5 mm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Duplicate rows of T2-weighted coronal images and anatomically-corresponding T1-weighted coronal images at 1 min and at the peak-signal time point after contrast-medium injection; the bottom row shows a similar set of axial images. Areas surrounded by squares are magnified in the insets; arrows mark the contrast-medium signal in the T1-weighted images. Each row corresponds to one mouse aged 18-21-days. E. Time courses of the normalized T1-weighted image intensities corresponding to the areas surrounded by numbered squares on the T2-weighted images. Scale bars, 1 mm; insets, 0.5 mm.


A . Coronal HE - stained brain sections of a P18 Mpdz + / + mouse ( out of a total of three ) showing 3rd ventricle , proximal distal sections of aqueduct of Sylvius ; these features are marked by arrows in insets . B . same , for P18 Mpdz - / - mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Coronal HE-stained brain sections of a P18 Mpdz+/+ mouse (out of a total of three) showing the 3rd ventricle, and the proximal and distal sections of the aqueduct of Sylvius; these features are marked by arrows in the insets. B. The same, for P18 Mpdz-/- mice.


C . Top views of brains of P7 Mpdz + / + Mpdz - / - mice that show sites of Evans blue injection , midline planes ( dashed ) sectioned to produce sagittal below . They show extent of Evans blue spread in ventricles surrounding tissue ( one out of three experiments ) . In , injection ; LV , lateral ventricle ; SA , Sylvian Aqueduct ; V3 , third ventricle ; V4 , fourth ventricle . Scale bars , 1 mm ; insets , 0 . 25 mm .

C. Top views of brains of P7 Mpdz+/+ and Mpdz-/- mice that show the sites of Evans blue injection, and the midline planes (dashed line) sectioned to produce the sagittal images below. They show the extent of Evans blue spread in the ventricles and surrounding tissue (one out of three experiments). In, injection; LV, lateral ventricle; SA, Sylvian Aqueduct; V3, third ventricle; V4, fourth ventricle. Scale bars, 1 mm; insets, 0.25 mm.


A . Immunofluorescence of 10 μm - thick sections of CP from third ventricle villi of Mpdz + / + Mpdz - / - mice were immunolabeled as shown . areas in square frames are magnified in bottom panels .

O O O O O O O O O O O O O O O O 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. Immunofluorescence images of 10 μm-thick sections of CP from third ventricle villi of Mpdz+/+ and Mpdz-/- mice were immunolabeled as shown. The areas in the square frames are magnified in the bottom panels.


CP sections from lateral ventricle villi immunolabeled for ZO1 ( B ) are representative of 2 Mpdz + / + mice 3 Mpdz - / - mice . Scale bars , top panels , 100 μm ; bottom panels , 10 μm .

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

CP sections from lateral ventricle villi immunolabeled for ZO1 (B) The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm.


CP sections from lateral ventricle villi immunolabeled for E - cadherin ( E - cad ; C ) . are representative of 2 Mpdz + / + mice 3 Mpdz - / - mice . Scale bars , top panels , 100 μm ; bottom panels , 10 μm .

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

CP sections from lateral ventricle villi immunolabeled for E-cadherin (E-cad; C). The images are representative of 2 Mpdz+/+ mice and 3 Mpdz-/- mice. Scale bars, top panels, 100 μm; bottom panels, 10 μm.


D . Immunoblots with indicated antibodies ( top ) their quantifications . densitometry measurements were normalized relative to signal of samples transduced by non - targeting shRNA ( Ctrl ) . Note that same β - actin immunoblot was used twice because Jam - C E - cadherin samples were immunoblotted on same ( mean ± SD , n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblots with the indicated antibodies (top) and their quantifications. The densitometry measurements were normalized relative to the signal of the samples transduced by non-targeting shRNA (Ctrl). Note that the same β-actin immunoblot was used twice because the Jam-C and E-cadherin samples were immunoblotted on the same membrane (mean ± SD, n=3).


A . of longitudinal sections of lateral ventricle CP villi of P18 - P21 Mpdz + / + Mpdz - / - mice . Scale bar , 2 μm . B . A large number of voids of varying sizes was evident in CPECs of Mpdz - / - mice . Higher magnification of areas in rectangles are shown underneath panels . Adherens junctions are denoted by horizontal lines arrows ; voids are indicated by arrows . Scale bar , 1 μm ; insets , 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TEM images of longitudinal sections of lateral ventricle CP villi of P18-P21 Mpdz+/+ and Mpdz-/- mice. Scale bar, 2 μm. B. A large number of voids of varying sizes was evident in the CPECs of Mpdz-/- mice. Higher magnification images of the areas in the rectangles are shown underneath the panels. Adherens junctions are denoted by horizontal lines and arrows; voids are indicated by arrows. Scale bar, 1 μm; insets, 200 nm.


C . Triplicate of tight junctions proximal to apical faces of CPECs in Mpdz + / + Mpdz - / - mice . Scale bar , 100

O O O 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. Triplicate images of tight junctions proximal to the apical faces of CPECs in Mpdz+/+ and Mpdz-/- mice. Scale bar, 100 nm


D . Mitochondria were smaller frequently lacked large portions of their cristae . Autophagosomes are present in some of them ( arrows ) . Scale bar , 200 nm . are representative of 2 Mpdz + / + mice 2 Mpdz - / - mice .

D. Mitochondria were smaller and frequently lacked large portions of their cristae. Autophagosomes are present in some of them (arrows). Scale bar, 200 nm. The images are representative of 2 Mpdz+/+ mice and 2 Mpdz-/- mice.


E . Time course standard deviations of impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non - targeting ( Ctrl ) shRNA . Each record represents 4 wells . MPDZ immunoblot of each group is shown in inset .

E. Time course and standard deviations of the impedance of confluent hpCPEC monolayers that were transduced by either MPDZ or non-targeting (Ctrl) shRNA. Each record represents 4 wells. MPDZ immunoblot of each cell group is shown in the inset.


