Fig 2A ) Evolution of rat weight of different treatment groups ( 12 animals in each group ) : control , L - BMAA ( 300 mg / kg / day during 5 consecutive days treated at weaning ) , VP2 . 51 ( 2 , 5 mg / kg during 15 days ) with a group treated at PT1 and another at PT30 , L - BMAA ( with the same treatment as the group of L - BMAA ) + VP2 . 51 ( with the same treatment groups VP2 . 51 ) with a group treated at PT1 and another at PT30 . B ) . Evolution of neurological evaluation in the treatment assay with VP2 . 51 ( 12 animals in each group ) . The control group and the groups treated exclusively with VP2 . 51 ( at PT1 and PT30 ) are not shown because its assessment coincides with the horizontal axis . The time when the inhibitor is added is indicated with arrows .

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Fig 2A) Evolution of rat weight of different treatment groups (12 animals in each group): control, L-BMAA (300 mg/kg/day during 5 consecutive days treated at weaning), VP2.51 (2,5 mg/kg during 15 days) with a group treated at PT1 and another at PT30, L-BMAA (with the same treatment as the group of L-BMAA) + VP2.51 (with the same treatment groups VP2.51) with a group treated at PT1 and another at PT30.B). Evolution of neurological evaluation in the treatment assay with VP2.51 (12 animals in each group). The control group and the groups treated exclusively with VP2.51 (at PT1 and PT30) are not shown because its assessment coincides with the horizontal axis. The time when the inhibitor is added is indicated with arrows.


Fig 3A ) Analysis of LC3 - II and P - mTOR by western - blot in the motor cortex of control and treated animals . Proteins were detected by Western blotting using β - tubulin for calibration of sample loading . The protein bands were quantified by Image Multigauge v3 . 0 software . Immunoblots are representative of one of three experiments with similar results . These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM . * , * * Indicate differences of p - value < 0 . 05 < 0 . 01 with the control group , respectively . # , # # # indicate differences of p - value < 0 . 05 < 0 . 001 with the group of animals treated with L - BMAA , respectively . B ) Analysis of LC3 - II and P - mTOR by western - blot in the lumbar spinal cord of control and treated animals . Proteins were detected by Western blotting using β - tubulin for calibration of sample loading . The protein bands were quantified by Image Multigauge v3 . 0 software . Immunoblots are representative of one of three experiments with similar results . These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM . * , * * * Indicate differences of p - value < 0 . 05 < 0 . 001 with the control group , respectively . # # Indicate differences of p - value < 0 . 01 with the group of animals treated with L - BMAA .

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

Fig 3A) Analysis of LC3-II and P-mTOR by western-blot in the motor cortex of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, ** Indicate differences of p-value <0.05 <0.01 with the control group, respectively. #, ### indicate differences of p-value <0.05 <0.001 with the group of animals treated with L-BMAA, respectively.B) Analysis of LC3-II and P-mTOR by western-blot in the lumbar spinal cord of control and treated animals. Proteins were detected by Western blotting using β-tubulin for calibration of sample loading. The protein bands were quantified by Image Multigauge v3.0 software. Immunoblots are representative of one of three experiments with similar results. These results were quantitated in arbitrary units and were represented in the accompanying graphs as mean ± SEM. *, *** Indicate differences of p-value <0.05 <0.001 with the control group, respectively. ## Indicate differences of p-value <0.01 with the group of animals treated with L-BMAA.


Fig 4B ) Cell viability measured in the experiment with or without inhibitors .

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Fig 4B) Cell viability measured in the experiment with or without inhibitors.


Figure 1 ( A ) Relaxed DNA ( 3 nM ) , condensin ( 0 , 60 , 120 , 240 nM ) and Topo I ( 1 unit ) were mixed and incubated with or without ATP ( 1 mM ) at 30ºC for 30 min . DNA electrophoresis contained 0 . 1 µg / ml chloroquine . In all gels , N denotes nicked circles ; Lkº , the input Lk distribution of relaxed DNA ; and LkC , the Lk distribution restrained by condensin . ( B ) 2D - gel of the preceding samples in lane 1 ( relaxed DNA , R ) , and lanes 5 and 6 ( condensin 120 nM ± ATP ) . Electrophoresis contained 0 . 1 and 1 µg / ml chloroquine in the first and second dimension , respectively . The histogram shows the relative intensity ( i ) of individual topoisomers of the Lk distributions resolved in lane 1 ( green ) and lane 6 ( red ) . Lkº and LkC denote the midpoint of each Lk distribution ; and ∆ Lk , the difference ( Lk units ) between them . ( C ) Experiment conducted as in A , but reducing the concentration of DNA ( 0 . 3 nM ) and condensin ( 0 , 1 . 5 , 3 , 6 nM ) . DNA electrophoresis contained 0 . 4 µg / ml chloroquine . ( D ) 2D - gel of the preceding sample in lane 1 ( relaxed DNA , R ) , and lanes 5 and 6 ( condensin 3 nM ± ATP ) . Electrophoresis contained 0 . 4 and 1 µg / ml chloroquine in the first and second dimension , respectively . The histogram shows relative Lk intensities ( i ) of lanes 1 and 6 , indicating Lkº , LkC and ∆ Lk . ( E ) Experiment conducted as in A , but mixing DNA ( 1 nM ) with intermediate condensin concentrations ( 10 to 100 nM ) . DNA electrophoresis contained 0 . 2 µg / ml chloroquine . The length of DNA ( bp ) available per condensin complex in each reaction is indicated .

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

Figure 1(A) Relaxed DNA (3 nM), condensin (0, 60, 120, 240 nM) and Topo I (1 unit) were mixed and incubated with or without ATP (1 mM) at 30ºC for 30 min. DNA electrophoresis contained 0.1 µg/ml chloroquine. In all gels, N denotes nicked circles; Lkº, the input Lk distribution of relaxed DNA; and LkC, the Lk distribution restrained by condensin. (B) 2D-gel of the preceding samples in lane 1 (relaxed DNA, R), and lanes 5 and 6 (condensin 120 nM ± ATP). Electrophoresis contained 0.1 and 1 µg/ml chloroquine in the first and second dimension, respectively. The histogram shows the relative intensity (i) of individual topoisomers of the Lk distributions resolved in lane 1 (green) and lane 6 (red). Lkº and LkC denote the midpoint of each Lk distribution; and ∆Lk, the difference (Lk units) between them. (C) Experiment conducted as in A, but reducing the concentration of DNA (0.3 nM) and condensin (0, 1.5, 3, 6 nM). DNA electrophoresis contained 0.4 µg/ml chloroquine. (D) 2D-gel of the preceding sample in lane 1(relaxed DNA, R), and lanes 5 and 6 (condensin 3 nM ± ATP). Electrophoresis contained 0.4 and 1 µg/ml chloroquine in the first and second dimension, respectively. The histogram shows relative Lk intensities (i) of lanes 1 and 6, indicating Lkº, LkC and ∆Lk.(E) Experiment conducted as in A, but mixing DNA (1 nM) with intermediate condensin concentrations (10 to 100 nM). DNA electrophoresis contained 0.2 µg/ml chloroquine. The length of DNA (bp) available per condensin complex in each reaction is indicated.