A . of CPEC sections from lateral ventricle villi of two Mpdz + / + two Mpdz - / - P14 - P16 mice injected with HRP . CPs were reacted with hydrogen peroxide DAB ex - vivo . particles are DAB deposits internalized by micropinocytosis . magnified fields to right show particles engulfed in macropinosomes . Note layered structure of particles , macropinosome that is open to ventricular space in Mpdz - / - section . Scale bars , 1 μm ; insets , 100 nm . B . Mean number of engulfed DAB particles per in Mpdz + / + Mpdz - / - mice ( mean ± SD , n = 22 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. TEM images of CPEC sections from lateral ventricle villi of two Mpdz+/+ and two Mpdz-/- P14-P16 mice injected with HRP. The CPs were reacted with hydrogen peroxide and DAB ex-vivo. The dark particles are DAB deposits internalized by micropinocytosis. The magnified fields to the right show individual particles engulfed in macropinosomes. Note the layered structure of the particles, and the macropinosome that is open to the ventricular space in the Mpdz-/- section. Scale bars, 1 μm; insets, 100 nm. B. Mean number of engulfed DAB particles per cell in Mpdz+/+ and Mpdz-/- mice (mean ± SD, n=22).


C . A CPEC section showing engulfment of a DAB particle by its basal ruffles in magnified field . Scale bars in panels C D , 1 μm ; insets , 200 nm . D . A CPEC section showing a preponderance of macropinosomes close to apical face of , a magnified field that contains several macropinosomes . E . Mean numbers of DAB - containing macropinosomes close to apical or basal sides of CPECs from Mpdz + / + or Mpdz - / - mice ( mean ± SD , n = 20 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A CPEC section showing the engulfment of a DAB particle by its basal ruffles in the magnified field. Scale bars in panels C and D, 1 μm; insets, 200 nm. D. A CPEC section showing a preponderance of macropinosomes close to the apical face of the cell, and a magnified field that contains several macropinosomes. E. Mean numbers of DAB-containing macropinosomes close to the apical or basal sides of CPECs from Mpdz+/+ or Mpdz-/- mice (mean ± SD, n=20).


A . Immunofluorescence of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + Mpdz - / - P14 - P16 mice immunolabeled by anti - LDLR . Scale bars , 50 ( top ) 25 ( bottom ) μm . B . Mean intensities ( normalized relative to highest recorded ) per in several LDLR - immunolabeled CP sections ( mean ± SD , n = 101 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- P14-P16 mice immunolabeled by anti-LDLR. Scale bars, 50 (top) and 25 (bottom) μm. B. Mean fluorescence intensities (normalized relative to the highest recorded intensity) per cell in several LDLR-immunolabeled CP sections (mean ± SD, n=101).


C . Immunoblots showing abundances of LDLR MPDZ in hCPECs transduced by MPDZ - targeting or non - targeting shRNA . Mean abundances are quantified by densitometry of LDLR bands , normalized relative to β - actin bands ( mean ± SD , n = 3 ) .

C. Immunoblots showing the abundances of LDLR and MPDZ in hCPECs transduced by MPDZ-targeting or non-targeting shRNA. Mean abundances are quantified by densitometry of the LDLR bands, normalized relative to the β-actin bands (mean ± SD, n=3).


D . of hCPECs transduced by either MPDZ or non - targeting ( Ctrl ) shRNA immunolabeled by anti - LDLR either before ( 0 min ) or after 8 min of constitutive endocytosis of LDLR . histograms below show mean intensities per hCPEC in each group , normalized relative to highest recorded ( mean ± SD , n = 41 ÷ 53 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fluorescence images of hCPECs transduced by either MPDZ or non-targeting (Ctrl) shRNA and immunolabeled by anti-LDLR either before (0 min) or after 8 min of constitutive endocytosis of LDLR. The histograms below show the mean fluorescence intensities per hCPEC in each cell group, normalized relative to the highest recorded intensity (mean ± SD, n=41÷53).


A . Mean in CSF of Mpdz + / + Mpdz - / - P14 - P21 mice ( mean ± SD , n = 2 ÷ 3 ) .

A. Mean protein concentration in the CSF of Mpdz+/+ and Mpdz-/- P14-P21 mice (mean ± SD, n=2÷3).


B . Volcano plot showing that contents in sera of P15 - P21 Mpdz + / + Mpdz - / - mice are similar to each other .

O O O O 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. Volcano plot showing that the protein contents in sera of P15-P21 Mpdz+/+ and Mpdz-/- mice are similar to each other.


C . A volcano plot showing that 23 are at least two - fold significantly more abundant in CSF of P15 - 21 Mpdz - / - mice ( red circles , names are indicated ) whereas only two were more abundant in CSF of Mpdz + / + mice ( green circles ) . CSF composition was analyzed in 3 Mpdz - / - 3 Mpdz + / + mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A volcano plot showing that 23 proteins are at least two-fold significantly more abundant in the CSF of P15-21 Mpdz-/- mice (red circles, protein names are indicated) whereas only two proteins were more abundant in the CSF of Mpdz+/+ mice (green circles). The CSF composition was analyzed in 3 Mpdz-/- and 3 Mpdz+/+ mice.


D . of 10 μm - thick sections from lateral ventricle CP villi of Mpdz + / + Mpdz - / - mice , immunolabeled for Nkcc1 . areas in marked squares are magnified below . Scale bars , top , 50 μm , bottom 20 μm .

D. Images of 10 μm-thick sections from lateral ventricle CP villi of Mpdz+/+ and Mpdz-/- mice, immunolabeled for Nkcc1. The areas in the marked squares are magnified below. Scale bars, top, 50 μm, bottom 20 μm.


E . Immunoblots of LDLR Mpdz , with β - actin as loading control . MPDZ immunoblot shows efficiency of shRNA - mediated knockdown . Bands from three immunoblots were quantified by normalization to β - actin bands ( mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O 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. Immunoblots of LDLR and Mpdz, with β-actin as loading control. The MPDZ immunoblot shows the efficiency of the shRNA-mediated knockdown. Bands from three immunoblots were quantified by normalization to β-actin bands (mean ± SD).


( A ) Immunoblots of mitochondrial fractions from siRNA ‐ transfected HEK293T ± CCCP .

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

(A) Immunoblots of mitochondrial fractions from siRNA‐transfected HEK293T cells±CCCP.


( B ) Immunoblots against indicated proteases in siRNA ‐ transfected .

(B) Immunoblots against indicated proteases in siRNA‐transfected cells.


( C ) of full ‐ PINK1 ( n = 4 ) knockdown efficiency ( n = 2 ) compared with non ‐ targeting ( NT ) siRNA from experiments performed as in B . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase tensin homologue ‐ induced kinase 1 ; siRNA , short interfering .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 full‐length PINK1 (n=4) and knockdown efficiency (n=2) compared with non‐targeting (NT) siRNA from experiments performed as in B. *P0.05; **P0.01; ***P0.001. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( A ) Live confocal of siRNA ‐ transfected HEK293T ± CCCP stained with MitoTracker ( MTR ) Green Deep Red ( DR ) ; Scale bar , 5 μm .

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

(A) Live confocal micrographs of siRNA‐transfected HEK293T cells±CCCP stained with MitoTracker (MTR) Green and Deep Red (DR); Scale bar, 5 μm.


( B ) MTR DR / Green from as in A . N = 3 ; * P0 . 05 compared with NT siRNA .

O 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) MTR DR/Green ratio from images as in A. N=3; *P0.05 compared with NT siRNA.


( C ) Mitochondrial interconnectivity from as in A , N = 3 .

(C) Mitochondrial interconnectivity from images as in A, N=3.


( D ) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA ‐ transfected . Antimycin A with NT siRNA was used as a control . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; siRNA , short interfering .

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) Oxygen consumption in state 3 respiration in mitochondrial fractions from siRNA‐transfected cells. Antimycin A with NT siRNA was used as a control. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; siRNA, short interfering RNA.


( A ) Immunoblots of mitochondrial ( M ) ( C ) fractions from siRNA ‐ transfected HEK293T ± CCCP for 3 . 5 h , ± MG132 for final hour .

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 B-CONTROLLED_VAR O O O O

(A) Immunoblots of mitochondrial (M) and cytosolic (C) fractions from siRNA‐transfected HEK293T cells±CCCP for 3.5 h, ±MG132 for the final hour.


( B ) of 52 ‐ kDa PINK1 from A ( with MG132 ) , n = 3 ; * P0 . 05 ; * * P0 . 001 compared with NT siRNA .

O O O O O O O O O O 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) Quantification of 52‐kDa PINK1 from A (with MG132), n=3; *P0.05; **P0.001 compared with NT siRNA.


( C ) Mitochondria from siRNA ‐ transfected treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate ( Resp . mix ) for 30 min at 37 ° C . al hour .

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

(C) Mitochondria from siRNA‐transfected cells treated with 10 μM CCCP or dimethylsulphoxide were incubated with or without respiration substrate (Resp. mix) for 30 min at 37°C.al hour.


( A ) Mitochondria from CCCP ‐ treated or siRNA ‐ transfected HEK293T were incubated with increasing concentrations of proteinase K ( PK ) .

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

(A) Mitochondria from CCCP‐treated or siRNA‐transfected HEK293T cells were incubated with increasing concentrations of proteinase K (PK).


( B ) Mitochondria from CCCP ‐ treated or MPPβ knockdown were incubated ± 0 . 1 M Na2CO3 before supernatant ( S ) pellet ( P ) were separated by centrifugation . CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; MPP , mitochondrial processing peptidase ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase tensin homologue ‐ induced kinase 1 ; siRNA , short interfering ; WB , western blot .

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

(B) Mitochondria from CCCP‐treated or MPPβ knockdown cells were incubated ±0.1 M Na2CO3 before supernatant (S) and pellet (P) were separated by centrifugation. CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; MPP, mitochondrial processing peptidase; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA; WB, western blot.


( A ) Live confocal of siRNA ‐ , GFP ‐ Parkin ‐ OCT ‐ DsRed ‐ transfected HEK293T ± CCCP ; Scale bars , 20 μm ( low magnification ) 5 μm ( high magnification ) .

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

(A) Live confocal micrographs of siRNA‐, GFP‐Parkin‐ and OCT‐DsRed‐transfected HEK293T cells ±CCCP; Scale bars, 20 μm (low magnification) and 5 μm (high magnification).


( B ) of transfected displaying a punctate mitochondrial pattern of GFP ‐ Parkin distribution , n = 3 ; * P0 . 05 ; * * P0 . 01 compared with NT siRNA .

O O O O O O O O O O O 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) Percentage of transfected cells displaying a punctate mitochondrial pattern of GFP‐Parkin distribution, n=3; *P0.05; **P0.01 compared with NT siRNA.


( C ) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA ‐ transfected ± CCCP .

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

(C) Immunoblots of endogenous Parkin in mitochondrial fractions from siRNA‐transfected cells ±CCCP.


( D ) Mitochondrial mass measured by flow cytometry of MTR Green in siRNA ‐ transfected treated ± CCCP for 24 h , n = 3 . A . U . , arbitrary unit ; CCCP , carbonyl cyanide m ‐ chlorophenyl hydrazone ; GFP , green ; MPP , mitochondrial processing peptidase ; MTR , MitoTracker ; NT , non ‐ targeting ; PARL , presenilin ‐ associated rhomboid ‐ like protease ; PINK1 , phosphatase tensin homologue ‐ induced kinase 1 ; siRNA , short interfering .

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 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

(D) Mitochondrial mass measured by flow cytometry of MTR Green fluorescence in siRNA‐transfected cells treated ±CCCP for 24 h, n=3. A.U., arbitrary unit; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; GFP, green fluorescent protein; MPP, mitochondrial processing peptidase; MTR, MitoTracker; NT, non‐targeting; PARL, presenilin‐associated rhomboid‐like protease; PINK1, phosphatase and tensin homologue‐induced kinase 1; siRNA, short interfering RNA.


( a ) Transverse slice of live E10 . 5 Venus : : HES5 homozygous knock - in mouse showing ventral HES5 domain in spinal cord ex vivo ( left panel ) ; Draq5 live stain with segmentation overlay ( right panel ) ; scale bar 30 μm .

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

(a) Transverse slice of live E10.5 Venus::HES5 homozygous knock-in mouse showing the ventral HES5 domain in spinal cord ex vivo (left panel); Draq5 live nuclear stain with nuclear segmentation overlay (right panel); scale bar 30 μm.


( a ) 20x snapshot of an ex - vivo slice culture of E10 . 5 spinal cord from Venus : : HES5 heterozygous knock - in mouse , transverse section ; delineated region ( blue ) correspond to data shown in ( b - c ) . D - dorsal , V - ventral . ( b ) Spatial profile of Venus : : HES5 averaged over 2 . 5hrs with 0 representing ventral end of kymograph ; black represents trend in Venus : : HES5 data across domain produced using an polynomial order 6 ( see Materials Methods ) . ( c ) Detrended spatial profile of Venus : : HES5 corresponding to data shown in ( b ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 20x snapshot of an ex-vivo slice culture of E10.5 spinal cord from Venus::HES5 heterozygous knock-in mouse, transverse section; delineated region (blue) correspond to data shown in (b-c). D- dorsal, V- ventral. (b) Spatial profile of Venus::HES5 intensity averaged over 2.5hrs with 0 distance representing the ventral end of kymograph; black line represents the trend in Venus::HES5 data across the domain produced using an polynomial order 6 (see Materials and Methods). (c) Detrended spatial profile of Venus::HES5 corresponding to data shown in (b).