Figure 2 ( A ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I ( 1 unit ) were incubated at 30ºC for indicated time periods ( 0 to 90 min ) in the presence of different concentrations of ATP ( 1 , 0 . 25 , 0 . 1 mM ) . ( B ) Plot of ∆ Lk values ( mean ± SD , 3 technical replicates ) constrained in the preceding experiment . ( C ) DNA , condensin and Topo I ( mixed as in A ) were incubated at 30ºC without nucleotides for 10 min ( lane 1 ) , with ATP 1mM for 10 min ( lane 2 ) , AMPPNP 2 mM for 10 min ( lane 3 ) , and AMPPNP 2mM for 10 min followed by ATP 1 mM for 10 min ( lane 4 ) . ( D ) DNA , condensin and Topo I ( mixed as in A ) were incubated at 30ºC without nucleotides for 10 min ( lane 1 ) , with Apyrase for 10 min ( lane 2 ) , Apyrase and ATP 1mM for 10 min ( lane 3 ) , ATP 1mM for 10 min ( lane 4 ) , no nucleotide for 10 min followed by Apyrase for 60 min ( lane 5 ) , ATP 1mM for 10 min followed by Apyrase for 60 min ( lane 6 ) . ( E ) DNA , condensin and Topo I ( mixed as in A ) were incubated at 30ºC without nucleotides for 20 min ( lane 1 ) , with ATP 1mM for 20 min ( lane 2 ) , AMPPNP 2 mM for 20 min ( lane 3 ) , ATP 1mM for 10 min followed by AMPPNP 2 mM for 10 min ( lane 4 ) . ( F ) 2D - gel of the samples in E ( lanes 1 to 4 ) and histogram of Lk intensities ( i ) of lanes 1 and 4 , indicating Lkº , LkC and ∆ Lk .

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

Figure 2(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for indicated time periods (0 to 90 min) in the presence of different concentrations of ATP (1, 0.25, 0.1 mM).(B) Plot of ∆Lk values (mean ±SD, 3 technical replicates) constrained in the preceding experiment.(C) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with ATP 1mM for 10 min (lane 2), AMPPNP 2 mM for 10 min (lane 3), and AMPPNP 2mM for 10 min followed by ATP 1 mM for 10 min (lane 4). (D) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 10 min (lane 1), with Apyrase for 10 min (lane 2), Apyrase and ATP 1mM for 10 min (lane 3), ATP 1mM for 10 min (lane 4), no nucleotide for 10 min followed by Apyrase for 60 min (lane 5), ATP 1mM for 10 min followed by Apyrase for 60 min (lane 6).(E) DNA, condensin and Topo I (mixed as in A) were incubated at 30ºC without nucleotides for 20 min (lane 1), with ATP 1mM for 20 min (lane 2), AMPPNP 2 mM for 20 min (lane 3), ATP 1mM for 10 min followed by AMPPNP 2 mM for 10 min (lane 4). (F) 2D-gel of the samples in E (lanes 1 to 4) and histogram of Lk intensities (i) of lanes 1 and 4, indicating Lkº, LkC and ∆Lk.


Figure 3 ( D ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I were mixed with / without nuclease P1 and incubated at 30ºC with no nucleotide for 30 min , ATP 1mM for 30 min , AMPPNP 2 mM for 30 min , ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min . ( E ) Experiment conducted as in D , but using ( - ) supercoiled DNA and without Topo I . Following the incubation with nucleotides for 30 min , nuclease P1 was added for 5 min .

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

Figure 3(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I were mixed with/without nuclease P1 and incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min.(E) Experiment conducted as in D, but using (-) supercoiled DNA and without Topo I. Following the incubation with nucleotides for 30 min, nuclease P1 was added for 5 min.


Figure 4 ( A ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I ( 1 unit ) were mixed with ss - oligos or ds - oligos ( 100 and 500 nM ) . Following incubation for 10 min at 30ºC , ATP ( 1 mM ) was added and incubations continued for 10 min . ( B ) Experiment as in A , but first adding ATP for 10 min and afterwards the oligonucleotides for 10 min . ( C ) Experiment as in B , but after the 10 min incubation with oligonucleotides , AMPPNP ( 2 mM ) was added and incubations continued for 10 min . ( D ) Relaxed DNA ( 0 . 3 nM ) , condensin ( 3 nM ) and Topo I ( 1 unit ) were incubated at 30ºC for 10 min . Reactions were then split into thirds to which NaCl concentration was raised to 50 , 150 and 300 mM . Salt - resistant condensin - DNA complexes were immobilized to His - Tag magnetic beads and the fractions of free ( F ) and bound ( B ) DNA recovered . The plot shows the fractions of bound DNA ( mean ± SD , 3 technical replicates ) . ( E ) As in D , but containing AMPPNP ( 2 mM ) . ( F ) As in D , but containing ATP ( 1 mM ) . ( G ) As in D , but containing ATP ( 1 mM ) for 10 min followed by AMPPNP ( 2 mM ) for 10 min . ( H ) As in D , but containing ATP ( 1 mM ) for 10 min followed by Apyrase for 10 min .

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Figure 4(A) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were mixed with ss-oligos or ds-oligos (100 and 500 nM). Following incubation for 10 min at 30ºC, ATP (1 mM) was added and incubations continued for 10 min. (B) Experiment as in A, but first adding ATP for 10 min and afterwards the oligonucleotides for 10 min. (C) Experiment as in B, but after the 10 min incubation with oligonucleotides, AMPPNP (2 mM) was added and incubations continued for 10 min.(D) Relaxed DNA (0.3 nM), condensin (3 nM) and Topo I (1 unit) were incubated at 30ºC for 10 min. Reactions were then split into thirds to which NaCl concentration was raised to 50, 150 and 300 mM. Salt-resistant condensin-DNA complexes were immobilized to His-Tag magnetic beads and the fractions of free (F) and bound (B) DNA recovered. The plot shows the fractions of bound DNA (mean ±SD, 3 technical replicates). (E) As in D, but containing AMPPNP (2 mM). (F) As in D, but containing ATP (1 mM). (G) As in D, but containing ATP (1 mM) for 10 min followed by AMPPNP (2 mM) for 10 min. (H) As in D, but containing ATP (1 mM) for 10 min followed by Apyrase for 10 min.


Figure 5 ( B ) Relaxed DNA ( 0 . 3 nM ) and condensin ( 3 nM ) were mixed with Topo I or Topo II ( 1 unit ) . Upon addition of ATP ( 1 mM ) , incubations proceeded at 30ºC for 30 min . ( C ) DNA , condensin , and Topo II were mixed as in B . Upon addition of ATP ( 1 mM ) , incubations proceeded at 30ºC for 10 min . Topo I was added to one half of the mixtures and incubations continued for 10 min . ( D ) DNA , condensin , and Topo II were mixed as in B . Following incubation at 30ºC for 10 min with or without ATP , DNA was recovered and nicked to reveal the occurrence of DNA knots . K3 to K6 denote knots with 3 to 6 irreducible DNA crossings . ( F ) Nicked DNA ( 0 . 3 nM ) was mixed without or with condensin ( 3 nM ) in the presence of ATP ( 1 mM ) . Following incubations at 30ºC for 20 min , mixtures were supplemented with T4 DNA Ligase and Topo I ( as indicated ) and incubation continued for 10 min .

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Figure 5(B) Relaxed DNA (0.3 nM) and condensin (3 nM) were mixed with Topo I or Topo II (1 unit). Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 30 min. (C) DNA, condensin, and Topo II were mixed as in B. Upon addition of ATP (1 mM), incubations proceeded at 30ºC for 10 min. Topo I was added to one half of the mixtures and incubations continued for 10 min. (D) DNA, condensin, and Topo II were mixed as in B. Following incubation at 30ºC for 10 min with or without ATP, DNA was recovered and nicked to reveal the occurrence of DNA knots. K3 to K6 denote knots with 3 to 6 irreducible DNA crossings.(F) Nicked DNA (0.3 nM) was mixed without or with condensin (3 nM) in the presence of ATP (1 mM). Following incubations at 30ºC for 20 min, mixtures were supplemented with T4 DNA Ligase and Topo I (as indicated) and incubation continued for 10 min.


Figure 6 ( B ) Relaxed DNA ( 0 . 3 nM ) was mixed with condensin ( 3 nM ) . Following incubation in absence or presence of the indicated nucleotides ( ATP 1mM for 30 min , AMPPNP 2 mM for 30 min , ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min ) , TopA was added to one half of each reaction and Topo I to the other half . Incubations continued for 10 min . ( D ) Experiment conducted as in B but starting with ( - ) supercoiled DNA instead of relaxed DNA . Following incubation in presence of the indicated nucleotides ( as in B ) Topo I was added to one half of each mixture and incubations continued for 10 min . ( E ) Experiment conducted as in D but adding TopA instead of Topo I .

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

Figure 6(B) Relaxed DNA (0.3 nM) was mixed with condensin (3 nM). Following incubation in absence or presence of the indicated nucleotides (ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min), TopA was added to one half of each reaction and Topo I to the other half. Incubations continued for 10 min.(D) Experiment conducted as in B but starting with (-) supercoiled DNA instead of relaxed DNA. Following incubation in presence of the indicated nucleotides (as in B) Topo I was added to one half of each mixture and incubations continued for 10 min. (E) Experiment conducted as in D but adding TopA instead of Topo I.


Figure 7 ( A ) Relaxed DNA ( 0 . 3 nM ) , ∆ Ycg1 condensin ( 3 nM ) and Topo I were incubated at 30ºC with no nucleotide for 30 min , ATP 1mM for 30 min , AMPPNP 2 mM for 30 min , ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min . The plot ( mean ± SD , 3 technical replicates ) compares the ∆ Lk values restrained by the condensin holo - complex and the ∆ Ycg1 condensin . ( B ) Salt resistance of the ∆ Ycg1 condensin - DNA complex . Experiments were conducted as in Fig 4D - H . The plot ( mean ± SD , 3 technical replicates ) compares the fractions of DNA bound to the holo - complex and to the ∆ Ycg1 tetramer .

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

Figure 7(A) Relaxed DNA (0.3 nM), ∆Ycg1 condensin (3 nM) and Topo I were incubated at 30ºC with no nucleotide for 30 min, ATP 1mM for 30 min, AMPPNP 2 mM for 30 min, ATP for 20 min followed by AMPPNP for 10 min or ATP for 20 min followed by Apyrase for 10 min. The plot (mean ±SD, 3 technical replicates) compares the ∆Lk values restrained by the condensin holo-complex and the ∆Ycg1 condensin.(B) Salt resistance of the ∆Ycg1 condensin-DNA complex. Experiments were conducted as in Fig 4D-H. The plot (mean ±SD, 3 technical replicates) compares the fractions of DNA bound to the holo-complex and to the ∆Ycg1 tetramer.


Figure 1 The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating ( n = 192 plants ) , expanding ( n = 16 plants ) and mature ( n = 16 ) leaf developmental stage . Expression levels in wild - type plants transferred to mannitol - induced stress were compared to those transferred to control conditions at the same developmental stage . Data information : Data are presented as mean ± SEM , n = 4 independent experiments . FC = Fold change . * = FDR < 0 . 05 , Unpaired two - sided Student ' s t - test .

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

Figure 1 The expression of the 20 genes encoding TFs was measured 24 h after mannitol treatment during the proliferating (n = 192 plants), expanding (n = 16 plants) and mature (n = 16) leaf developmental stage. Expression levels in wild-type plants transferred to mannitol-induced stress were compared to those transferred to control conditions at the same developmental stage. Data information: Data are presented as mean ± SEM, n = 4 independent experiments. FC = Fold change. * = FDR < 0.05, Unpaired two-sided Student's t-test.


Figure 2A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . The first group contains TFs that reached the log2 ( FC ) threshold 40 min after mannitol treatment ( A ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 . A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the second group reached the threshold after 1 h ( B ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 . A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the third group after 2 h ( C ) The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 . A , B , C , D Based on a threshold of log2 [ fold change ( FC ) ] > 1 , the 20 TFs were categorized into four groups . the fourth group after 4 h ( D ) . The arrow indicates the initial upregulation of every group . Data information : Data are presented as mean ± SEM . n = 4 independent experiments . FDR values are available in Table EV1 .

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

Figure 2A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. The first group contains TFs that reached the log2(FC) threshold 40 min after mannitol treatment (A) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the second group reached the threshold after 1 h (B) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the third group after 2 h (C) The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1. A, B, C, D Based on a threshold of log2[fold change (FC)] > 1, the 20 TFs were categorized into four groups. the fourth group after 4 h (D). The arrow indicates the initial upregulation of every group. Data information: Data are presented as mean ± SEM. n = 4 independent experiments. FDR values are available in Table EV1.


Figure 3 C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network , the activators ( green ) ERF6 , ERF59 , ERF98 and the repressors ( red ) ERF8 and ERF9 . Color code represents FDR - corrected p - values with thresholds at FDR = 0 . 01 , 0 . 05 and 0 . 1 . Data information : In A , data are extrapolated from estimated averages , n = 3 independent experiments , FDR - corrected p - values < 0 . 1 ( mixed model analysis , user - defined Wald tests ) . The thickness of the arrows represents the FDR value . In B , data are presented as averages , n = 3 independent experiments . The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment . In C , data are represented as FDR - corrected p - values , n = 3 independent experiments .

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

Figure 3 C Heatmap of the significant regulations upon induction of overexpression of the five members of the core network, the activators (green) ERF6, ERF59, ERF98 and the repressors (red) ERF8 and ERF9. Color code represents FDR-corrected p-values with thresholds at FDR=0.01, 0.05 and 0.1. Data information: In A, data are extrapolated from estimated averages, n = 3 independent experiments, FDR-corrected p-values < 0.1 (mixed model analysis, user-defined Wald tests). The thickness of the arrows represents the FDR value. In B, data are presented as averages, n = 3 independent experiments. The intensity of the color of the arrows represents the strength of the regulation according to the TEA values and the thickness the FDR value of the nCounter Nanostring experiment. In C, data are represented as FDR-corrected p-values, n = 3 independent experiments.