( a ) Start : Finish Venus : : HES5 in E10 . 5 Venus : : HES5 heterozygous spinal cord slices treated with control ( DMSO ) Notch inhibitor DBZ ( 2 μM ) observed over 16h ; bars indicate mean standard deviation of DMS0 ( n = 3 experiments ) DBZ ( n = 4 experiments ) ; 2 - t - * * * * p = 0 . 0001 .

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

(a) Start:Finish Venus::HES5 intensity ratio in E10.5 Venus::HES5 heterozygous spinal cord slices treated with control (DMSO) and Notch inhibitor DBZ (2 μM) observed over 16h; bars indicate mean and standard deviation of DMS0 (n=3 experiments) and DBZ (n=4 experiments); 2-tailed t-test ****p=0.0001.


( a ) Transverse cryosection of E10 . 5 Venus : : HES5 spinal cord . Venus : : HES5 endogenous signal , OLIG2 - motorneuron progenitor marker , NGN2 - early marker of neuronal commitment ; scale bar 20 μm . ( b ) Relative intensities of Venus : : HES5 NGN2 in motorneuron interneuron progenitors ; bars show mean SD of at least 494 per domain from 5 slices in 2 experiments ; Kruskal - Wallis with Dunn ' s multiple comparison adjusted p - values * * p = 0 . 0032 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Transverse cryosection of E10.5 Venus::HES5 spinal cord. Venus::HES5 endogenous signal, OLIG2 - motorneuron progenitor marker, NGN2 - early marker of neuronal commitment; scale bar 20 μm. (b) Relative nuclear intensities of Venus::HES5 and NGN2 in motorneuron and interneuron progenitors; bars show mean and SD of at least 494 cells per domain from 5 slices in 2 experiments; Kruskal-Wallis with Dunn's multiple comparison test adjusted p-values **p=0.0032, ***p<0.001.


( a ) Detrended spatial profile of NGN2 : : mScarlet - I from transverse slice of E10 . 5 spinal cord from heterozygous knock - in mouse in ventral - dorsal direction ; red indicates motorneuron ( MN ) domain , blue interneuron domain ( IN ) .

(a) Detrended spatial profile of NGN2::mScarlet-I intensity from transverse slice of E10.5 spinal cord from heterozygous knock-in mouse in ventral-dorsal direction;red indicates motorneuron (MN) domain, blue interneuron domain (IN).


A Combined IF FISH of RAD54 ( IF ) telomeres ( FISH ) in ALT non - ALT lines . arrows indicate RAD54 foci that colocalize with telomeres . Scale bars = 10 µm B of data in A . A was counted positive if it contained 1 or more colocalization event between RAD54 telomere . At least 100 were counted per per repeat . For SaOS2 , NOS , SJSA1 , HeLa LT n = 3 . For Cal72 , U2OS , HuO9 n = 4 . Values shown are mean ± sd

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd


Combined IF FISH of RAD54 ( IF ) , PML ( IF ) telomeres ( FISH ) in ALT lines SaOS2 HuO9 . Scale bars = 10

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

Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µm


Western blot showing expression of TRF1 - FOK1 - mCherry fusion following doxycycline induction , both WT D450A ( nuclease dead ) . were treated with 40 ng / mL doxycycline for 16 hrs then 1µM Shield - 1 1µM 4 - OHT for 4

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

Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs


Combined IF FISH for RAD54 ( IF ) telomeres ( FISH ) in U2OS - TRF1 - FOK1 - WT or U2OS - TRF1 - FOK1 - D450A ( nuclease dead ) . were treated as in E immediately prior to . Scale bars = 10 µm of data from F . RAD54 foci were detected using particle in Fiji . At least a total of 300 were counted from 3 repeated experiments . Values shown are mean ± . Values were compared using Kruskal - Wallis followed by Dunn ' s multiple comparison . of data from F . RAD54 foci were detected using particle in Fiji . were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres . Values shown are mean ± of n = 3 biological replicates . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison .

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

Combined IF and DNA FISH for RAD54 (IF) and telomeres (FISH) in U2OS-TRF1-FOK1-WT or U2OS-TRF1-FOK1-D450A (nuclease dead) cells. Cells were treated as in E immediately prior to staining. Scale bars = 10 µm Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. At least a total of 300 cells were counted from 3 repeated experiments. Values shown are mean±sem. Values were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test. Quantification of data from F. RAD54 foci were detected using particle analysis in Fiji. Cells were counted as positive if they counted 8 or more RAD54 foci that colocalized with telomeres. Values shown are mean±sem of n=3 biological replicates. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Combined IF FISH for RAD51 ( IF ) telomeres ( FISH ) in SaOS2 or HuO9 . were transfected with 20nM siRAD54 # 2 for 48 hours immediately prior to . Scale bars = 10 µm of A . A was counted as positive it is contained at least 1 colocalization event between RAD51 telomeres . At least 100 were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison .

Combined IF and DNA FISH for RAD51 (IF) and telomeres (FISH) in SaOS2 or HuO9 cells. Cells were transfected with 20nM siRAD54#2 for 48 hours immediately prior to staining. Scale bars = 10 µm Quantification of A. A cell was counted as positive it is contained at least 1 colocalization event between RAD51 and telomeres. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


ChIP for telomeric associated with RAD51 . dot blot probed with DIG labeled telomere probe or Alu probe . HuO9 were transfected with 20nM siRAD54 # 2 , or 15µg myc - TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody ( Mock , siRAD54 conditions ) or myc antibody ( myc - TRF2 positive control ) . of ( CCCTAA ) 4 dot blot performed in C . IP values were normalized to loading ( IP / input ) then normalized to mock control . Values shown are mean ± sd of n = 3 biological replicates . Values were compared using two - unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

ChIP for telomeric DNA associated with RAD51. DNA dot blot probed with DIG labeled telomere probe or Alu probe. HuO9 cells were transfected with 20nM siRAD54 #2, or 15µg myc-TRF2 for 72 hours prior to immunoprecipitation with RAD51 antibody (Mock, siRAD54 conditions) or myc antibody (myc-TRF2 positive control). Quantification of (CCCTAA)4 dot blot performed in C. IP values were normalized to loading (IP/input) and then normalized to mock control. Values shown are mean±sd of n=3 biological replicates. Values were compared using two-tailed unpaired student's t-test.


Western blot of samples used for ChIP in C - D .

Western blot of samples used for ChIP in C-D.


Combined IF FISH for GFP ( IF ) telomeres ( FISH ) in SaOS2 HuO9 . were forward transfected with Polη - GFP , then after 24 hours transfected with 20nM siRAD54 # 2 . were stained for GFP telomeres 48 hours after siRNA transfection . Scale bars = 10 µm of data in F . GFP negative were excluded from . were considered positive if they showed 1 or more colocalization events between GFP ( Polη ) telomeres . At least 50 GFP positive were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Values were compared using standard two - way ANOVA followed by Sidak ' s multiple comparison .

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

Combined IF and DNA FISH for GFP (IF) and telomeres (FISH) in SaOS2 and HuO9 cells. Cells were forward transfected with Polη-GFP, and then after 24 hours transfected with 20nM siRAD54#2. Cells were stained for GFP and telomeres 48 hours after siRNA transfection. Scale bars = 10 µm Quantification of data in F. GFP negative cells were excluded from analysis. Cells were considered positive if they showed 1 or more colocalization events between GFP (Polη) and telomeres. At least 50 GFP positive cells were counted per condition per repeat, n=3. Values shown are mean±sd. Values were compared using standard two-way ANOVA followed by Sidak's multiple comparison test.


Representative of combined EdU ( Click - it ) FISH for telomeres . HuO9 or SaOS2 were transfected with 20nM siRAD54 # 2 ( representative for siRAD54 # 1 not shown ) for 48 hours followed by a 1 . 5 hr of EdU . Pan - EdU stain represents S - phase . arrows indicate non - S - phase containing EdU colocalizing with telomeres . Scale bars = 10 µm of data shown in A . HuO9 or SaOS2 were mock transfected or transfected with 20nM siRAD54 # 1 or 20nM siRAD54 # 2 for 48 hours followed by a 1 . 5 hr of EdU . with pan - EdU signal or greater than 8 EdU foci were excluded from as S - phase . Non - S - phase were considered positive if they contained at least one EdU foci colocalizing with telomeres . Data were normalized to mock condition for each repeat . At least 100 non - S - phase were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Dunnett ' s multiple comparison .

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

Representative images of combined EdU staining (Click-it) and DNA FISH for telomeres. HuO9 or SaOS2 cells were transfected with 20nM siRAD54#2 (representative images for siRAD54#1 not shown) for 48 hours followed by a 1.5 hr pulse of EdU. Pan-nuclear EdU stain represents S-phase cells. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in A. HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. Cells with pan-nuclear EdU signal or greater than 8 EdU foci were excluded from analysis as S-phase cells. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.


Representative dot blot from C - circle on HuO9 , either mock transfected or transfected with 20nM siRAD54 # 2 for 48 hours . of C - circle in C . Signal was quantified with densitometry , background ( - Φ29 ) was subtracted signal was normalized to mock . N = 5 biological replicates values shown are mean ± sd . Conditions were analyzed using unpaired two - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative DNA dot blot from C-circle assay on HuO9 cells, either mock transfected or transfected with 20nM siRAD54#2 for 48 hours. Quantification of C-circle assay in C. Signal was quantified with densitometry, background (- Φ29) was subtracted and signal was normalized to mock. N=5 biological replicates values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.


Representative of combined IF FISH for PML ( IF ) telomeres ( FISH ) . HuO9 or SaOS2 were mock transfected or transfected with 20nM siRAD54 # 2 for 48 hours . arrows indicate large colocalization events . Blue arrows indicate small colocalization events . Scale bars = 10 µm HuO9 or SaOS2 were mock transfected or transfected with 20nM siRAD54 # 1 or 20nM siRAD54 # 2 for 48 hours . were considered positive if they contained 1 large or 3 small colocalization events . At least 100 were counted per condition per repeat , n = 3 . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Dunnett ' s multiple comparison .

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

Representative images of combined IF and DNA FISH for PML (IF) and telomeres (FISH). HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#2 for 48 hours. White arrows indicate large colocalization events. Blue arrows indicate small colocalization events. Scale bars = 10 µm Quantification HuO9 or SaOS2 cells were mock transfected or transfected with 20nM siRAD54#1 or 20nM siRAD54#2 for 48 hours. Cells were considered positive if they contained 1 large or 3 small colocalization events. At least 100 cells were counted per condition per repeat, n=3. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Dunnett's multiple comparison test.


Representative of combined EdU ( Click - it ) FISH for telomeric . HuO9 were forward transfected with 2µg empty vector ( EV ) , RAD54 - WT , RAD54 - K189R , or S49E construct . After 24 hours , were transfected with 20 nM siRAD54 # 2 for 48 hours followed by a 1 . 5 hr of EdU . arrows indicate non - S - phase containing EdU colocalizing with telomeres . Scale bars = 10 µm of data shown in G . Non - S - phase were considered positive if they contained at least one EdU foci colocalizing with telomeres . Data were normalized to EV - Mock condition for each repeat . At least 100 non - S - phase were counted per condition per repeat , n = 4 for EV , WT , S49E , n = 3 for K189R . Values shown are mean ± sd . Data were analyzed using two - way ANOVA followed by Sidak ' s multiple comparison .

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

Representative images of combined EdU staining (Click-it) and DNA FISH for telomeric DNA. HuO9 cells were forward transfected with 2µg empty vector (EV), RAD54-WT, RAD54-K189R, or S49E construct. After 24 hours, cells were transfected with 20 nM siRAD54#2 for 48 hours followed by a 1.5 hr pulse of EdU. White arrows indicate non-S-phase cells containing EdU colocalizing with telomeres. Scale bars = 10 µm Quantification of data shown in G. Non-S-phase cells were considered positive if they contained at least one EdU foci colocalizing with telomeres. Data were normalized to EV-Mock condition for each repeat. At least 100 non-S-phase cells were counted per condition per repeat, n=4 for EV, WT, S49E, n=3 for K189R. Values shown are mean±sd. Data were analyzed using two-way ANOVA followed by Sidak's multiple comparison test.


Representative of combined IF FISH for BLM ( IF ) telomeres ( FISH ) . HuO9 were transfected with 20nM siRAD54 # 2 for 48 hours . Scale bars = 10 µm of data shown in A . were counted positive if they contained at least 1 colocalization event between BLM telomeres . Data were normalized to mock condition of each repeat . At least 100 per condition per repeat were counted , n = 3 . Values shown are mean ± sd . Conditions were analyzed using unpaired two - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of combined IF and DNA FISH for BLM (IF) and telomeres (FISH). HuO9 cells were transfected with 20nM siRAD54#2 for 48 hours. Scale bars = 10 µm Quantification of data shown in A. Cells were counted positive if they contained at least 1 colocalization event between BLM and telomeres. Data were normalized to mock condition of each repeat. At least 100 cells per condition per repeat were counted, n=3. Values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.