Figure 4A At 22 days after stratification ( DAS ) , leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves ( n = 10 plants ) . The rosette area is presented relative to the corresponding wild type . B Average area of the individual leaves of rap2 . 6L and myb51 , two knock - out lines with a smaller average rosette area . C Rosette area of two or three independent GOF lines per TF ( calculated as the projected area or the sum of the leaves , n = 10 plants ) germinated and grown on DEX - containing medium . Measurements were performed at 22 DAS relative to the control line . " Independent line 1 " is the line with the highest overexpression level . D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS . Scale bar is 1 cm . E , F Pavement cell number and area of the third leaf at 22 DAS of LOF ( E ) that showed a significant rosette area phenotype . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . In E and F , data are presented as mean ± SEM , n = 3 independent experiments , . = P < 0 . 1 , * = P < 0 . 05 , * * = P < 0 . 01 , * * * = P < 0 . 001 ( Tukey ' s test ) . E , F Pavement cell number and area of the third leaf at 22 DAS of GOF lines ( F ) that showed a significant rosette area phenotype . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . In E and F , data are presented as mean ± SEM , n = 3 independent experiments , . = P < 0 . 1 , * = P < 0 . 05 , * * = P < 0 . 01 , * * * = P < 0 . 001 ( Tukey ' s test ) .

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

Figure 4A At 22 days after stratification (DAS), leaf series of the LOF lines were made and the rosette area was calculated as the sum of the area of all individual leaves (n = 10 plants). The rosette area is presented relative to the corresponding wild type.B Average area of the individual leaves of rap2.6L and myb51, two knock-out lines with a smaller average rosette area.C Rosette area of two or three independent GOF lines per TF (calculated as the projected area or the sum of the leaves, n = 10 plants) germinated and grown on DEX-containing medium. Measurements were performed at 22 DAS relative to the control line. "Independent line 1" is the line with the highest overexpression level.D A representative picture of the rosette of GOF lines with significant growth phenotypes in both independent lines at 22 DAS. Scale bar is 1 cm.E, F Pavement cell number and area of the third leaf at 22 DAS of LOF (E) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test). E, F Pavement cell number and area of the third leaf at 22 DAS of GOF lines (F) that showed a significant rosette area phenotype. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test). In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). In E and F, data are presented as mean ± SEM, n = 3 independent experiments, . = P<0.1, * = P<0.05, ** = P<0.01, *** = P<0.001 (Tukey's test).


Figure 5A The expression level of two GA - degradation and two GA - biosynthesis enzymes in expanding leaf tissue ( third leaf at 15 DAS ) of wild - type plants 20 min , 40 min , 1 h , 2 h , 4 h , 8 h , 12 h , 16 h , and 24 h after mannitol treatment . The fold changes ( FC ) were calculated relative to control conditions . Data information : In A , data are presented as mean ± SEM , n = 4 independent experiments , * = FDR < 0 . 05 ( unpaired two - sided Student ' s t - test ) B The expression of GA2 - OX6 and GA20 - OX1 in expanding leaf tissue ( third leaf - 15 DAS ) , 8 h after transfer to DEX - containing medium to induce overexpression . The FC was calculated relative to control conditions . Data information : In B , data are presented as mean ± SEM , n = 3 independent experiments , * = FDR < 0 . 1 ( mixed model analysis , user - defined Wald tests ) . C The effect of ERF6 , ERF9 , ERF98 and WRKY15 on the GA2 - OX6 promoter determined with transient expression assays . The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Data information : In C , data are presented as mean ± SEM , n = 3 independent experiments .

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

Figure 5A The expression level of two GA-degradation and two GA-biosynthesis enzymes in expanding leaf tissue (third leaf at 15 DAS) of wild-type plants 20 min, 40 min, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h after mannitol treatment. The fold changes (FC) were calculated relative to control conditions.Data information: In A, data are presented as mean ± SEM, n = 4 independent experiments, *= FDR<0.05 (unpaired two-sided Student's t-test)B The expression of GA2-OX6 and GA20-OX1 in expanding leaf tissue (third leaf - 15 DAS), 8 h after transfer to DEX-containing medium to induce overexpression. The FC was calculated relative to control conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, * = FDR < 0.1 (mixed model analysis, user-defined Wald tests). C The effect of ERF6, ERF9, ERF98 and WRKY15 on the GA2-OX6 promoter determined with transient expression assays. The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Data information: In C, data are presented as mean ± SEM, n = 3 independent experiments.


Figure 6A At 22 DAS , leaf series were made of the LOF lines ( n = 10 plants ) grown under control conditions or mild osmotic stress conditions , and the reduction under mild osmotic stress was calculated . The rosette area reduction is presented relative to the corresponding wild type . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) . B Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions . Data information : In B , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( mixed model , partial F - tests ) . C The rosette area of two or three independent GOF lines ( n = 10 plants ) germinated on DEX - containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line . " Independent line 1 " is the line with the highest overexpression level . Data information : In A and C , data are presented as mean ± SEM , n = 3 independent experiments , * = P < 0 . 05 ( Tukey ' s test ) .

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

Figure 6A At 22 DAS, leaf series were made of the LOF lines (n = 10 plants) grown under control conditions or mild osmotic stress conditions, and the reduction under mild osmotic stress was calculated. The rosette area reduction is presented relative to the corresponding wild type.Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test).B Leaf series of stz and erf11 mutants grown on control and mild osmotic stress conditions.Data information: In B, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (mixed model, partial F-tests). C The rosette area of two or three independent GOF lines (n = 10 plants) germinated on DEX-containing control or mild osmotic stress medium was measured at 22 DAS and the reduction by mild osmotic stress was calculated relative to the control line. "Independent line 1" is the line with the highest overexpression level. Data information: In A and C, data are presented as mean ± SEM, n = 3 independent experiments, *=P<0.05 (Tukey's test).


Figure 7A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 ( A ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments . A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF5 ( B ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments . A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes MYB51 ( C ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments . A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes , RAP2 . 6L ( D ) The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments . A , B , C , D , E The effect of the individual and the combination of two TFs on the expression of target genes ERF6 ( E ) . The relative luminescence was calculated relative to the control , 35S : : GUS ( n = 4 biological repeats ) . Green represents activation , red repression and gray absence of regulation . Data information : Data are presented as mean ± SEM , n = 3 independent experiments .

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

Figure 7A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF11 (A) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments. A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF5 (B) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments. A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes MYB51 (C) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments. A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes, RAP2.6L (D) The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments. A, B, C, D, E The effect of the individual and the combination of two TFs on the expression of target genes ERF6 (E). The relative luminescence was calculated relative to the control, 35S::GUS (n = 4 biological repeats). Green represents activation, red repression and gray absence of regulation. Data information: Data are presented as mean ± SEM, n = 3 independent experiments.


Figure 8The gain - of - function lines of all members of the core network were crossed either with each other , referred to as double cross , or with the control line ( GFP - GR ) , referred to as single cross . The projected rosette area of the F1 double and single cross and the GFP - GR , germinated and grown on DEX , was measured at 22 DAS . A representative picture of the single and double crosses is depicted . The values represent the relative increase or decrease in rosette size of the double crosses compared to their parental single crosses . The outcome of every double cross is classified into three groups : additive ( green connections ) , negative ( blue connection ) and synergistic ( orange connections ) phenotype . The precise measurements can be found in Fig EV2 .

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

Figure 8The gain-of-function lines of all members of the core network were crossed either with each other, referred to as double cross, or with the control line (GFP-GR), referred to as single cross. The projected rosette area of the F1 double and single cross and the GFP-GR, germinated and grown on DEX, was measured at 22 DAS. A representative picture of the single and double crosses is depicted. The values represent the relative increase or decrease in rosette size of the double crosses compared to their parental single crosses. The outcome of every double cross is classified into three groups: additive (green connections), negative (blue connection) and synergistic (orange connections) phenotype. The precise measurements can be found in Fig EV2.