Representative of IF - FISH for MUS81 telomeres . HuO9 were transfected with 20nM siRAD54 # 2 for 48 hours prior to . Scale bars = 10 µm of data shown in C . were counted positive if they contained at least 1 colocalization event between MUS81 telomeres . At least 100 per condition were counted per repeat . n = 5 , values shown are mean ± sd . Conditions were analyzed using unpaired two - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of IF-DNA FISH for MUS81 and telomeres. HuO9 cells were transfected with 20nM siRAD54 #2 for 48 hours prior to staining. Scale bars = 10 µm Quantification of data shown in C. Cells were counted positive if they contained at least 1 colocalization event between MUS81 and telomeres. At least 100 cells per condition were counted per repeat. n=5, values shown are mean±sd. Conditions were analyzed using unpaired two-tailed student's t-test.


Representative of HuO9 SaOS2 chromosomes with or without an exchange event . Colocalization of green red signal indicates an exchange event .

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

Representative images of HuO9 and SaOS2 chromosomes with or without an exchange event. Colocalization of green and red signal indicates an exchange event.


of telomere sister chromatid exchange events . were transfected with 20nM of siRNA for 48 hours prior to addition of BrdU / BrdC . Metaphases were imaged by confocal microscopy each data point represents a metaphase . For each condition , there were 3 biological repeats with a total of at least 1300 chromosome ends quantified . Values shown are mean ± . Conditions were analyzed using two - way ANOVA followed by Sidak ' s .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 telomere sister chromatid exchange events. Cells were transfected with 20nM of siRNA for 48 hours prior to the addition of BrdU/BrdC. Metaphases were imaged by confocal microscopy and each data point represents a metaphase. For each condition, there were 3 biological repeats with a total of at least 1300 chromosome ends quantified. Values shown are mean±sem. Conditions were analyzed using two-way ANOVA followed by Sidak's test.


Representative of UFB detection for PICH in HuO9 . were transfected with 20nM of noted siRNAs for 72 hrs . Scale bars = 10

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

Representative images of UFB detection staining for PICH in HuO9 cells. Cells were transfected with 20nM of noted siRNAs for 72 hrs. Scale bars = 10 µm


of data shown in G . Anaphases were scored from 4 independent experiments ( mock ) , or 3 independent experiments ( remaining conditions ) . Anaphases were considered positive for UFB if there was PICH between DAPI bodies . Single knockdown conditions had > 50 anaphases scored . Combined knockdown conditions had > 88 anaphases scored . Values shown are mean ± sd . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison . of data shown in G . Number of PICH stained UFBs counted per anaphase . Values shown are total over 3 independent experiments , shown mean ± . Total number of anaphases per condition were as follows : Mock n = 134 , siBLM / siSLX4 n = 90 , siRAD54 / siSLX4 n = 96 , siRAD54 / siBLM / siSLX4 n = 88 . Conditions were compared using Kruskal - Wallis followed by Dunn ' s multiple comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 data shown in G. Anaphases were scored from 4 independent experiments (mock), or 3 independent experiments (remaining conditions). Anaphases were considered positive for UFB if there was PICH staining between DAPI bodies. Single knockdown conditions had >50 anaphases scored. Combined knockdown conditions had >88 anaphases scored. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of data shown in G. Number of PICH stained UFBs counted per anaphase. Values shown are total counts over 3 independent experiments, shown mean±sem. Total number of anaphases per condition were as follows: Mock n=134, siBLM/siSLX4 n=90, siRAD54/siSLX4 n=96, siRAD54/siBLM/siSLX4 n=88. Conditions were compared using Kruskal-Wallis test followed by Dunn's multiple comparison test.


Representative of UFB for TRF2 in HuO9 . were transfected with 20nM siRAD54 # 2 alone or cotransfected siRAD54 # 2 siSLX4 for 72 hours prior to . Scale bars = 10

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

Representative images of UFB staining for TRF2 in HuO9 cells. Cells were transfected with 20nM siRAD54#2 alone or cotransfected siRAD54#2 and siSLX4 for 72 hours prior to staining. Scale bars = 10 µm


of data shown in J . Anaphases were scored from 3 independent experiments . A total of 90 anaphases were scored per condition . Values shown are mean ± sd . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison . of micronuclei from HuO9 transfected with siRAD54 # 2 alone or siRAD54 # 2 cotransfected with siSLX4 for 48 hours . Micronuclei containing telomeric were identified using DAPI telomere FISH . Micronuclei were normalized to number of nuclei counted . Values shown are mean ± sd of n = 3 biological replicates . Conditions were compared using one - way ANOVA followed by Dunnett ' s multiple comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 data shown in J. Anaphases were scored from 3 independent experiments. A total of 90 anaphases were scored per condition. Values shown are mean±sd. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test. Quantification of micronuclei from HuO9 cells transfected with siRAD54#2 alone or siRAD54#2 cotransfected with siSLX4 for 48 hours. Micronuclei containing telomeric DNA were identified using DAPI and telomere FISH. Micronuclei were normalized to the number of nuclei counted. Values shown are mean±sd of n=3 biological replicates. Conditions were compared using one-way ANOVA followed by Dunnett's multiple comparison test.


( A ) Immunostaining of API in vacuole of SEY6210 ( wild - type ) showing a dispersed pattern .

(A) Immunostaining of API in the vacuole of SEY6210 (wild-type) cell showing a dispersed pattern.


( B - D ) Immunostaining of TVY1 ( pep4 ) showing API in Cvt . arrow points to a Cvt . Higher magnification are shown in C D .

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

(B-D) Immunostaining of TVY1 (pep4) cells showing API in the Cvt complex. The arrow points to a Cvt complex. Higher magnification images are shown in C and D.


E ) Freeze - substitution fixation image of Cvt in TVY1 .

E) Freeze-substitution fixation image of Cvt complex in TVY1 cell.


( F ) Freeze - substitution fixation image of a Cvt in SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium .

O 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) Freeze-substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium.


( G ) Immunostaining of SEY6210 containing a 2μ APE1 plasmid grown in SD medium . N , Nucleus ; V , vacuole .

(G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.


( A B ) Immunostaining image showing Cvt vesicles ( marked with arrows in A ) in cytosol . Arrowhead double arrowheads show inner outer of Cvt vesicle , respectively .

O O O O O O O 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 and B) Immunostaining image showing Cvt vesicles (marked with arrows in A) in the cytosol. Arrowhead and double arrowheads show the inner and outer membrane of Cvt vesicle, respectively.


( C ) Freeze - substitution fixation image of Cvt vesicles ( marked with arrows ) .

(C) Freeze-substitution fixation image of Cvt vesicles (marked with arrows).


( D ) Immunostaining of Cvt vesicle ( marked with arrows ) in cytosol Cvt ( marked with double arrows ) in vacuole .

(D) Immunostaining of Cvt vesicle (marked with arrows) in the cytosol and Cvt body (marked with double arrows) in the vacuole.


( E F ) Freeze - substitution fixation image of Cvt bodies in vacuole ( marked with arrows ) . A Cvt vesicle is marked with an arrowhead .

O O O O O 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 and F) Freeze-substitution fixation image of Cvt bodies in the vacuole (marked with arrows). A Cvt vesicle is marked with an arrowhead.


( G H ) Freeze - substitution fixation image of Cvt vesicle contacting fusing to a vacuole . V , Vacuole ; VM , vacuolar .

O O 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 and H) Freeze-substitution fixation image of Cvt vesicle contacting and fusing to a vacuole. V, Vacuole; VM, vacuolar membrane.


Cvt complexes in TVY1 ( pep4 ) under nitrogen starvation conditions . Logarithmically growing in YEPD were transferred to nitrogen starvation medium ( SD [ - N ] ) at 30 ° C for 30 min ( A B ) or 60 min ( C D ) prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image showing a Cvt in an autophagosome ( marked by an arrow ) in cytosol . ( B ) Freeze - substitution fixation image showing a Cvt in an autophagic in vacuole . ( C ) Immunostaining of a Cvt in an autophagic in vacuole . ( D ) Immunostaining of Cvt vesicles ( marked with arrows ) in vacuole . AB , Autophagic ; AP , autophagosome ; V , vacuole .

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

Cvt complexes in TVY1 (pep4) cells under nitrogen starvation conditions. Logarithmically growing cells in YEPD were transferred to nitrogen starvation medium (SD[-N]) at 30°C for 30 min (A and B) or 60 min (C and D) and prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image showing a Cvt complex in an autophagosome (marked by an arrow) in the cytosol. (B) Freeze-substitution fixation image showing a Cvt complex in an autophagic body in the vacuole. (C) Immunostaining of a Cvt complex in an autophagic body in the vacuole. (D) Immunostaining of Cvt vesicles (marked with arrows) in the vacuole. AB, Autophagic body; AP, autophagosome; V, vacuole.


Transport of Cvt in SEY6210 ( wild - type ) harboring 2μ plasmid encoding API under growing nitrogen starvation conditions . Vegetative grown in SD medium were observed ( A - E ) . For starvation , logarithmically growing in SD were transferred to SD ( - N ) for 25 min ( F - H ) or 1 h ( I - K ) in presence of 1 mM PMSF . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Freeze - substitution fixation image of Cvt vesicle ( marked with an arrow ) . ( B ) Immunostaining of a Cvt vesicle ( marked with an arrow ) . ( C D ) Higher magnification image of Cvt vesicle . Arrowhead double arrowheads show inner outer of a Cvt vesicle , respectively . ( E ) Freeze - substitution fixation image of a sac enclosing small portion of a large Cvt ( marked with an arrow ) . ( F ) Freeze - substitution image of wrapping of a Cvt by isolation ( marked with an arrow ) . ( G ) Freeze - substitution image of a Cvt vesicle ( marked with an arrow ) fusing to a vacuole . ( H ) Freeze - substitution image of autophagic containing a Cvt . ( I ) Immunostaining image depicting wrapping of a Cvt . arrow marks enwrapping . ( J ) Immunostaining of an autophagosome containing a Cvt . ( K ) Immunostaining of an autophagic containing a Cvt in vacuole . V , vacuole .

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

Transport of Cvt complex in SEY6210 (wild-type) harboring 2μ plasmid encoding API under growing and nitrogen starvation conditions. Vegetative cells grown in SD medium were observed (A-E). For starvation, logarithmically growing cells in SD were transferred to SD(-N) for 25 min (F-H) or 1 h (I-K) in the presence of 1 mM PMSF. Samples were prepared for electron microscopy as described in Fig. 1. (A) Freeze-substitution fixation image of Cvt vesicle (marked with an arrow). (B) Immunostaining of a Cvt vesicle (marked with an arrow). (C and D) Higher magnification image of Cvt vesicle. Arrowhead and double arrowheads show the inner and outer membrane of a Cvt vesicle, respectively. (E) Freeze-substitution fixation image of a membrane sac enclosing small portion of a large Cvt complex (marked with an arrow). (F) Freeze-substitution image of the wrapping of a Cvt complex by the isolation membrane (marked with an arrow). (G) Freeze-substitution image of a Cvt vesicle (marked with an arrow) fusing to a vacuole. (H) Freeze-substitution image of autophagic body containing a Cvt complex. (I) Immunostaining image depicting the wrapping of a Cvt complex. The arrow marks the enwrapping membrane. (J) Immunostaining of an autophagosome containing a Cvt complex. (K) Immunostaining of an autophagic body containing a Cvt complex in the vacuole. V, vacuole.


Mutant allows for differentiation between vacuolar delivery by Cvt vesicles autophagosomes . ( A ) SEY6210 ( wild - type ) , cvt3 , cvt7 yeast were grown in SD to 1 OD600 U / ml transferred to SD ( - N ) . Aliquots were collected after 0 h ( + ) 4 h ( − ) incubation subjected to immunoblotting . positions of precursor ( pro ) mature ( m ) API are indicated . ( B ) cvt3 cvt7 were labeled for 10 min . After addition of cold cysteine methionine , were harvested , washed , resuspended in either SD or SD ( - N ) chased for indicated times . API was recovered by immunoprecipitation , resolved on SDS polyacrylamide gels , quantified using a phosphorimager ( STORM ; Molecular Dynamics , Sunnyvale , CA ) .

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

Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).


of Cvt in cvt3 mutant under vegetative starvation conditions . Vegetative were grown in SD medium at 30 ° C to log phase . For starvation , logarithmically growing in SD medium were transferred to SD ( - N ) for 5 h . Samples were prepared for electron microscopy as described in Fig . 1 . ( A ) Immunostaining image depicting Cvt in cytosol . Cvt vesicles are not detected in this . ( B ) Freeze - substitution fixation image showing Cvt in autophagosome ( marked with an arrow ) under starvation conditions , indicating that autophagy can still proceed . AP , Autophagosome ; N , nucleus ; V , vacuole .