Figure 1C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in control and PHSL1 - CDC20 cells . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of four independent experiments . D . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 strains containing the indicated mutations . Meiotic events were analyzed as in ( C ) . Data information : data are representative of two independent experiments .

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

Figure 1C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in control and PHSL1-CDC20 cells. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of four independent experiments.D. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 strains containing the indicated mutations. Meiotic events were analyzed as in (C). Data information: data are representative of two independent experiments.


Figure 2A . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and spo13 mutants . Top , time - lapse series . Bottom , meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . Data information : data are representative of two independent experiments . C . Imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP or Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ control cells and cells containing clb1 ∆ or cdc28 - as1 , which have been treated with 1NM - PP1 at metaphase I ( 8 h in SPM ) . Meiotic events ( bottom ) were analyzed as in ( A ) . Data information : data are representative of two independent experiments .

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

Figure 2A. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and spo13 mutants. Top, time-lapse series. Bottom, meiotic events were quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. Data information: data are representative of two independent experiments.C. Imaging of SPBs (Cnm67-RFP) and Spc72-GFP or Mpc70-GFP in PHSL1-CDC20 ama1∆ control cells and cells containing clb1∆ or cdc28-as1, which have been treated with 1NM-PP1 at metaphase I (8 h in SPM). Meiotic events (bottom) were analyzed as in (A). Data information: data are representative of two independent experiments.


Figure 3A . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus hrr25 - as . Cells were shifted from 24 to 36 ° C at 4 h in SPM and treated with 1NM - PP1 to inhibit Hrr25 - as . Top , time - lapse series . Bottom , Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I ( t = 0 ) . Data information : data are representative of three independent experiments . C . Imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP in PHSL1 - CDC20 ama1 ∆ spo13 ∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM . Mpc70 loading ( bottom ) was analyzed as in ( A ) . PEST - SPC72 expression delays the time of Mpc70 loading by 98 min ( 95 % CI , 67 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) . Data information : data are representative of three independent experiments .

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

Figure 3A. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus hrr25-as. Cells were shifted from 24 to 36°C at 4 h in SPM and treated with 1NM-PP1 to inhibit Hrr25-as. Top, time-lapse series. Bottom, Mpc70 loading to SPBs was quantified in cells synchronized in silico to SPB separation at entry into metaphase I (t = 0). Data information: data are representative of three independent experiments.C. Imaging of SPBs (Cnm67-RFP) and Mpc70-GFP in PHSL1-CDC20 ama1∆ spo13∆ control cells and cells expressing an additional copy of SPC72 from the PEST promoter at 4 h in SPM. Mpc70 loading (bottom) was analyzed as in (A). PEST-SPC72 expression delays the time of Mpc70 loading by 98 min (95% CI, 67-129; P < 0.0001; Welch's t-test). Data information: data are representative of three independent experiments.


Figure 4 ( B ) Analysis of cells containing wild - type Spo13 , Spo13 - mD , or Spo13 - mD - m2 . Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II ( t = 0 ) . Graphs show overlays of SPC72 - GFP and MPC70 - GFP strains . spo13 - mD delays Spc72 removal and Mpc70 loading by 30 min ( 95 % CI , 26 - 34 ; P < 0 . 0001 ) and 60 min ( 95 % CI , 55 - 66 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively . ( C ) Analysis of cells expressing Clb1 - mDK and different versions of Spo13 . Meiotic events were quantified as in ( B ) . spo13 - mD plus PEST - clb1 - mDK delays Spc72 removal and Mpc70 loading by 72 min ( 95 % CI , 63 - 80 ; P < 0 . 0001 ) and 114 min ( 95 % CI , 99 - 129 ; P < 0 . 0001 ; Welch ' s t - test ) , respectively .

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Figure 4(B) Analysis of cells containing wild-type Spo13, Spo13-mD, or Spo13-mD-m2. Meiotic events were quantified in cells synchronized in silico to SPB reduplication at entry into metaphase II (t = 0). Graphs show overlays of SPC72-GFP and MPC70-GFP strains. spo13-mD delays Spc72 removal and Mpc70 loading by 30 min (95% CI, 26-34; P < 0.0001) and 60 min (95% CI, 55-66; P < 0.0001; Welch's t-test), respectively. (C) Analysis of cells expressing Clb1-mDK and different versions of Spo13. Meiotic events were quantified as in (B). spo13-mD plus PEST-clb1-mDK delays Spc72 removal and Mpc70 loading by 72 min (95% CI, 63-80; P < 0.0001) and 114 min (95% CI, 99-129; P < 0.0001; Welch's t-test), respectively.


Figure 5C . Cdc5 interacts with Ime2 . Cdc5 - ha3 , Ime2 , and Spo13 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates from the indicated strains . Data information : data are representative of two independent experiments . D . Cdc5 ' s polo - box domain ( PBD ) binds to Ime2 . Ha3 - tagged versions of the wild - type PBD or the phosphopeptide - binding mutant PBD - FAA were expressed at t = 5 . 5 h in SPM in cells depleted of Cdc20 and Cdc5 . PBD - ha3 and Ime2 were detected by immunoblotting in whole - cell extracts and α - Ha immunoprecipitates . Data information : data are representative of two independent experiments . E . Cdc5 - dependent modification of Ime2 . PHSL1 - CDC20 ama1 ∆ control cells and cells containing spo13 ∆ or cdc5 - as were treated with CMK ( to inhibit Cdc5 - as ) at t = 8 h in SPM ( arrow heads ) . The panel shows immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5 - as . The arrow marks the modified form of Ime2 . Data information : data are representative of two independent experiments .

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Figure 5C. Cdc5 interacts with Ime2. Cdc5-ha3, Ime2, and Spo13 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates from the indicated strains. Data information: data are representative of two independent experiments.D. Cdc5's polo-box domain (PBD) binds to Ime2. Ha3-tagged versions of the wild-type PBD or the phosphopeptide-binding mutant PBD-FAA were expressed at t = 5.5 h in SPM in cells depleted of Cdc20 and Cdc5. PBD-ha3 and Ime2 were detected by immunoblotting in whole-cell extracts and α-Ha immunoprecipitates. Data information: data are representative of two independent experiments.E. Cdc5-dependent modification of Ime2. PHSL1-CDC20 ama1∆ control cells and cells containing spo13∆ or cdc5-as were treated with CMK (to inhibit Cdc5-as) at t = 8 h in SPM (arrow heads). The panel shows immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. Increased gel mobility of Ndt80 at t ≥ 10 h confirms inhibition of Cdc5-as. The arrow marks the modified form of Ime2. Data information: data are representative of two independent experiments.


Figure 6C , D . Immunoblot analysis of whole - cell extracts . C , sample from proliferating cells . ( C ) Protein levels in the cultures shown in ( A ) . ( D ) Protein levels in the cultures shown in ( B ) .

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Figure 6C, D. Immunoblot analysis of whole-cell extracts. C, sample from proliferating cells. (C) Protein levels in the cultures shown in (A). (D) Protein levels in the cultures shown in (B).


Figure 7A , B . Mitotic cdc20 - 3 cells were shifted to 36 ° C for 80 min . At t = 0 , cells were treated with estradiol to induce expression from the PEST promoter . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Spc72 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Spc72 - GFP at SPBs . ( A ) Expression of Ime2 - ∆ C induces removal of Spc72 from SPBs , which is inhibited by co - expression of Spo13 . ( B ) Expression of Cdc5 plus Ime2 - ∆ C causes rapid removal of Spc72 , which is inhibited by co - expression of Spo13 . Data information : data are representative of three independent experiments . C . Mitotic cdc20 - 3 cells containing PEST - CDC5 plus PEST - IME2 - ∆ C and / or PEST - NDT80 were shifted to 36 ° C for 30 min and subsequently treated with estradiol ( t = 0 ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Frame width , 19 μm . Bottom , quantification of cells with Mpc70 - GFP at SPBs . Data information : data are representative of two independent experiments .

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Figure 7A, B. Mitotic cdc20-3 cells were shifted to 36°C for 80 min. At t = 0, cells were treated with estradiol to induce expression from the PEST promoter. Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Spc72-GFP. Frame width, 19 μm. Bottom, quantification of cells with Spc72-GFP at SPBs. (A) Expression of Ime2-∆C induces removal of Spc72 from SPBs, which is inhibited by co-expression of Spo13. (B) Expression of Cdc5 plus Ime2-∆C causes rapid removal of Spc72, which is inhibited by co-expression of Spo13. Data information: data are representative of three independent experiments.C. Mitotic cdc20-3 cells containing PEST-CDC5 plus PEST-IME2-∆C and/or PEST-NDT80 were shifted to 36°C for 30 min and subsequently treated with estradiol (t = 0). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Frame width, 19 μm. Bottom, quantification of cells with Mpc70-GFP at SPBs. Data information: data are representative of two independent experiments.


Figure 8C . PHSL1 - CDC20 ama1 ∆ cdc28 - as2 control cells and cells containing spo13 ∆ or cdc5 - as were treated with 1Na - PP1 at t = 3 h in SPM to inhibit Cdc28 - as2 . At t = 9 h in SPM , Cdc28 - as2 was activated by washing cells with cSPM lacking 1Na - PP1 ( washout ) . cdc5 - as cells were washed with cSPM plus CMK to activate Cdc28 - as2 and inhibit Cdc5 - as . Top , time - lapse series from the imaging of Mpc70 - GFP and RFP - tubulin . The weak , nuclear signal originates from Ndt80 - GFP . Bottom , quantification of spindle formation and Mpc70 loading to SPBs . D . PHSL1 - CDC20 ama1 ∆ cells containing SPC72 , spc72 - 7 , or spc72 - 7 plus cdc5 - as were shifted from 24 to 36 ° C at t = 4 . 2 h in SPM ( to inactivate Spc72 - 7 , arrow heads ) and treated with CMK at t = 6 h in SPM ( to inhibit Cdc5 - as ) . Top , time - lapse series from the imaging of SPBs ( Cnm67 - RFP ) and Mpc70 - GFP . Bottom , quantification of SPB separation and Mpc70 loading to SPBs . Inhibition of Cdc5 - as in spc72 - 7 cells advances the time of Mpc70 loading by 119 min ( 95 % CI , 76 - 162 ; P < 0 . 0001 ; Welch ' s t - test ) . Data are representative of two independent experiments .

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

Figure 8C. PHSL1-CDC20 ama1∆ cdc28-as2 control cells and cells containing spo13∆ or cdc5-as were treated with 1Na-PP1 at t = 3 h in SPM to inhibit Cdc28-as2. At t = 9 h in SPM, Cdc28-as2 was activated by washing cells with cSPM lacking 1Na-PP1 (washout). cdc5-as cells were washed with cSPM plus CMK to activate Cdc28-as2 and inhibit Cdc5-as. Top, time-lapse series from the imaging of Mpc70-GFP and RFP-tubulin. The weak, nuclear signal originates from Ndt80-GFP. Bottom, quantification of spindle formation and Mpc70 loading to SPBs. D. PHSL1-CDC20 ama1∆ cells containing SPC72, spc72-7, or spc72-7 plus cdc5-as were shifted from 24 to 36°C at t = 4.2 h in SPM (to inactivate Spc72-7, arrow heads) and treated with CMK at t = 6 h in SPM (to inhibit Cdc5-as). Top, time-lapse series from the imaging of SPBs (Cnm67-RFP) and Mpc70-GFP. Bottom, quantification of SPB separation and Mpc70 loading to SPBs. Inhibition of Cdc5-as in spc72-7 cells advances the time of Mpc70 loading by 119 min (95% CI, 76-162; P < 0.0001; Welch's t-test). Data are representative of two independent experiments.


Figure 9A . Inhibition of Cdk1 at anaphase I . CDC20 - mAR and CDC20 - mAR cdc28 - as1 cells were released from the metaphase I - arrest with CuSO4 at 7 h in SPM ( t = 0 ) and treated with 1NM - PP1 at t = 40 min to inhibit Cdc28 - as1 ( arrows ) . Top , time - lapse series from the imaging of Mpc70 - GFP , SPBs ( Cnm67 - RFP ) , and nuclei ( TetR - RFP ) . Bottom , quantification of meiotic events .

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Figure 9A. Inhibition of Cdk1 at anaphase I. CDC20-mAR and CDC20-mAR cdc28-as1 cells were released from the metaphase I-arrest with CuSO4 at 7 h in SPM (t = 0) and treated with 1NM-PP1 at t = 40 min to inhibit Cdc28-as1 (arrows). Top, time-lapse series from the imaging of Mpc70-GFP, SPBs (Cnm67-RFP), and nuclei (TetR-RFP). Bottom, quantification of meiotic events.


Figure 1 ( D , E ) representative confocal micrographs and quantification of LC3 - II accumulation in CTNSWT and CTNS - / - cells in presence and absence of 25 nM bafilomycin ( BafA1 ) for 4 hrs , respectively ( n = 3 ) . Scale bars are 20 µm . ( F , G , H ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio and SQSTM1 protein levels in CTNSWT , CTNS - / - , and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h , respectively ( n = 3 ) . ( I , J ) Representative confocal micrographs and quantification of DQ - BSA and BSA in CTNSWT , CTNS - / - , and CTNSPatient cells , respectively ( n = 7 - 14 quantified images ) . Scale bars are 20 µm .

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Figure 1(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.


Figure 2 ( D ) List of metabolites that were shared and significantly altered in both the CTNS - / - and CTNSPatient cells compared to control cells ( n = 6 ) . ( I ) List of proteins that were significantly upregulated and downregulated in CTNS - / - cells compared to control cells ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , LIPA ; Lysosomal acid lipase , ACP2 ; Lysosomal acid phosphatase , CTSS ; Cathepsin S , CTSC ; Dipeptidyl peptidase , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , SORT1 ; Sortilin , CAT ; Catalase , CTSA ; Lysosomal protective protein , and SOD ; Superoxide dismutase .

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Figure 2(D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6).(I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.