Analysis of the Cvt complex in the cvt3 mutant under vegetative and starvation conditions. Vegetative cells were grown in SD medium at 30°C to log phase. For starvation, logarithmically growing cells in SD medium were transferred to SD(-N) for 5 h. Samples were prepared for electron microscopy as described in Fig. 1. (A) Immunostaining image depicting Cvt complex in the cytosol. Cvt vesicles are not detected in this strain. (B) Freeze-substitution fixation image showing Cvt complex in autophagosome (marked with an arrow) under starvation conditions, indicating that autophagy can still proceed. AP, Autophagosome; N, nucleus; V, vacuole.


Representative of combined IF for H3K27me3 or H2AK119ub ( green ) with FISH for Xist ( red ) in Xist - TetOP lines ( for clone 1 of each mutant type ) upon D2 in DOX conditions ; Blue - DAPI ; Scale bar : 10 µm .

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

Representative images of combined IF for H3K27me3 or H2AK119ub (green) with RNA FISH for Xist (red) in Xist-TetOP lines (for clone 1 of each mutant type) upon D2 in DOX conditions; Blue - DAPI staining; Scale bar: 10 µm.


Graph represents mean % + S . E . M . of Xist - coated X chromosomes enriched for H3K27me3 or H2AK119ub in different Xist - TetOP mutants ( for clone 1 of each mutant type ) from 2 - to - 4 independent experiments ; A minimum of 50 Xist - coated X chromosomes were counted per experiment ; Only p - values corresponding to significant differences from unpaired Student ' s t - comparing mutants to Xist FL are indicated as * ( p - value < 0 . 05 ) .

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

Graph represents the mean % + S.E.M. of Xist-coated X chromosomes enriched for H3K27me3 or H2AK119ub in the different Xist-TetOP mutants (for clone 1 of each mutant type) from 2-to-4 independent experiments; A minimum of 50 Xist-coated X chromosomes were counted per experiment; Only p-values corresponding to significant differences from unpaired Student's t-test comparing mutants to Xist FL are indicated as * (p-value < 0.05).


Top 20 hits from ChIRP - MS of Xist FL ; ranking was on fold - enrichment of Xist FL DOX versus Xist FL noDOX ; Weakly annotated isoforms with an Annotation score in UniprotKB < 3 ( out of 5 ) were excluded ; Fold - enrichment for Xist ΔB + C is also displayed for comparison ; green boxes correspond to as Xist interactors [ 13 ] ; in red ( RNF2 / RING1B ) represents a not found in Xist ΔB + C interactome ; in brown ( TRIM71 ) is less enriched in Xist ΔB + C than in Xist FL .

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

Top 20 protein hits from the ChIRP-MS of Xist FL; The ranking was based on fold-enrichment of Xist FL DOX versus Xist FL noDOX; Weakly annotated protein isoforms with an Annotation score in UniprotKB < 3 (out of 5) were excluded; Fold-enrichment for Xist ΔB+C is also displayed for comparison; Light green boxes correspond to proteins as Xist interactors [13]; protein in red (RNF2/RING1B) represents a protein not found in the Xist ΔB+C interactome; Protein in light brown (TRIM71) is less enriched in Xist ΔB+C than in Xist FL.


Scatter plot displaying differences in peptide between Xist FL Xist ΔB + C for 74 out of 81 Xist - interactors with a minimum of fold - change of 2 . 5 in Xist FL or Xist ΔB + C ; Shown is log2 fold change of peptide of each mutant in DOX conditions compared with Xist FL in noDOX conditions ; retrieved by both Xist FL Xist ΔB + C ChIRPs with a proposed role in XCI such as SPEN , RBM15 , WTAP , YTHDC1 hnRNPU are indicated ; brown dots mark more represented in Xist FL than in Xist ΔB + C ChIRPs , while red dots display which are only retrieved by Xist FL ChIRP .

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

Scatter plot displaying the differences in peptide counts between Xist FL and Xist ΔB+C for the 74 out of 81 Xist-interactors with a minimum of fold-change of 2.5 in Xist FL or Xist ΔB+C; Shown is the log2 fold change of peptide counts of each mutant in DOX conditions compared with the Xist FL in noDOX conditions; proteins retrieved by both Xist FL and Xist ΔB+C ChIRPs with a proposed role in XCI such as SPEN, RBM15, WTAP, YTHDC1 and hnRNPU are indicated; light brown dots mark proteins more represented in Xist FL than in Xist ΔB+C ChIRPs, while red dots display proteins which are only retrieved by Xist FL ChIRP.


Plots showing H3K27me3 H2AK119ub accumulation over X chromosome in Xist FL Xist ΔB + C with upon DOX induction at day 2 ( D2 ) of differentiation ; Each dot represents a single 10Kb window its enrichment relative to noDOX condition ; Black is a loess regression on windows ; Xist locus is represented by a blue long , active genes by green lines .

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

Plots showing H3K27me3 and H2AK119ub accumulation over the X chromosome in Xist FL and Xist ΔB+C with upon DOX induction at day 2 (D2) of differentiation; Each dot represents a single 10Kb window and its enrichment relative to noDOX condition; Black line is a loess regression on all windows; Xist locus is represented by a blue long line, active genes by green lines.


Violin plots quantifying H3K27me3 H2AK119ub enrichment over intergenic regions , active promoters active gene bodies in X chromosome in Xist FL Xist ΔB + C lines at D2 upon DOX induction ; Shown is log2 fold change of DOX vs noDOX conditions ; violin plots represent distribution of values , horizontal band is median , lower upper hinges correspond to 25th 75th percentiles ; n = indicates number of regions / genes analyzed ; p - values were calculated using paired Wilcoxon , comparing Xist FL Xist ΔB + C lines .

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

Violin plots quantifying H3K27me3 and H2AK119ub enrichment over intergenic regions, active promoters and active gene bodies in the X chromosome in Xist FL and Xist ΔB+C cell lines at D2 upon DOX induction; Shown is the log2 fold change of DOX vs noDOX conditions; violin plots represent the distribution of the values, the horizontal band is the median, the lower and upper hinges correspond to the 25th and 75th percentiles; n = indicates the number of regions/genes analyzed; p-values were calculated using paired Wilcoxon test, comparing Xist FL and Xist ΔB+C cell lines.