Figure 3 ( B , C , D ) ROS production ( ROS / mg protein ) in CTNSWT , CTNS ‑ / - , and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG ( 4 hrs ) , respectively ( n = 3 ) . Representative confocal micrographs ( scale bars are 20 µm ) of caspase 3 / 7 activation in DMKG ( 2 mM ) - treated CTNSWT , CTNS ‑ / - , and CTNSPatient cells for 24 hrs ( n = 3 ) . ( H , I ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio in CTNSWT , CTNS ‑ / - , and CTNSPatient cells cultured in the presence or in the absence of BafA1 ( 25 nM ) and DMKG ( 2 mM ) for 4 hrs , respectively ( n = 3 ) .

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Figure 3(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).


Figure 4 ( E ) , Proteomic analysis of CTNS - / - cells treated with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide , respectively ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , GLUD1 ; GLUD2 ; Mitochondrial glutamate dehydrogenase 1 / 2 , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , GSTK1 ; Glutathione S - transferase kappa 1 , COX6B1 ; Cytochrome c oxidase subunit 6B1 , ACACA ; Acetyl - CoA carboxylase 1 - Biotin carboxylase , GLS ; Glutaminase kidney isoform , mitochondrial , CASP3 ; Caspase - 3 , NPC1 ; Niemann - Pick C1 protein .

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Figure 4(E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.


Figure 5 ( D , E ) Representative confocal micrographs and quantification of DQ - BSA in CTNSWT , and CTNS - / - cells upon treatment with bicalutamide . Scale bars are 20 µm ( n = 8 - 14 quantified images ) . ( F ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in CTNS - / - in the absence of the drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 6 ) , bicalutamide ( n = 6 ) , and a combination of cysteamine and bicalutamide ( n = 6 ) . ( G ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in CTNSPatient in the absence of the drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 4 ) , bicalutamide ( n = 4 ) , and a combination of cysteamine and bicalutamide ( n = 6 ) .

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Figure 5(D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images).(F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6). (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).


Figure 6 ( A ) Immunocytochemistry of patient - derived cystinotic tubuloids ( CNTSPatient - 1 and CTNSPatient - 2 ) and healthy kidney tissue - derived control tubuloids ( CNTSWT - 1 and CTNSWT - 2 ) for PAX8 , TP63 and F - actin . Scale bar = 100 µm . ( C , D ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in two different patient - derived cystinotic tubuloids in the absence of the drugs ( NT ) or upon treatment with cysteamine ( 100 μM ) , bicalutamide ( 35 μM ) or cysteamine ( 100 μM ) - bicalutamide ( 35 μM ) combination treatment ( n = 3 ) . ( E ) αKG levels measured in patient - derived cystinotic tubuloids ( CNTSPatient - 1 and CTNSPatient - 2 ) in the absence of the drugs ( NT ) or upon treatment with cysteamine , bicalutamide or cysteamine - bicalutamide combination treatment using metabolomics ( n = 3 ) . ( I ) Survival rates in ctns - / - zebrafish upon treatment with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos per group ) . ( K ) Hatching rates in surviving ctns - / - zebrafish evaluated at 72 and 96 hrs post fertilization ( hpf ) with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos per group ) .

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Figure 6(A) Immunocytochemistry of patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) and healthy kidney tissue-derived control tubuloids (CNTSWT-1 and CTNSWT-2) for PAX8, TP63 and F-actin. Scale bar = 100 µm.(C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3). (E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3). (I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).(K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).


Figure 1A Time course images of the hippocampal CA1 region of Siglechdtr / dtr mice after DT administration . Sections were stained with an anti - Iba1 antibody ( green ) and DAPI ( cyan ) . Arrow : live microglia with normal nucleus . Double arrow : apoptotic microglia with pyknotic nucleus . Arrowhead : microglial debris with no nucleus . Scale bar , 50 µm . B Number of apoptotic microglia with pyknotic or fragmented nucleus ( n = 5 animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . C Number of live microglia with normal nucleus ( n = 5 animals per group , one - way ANOVA with post hoc Tukey ' s test ) . D Number of pieces of microglial debris ( Iba1 + spheres with a diameter > 2 µm and no nucleus ) ( n = 5 animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr / dtr mice 2 days after DT administration . Sections were stained with an anti - Iba1 antibody ( green ) and DAPI ( cyan ) . A 3D image ( rightmost panel ) was reconstructed using Imaris software . Arrow : live microglia . Arrowhead : microglial debris . Scale bar , 10 µm .

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Figure 1A Time course images of the hippocampal CA1 region of Siglechdtr/dtr mice after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). Arrow: live microglia with normal nucleus. Double arrow: apoptotic microglia with pyknotic nucleus. Arrowhead: microglial debris with no nucleus. Scale bar, 50 µm. B Number of apoptotic microglia with pyknotic or fragmented nucleus (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). C Number of live microglia with normal nucleus (n = 5 animals per group, one-way ANOVA with post hoc Tukey's test). D Number of pieces of microglial debris (Iba1+ spheres with a diameter > 2 µm and no nucleus) (n = 5 animals per group, Kruskal-Wallis test with post hoc Dunn's test). E A representative image of a survived microglial cell in the hippocampal CA1 region of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with an anti-Iba1 antibody (green) and DAPI (cyan). A 3D image (rightmost panel) was reconstructed using Imaris software. Arrow: live microglia. Arrowhead: microglial debris. Scale bar, 10 µm.


Figure 2A Histological analysis of RFP + cells in Siglechdtr / dtr : Ccr2RFP / + mice . Lumber spinal cord after EAE induction , and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed . Iba1 immunoreactivity ( green ) and RFP fluorescence ( red ) are shown . Images were acquired using the same laser power and sensitivity , and image processing was the same for all RFP signals ( red ) . Scale bar , 50 µm . B Flow cytometric analysis of RFP + Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr / dtr : Ccr2RFP / + mice 2 days after PBS or DT administration . Representative data and a quantification graph ( n = 5 animals per group , two - tailed unpaired Student ' s t - test ) are shown . C Immunohistochemical localization of CD206 + perivascular macrophages ( arrows ) in hippocampal CA1 of Siglechdtr / dtr mice . Sections prepared 2 days after administration of PBS or DT were stained with anti - Iba1 ( green ) , anti - CD206 ( red ) and anti - CD31 ( blue ) antibodies . Scale bar , 50 µm . D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr / dtr mice 2 days after PBS or DT administration ( n = 5 animals per group , two - tailed unpaired Student ' s t - test ) .

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Figure 2A Histological analysis of RFP+ cells in Siglechdtr/dtr:Ccr2RFP/+ mice. Lumber spinal cord after EAE induction, and hippocampal CA1 2 or 4 days after PBS or DT injection were analyzed. Iba1 immunoreactivity (green) and RFP fluorescence (red) are shown. Images were acquired using the same laser power and sensitivity, and image processing was the same for all RFP signals (red). Scale bar, 50 µm.B Flow cytometric analysis of RFP+Ly6Chigh inflammatory monocytes in peripheral blood of Siglechdtr/dtr:Ccr2RFP/+ mice 2 days after PBS or DT administration. Representative data and a quantification graph (n = 5 animals per group, two-tailed unpaired Student's t-test) are shown.C Immunohistochemical localization of CD206+ perivascular macrophages (arrows) in hippocampal CA1 of Siglechdtr/dtr mice. Sections prepared 2 days after administration of PBS or DT were stained with anti-Iba1 (green), anti-CD206 (red) and anti-CD31 (blue) antibodies. Scale bar, 50 µm. D Number of perivascular macrophages in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals per group, two-tailed unpaired Student's t-test).


Figure 3A qPCR analysis of marker molecules for CNS cell types . The hippocampus of WT and Siglechdtr / dtr mice 2 days after PBS or DT administration was analyzed ( n = 3 animals per group , one - way ANOVA with post hoc Tukey ' s test ) . Results are normalized to Gapdh , and are shown as ratios to the value of WT mice injected with PBS . B Immunohistochemical detection of astrocytes in Siglechdtr / dtr mice . Hippocampal CA1 sections were prepared 2 days after PBS or DT administration , and stained with an anti - GFAP antibody . Images were acquired using the same laser power and sensitivity , and image processing was the same . Scale bar , 30 µm . C Astrocyte number in hippocampal CA1 of Siglechdtr / dtr mice 2 days after PBS or DT administration ( n = 5 animals , two - tailed unpaired Student ' s t - test ) .

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Figure 3A qPCR analysis of marker molecules for CNS cell types. The hippocampus of WT and Siglechdtr/dtr mice 2 days after PBS or DT administration was analyzed (n = 3 animals per group, one-way ANOVA with post hoc Tukey's test). Results are normalized to Gapdh, and are shown as ratios to the value of WT mice injected with PBS.B Immunohistochemical detection of astrocytes in Siglechdtr/dtr mice. Hippocampal CA1 sections were prepared 2 days after PBS or DT administration, and stained with an anti-GFAP antibody. Images were acquired using the same laser power and sensitivity, and image processing was the same. Scale bar, 30 µm. C Astrocyte number in hippocampal CA1 of Siglechdtr/dtr mice 2 days after PBS or DT administration (n = 5 animals, two-tailed unpaired Student's t-test).


Figure 4A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1 . Sections were prepared from Siglechdtr / dtr mice 2 days after PBS or DT administration , and stained with anti - GFAP ( green ) and anti - CD11b ( red ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Scale bar , 10 µm . B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr / dtr mice 2 days after DT administration . A phagocytic astrocyte , which engulfed degenerated microglial components , was identified by immunohistochemistry using anti - S100β ( green ) and anti - CD11b ( red ) antibodies , and DAPI ( cyan ) ( upper and middle panels ) . Then , a phagocytic cup of the same cell was analyzed by electron microscopy ( lower panel ) . Degenerated microglial cytoplasm / plasma membrane ( arrow ) and nucleus ( double arrow ) in a phagocytic cup of the astrocyte , and a piece of microglial debris internalized in astrocytic cytoplasm ( arrowhead ) are indicated . Scale bar , 50 µm ( upper panel ) , 10 µm ( upper panel [ inset ] and middle panel ) , and 2 µm ( lower panel ) . C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr / dtr mice 2 days after DT administration . Sections were stained with anti - S100β ( green ) and anti - CD11b ( red ) antibodies , and confocal images were reconstructed using Imaris software . The lower two panels are higher magnification images of contacted microglial debris . Arrow : microglial debris contacted by astrocyte processes ( coverage : approximately 30 or 50 % ) . Arrowhead : microglial debris internalized in astrocyte cytoplasm . Scale bar , 10 µm ( upper panel ) and 2 µm ( lower panels ) . D , E Quantification of the pieces of debris ( CD11b + spheres with > 0 . 5 µm diameter ) contacted with ( coverage > 30 % ) ( D ) and internalized by ( E ) a single astrocyte within 30 µm - thick sections ( n = 75 cells from five animals per group ) . The number of debris pieces was counted in 3D images reconstructed using Imaris software . All confocal images were acquired using the same laser power and sensitivity , and image processing was the same .

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Figure 4A Representative images of microglial debris surrounded by astrocyte processes in hippocampal CA1. Sections were prepared from Siglechdtr/dtr mice 2 days after PBS or DT administration, and stained with anti-GFAP (green) and anti-CD11b (red) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Scale bar, 10 µm.B CLEM analysis of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. A phagocytic astrocyte, which engulfed degenerated microglial components, was identified by immunohistochemistry using anti-S100β (green) and anti-CD11b (red) antibodies, and DAPI (cyan) (upper and middle panels). Then, a phagocytic cup of the same cell was analyzed by electron microscopy (lower panel). Degenerated microglial cytoplasm/plasma membrane (arrow) and nucleus (double arrow) in a phagocytic cup of the astrocyte, and a piece of microglial debris internalized in astrocytic cytoplasm (arrowhead) are indicated. Scale bar, 50 µm (upper panel), 10 µm (upper panel [inset] and middle panel), and 2 µm (lower panel).C A representative image of a phagocytic astrocyte in hippocampal CA1 of Siglechdtr/dtr mice 2 days after DT administration. Sections were stained with anti-S100β (green) and anti-CD11b (red) antibodies, and confocal images were reconstructed using Imaris software. The lower two panels are higher magnification images of contacted microglial debris. Arrow: microglial debris contacted by astrocyte processes (coverage: approximately 30 or 50%). Arrowhead: microglial debris internalized in astrocyte cytoplasm. Scale bar, 10 µm (upper panel) and 2 µm (lower panels). D, E Quantification of the pieces of debris (CD11b+ spheres with > 0.5 µm diameter) contacted with (coverage > 30%) (D) and internalized by (E) a single astrocyte within 30 µm-thick sections (n = 75 cells from five animals per group). The number of debris pieces was counted in 3D images reconstructed using Imaris software. All confocal images were acquired using the same laser power and sensitivity, and image processing was the same.


Figure 5B Expression level of phagocytic receptors and their extracellular adaptor molecules ( n = 3 independent experiments ) . Values show the mean ± SEM . N . S . : no significance ; * * * q < 0 . 001 . The probability values for differential expression analysis were computed with edgeR followed by a correction of multiple testing with q - value .

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Figure 5B Expression level of phagocytic receptors and their extracellular adaptor molecules (n = 3 independent experiments). Values show the mean ± SEM. N.S.: no significance; ***q < 0.001. The probability values for differential expression analysis were computed with edgeR followed by a correction of multiple testing with q-value.


Figure 6Cultured astrocytes engulf microglial debris . CAG - EGFP mouse - derived primary astrocytes were co - cultured with or without fluorescence - labelled apoptotic microglia . ( A ) Fluorescence images of GFP ( green ) , microglial debris ( red ) and GFAP immunoreactivity ( blue ) . Images were acquired using the same laser power and sensitivity , and image processing was the same in the two images ( red ) . Scale bar , 50 µm . ( B ) Fluorescence images of GFP ( green ) , microglial debris ( red ) and LAMP - 1 immunoreactivity ( cyan ) . Scale bar , 30 µm and 5 µm ( inset ) . C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes . Fluorescence of internalized microglial debris was measured in GLAST + astrocytes co - cultured with or without fluorescence - labelled apoptotic microglia . D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes . Tyro3 , Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting . Arrows indicate the corresponding protein band ( s ) . The secondary antibody against goat IgG generates a ~ 115 kDa non - specific band . The ~ 115 kDa band in the Axl image is the Axl - specific band overlapping the non - specific band . The ~ 115 kDa band in the Mertk image is the non - specific band . Axl and Mertk expression was knocked down by two siRNAs . GAPDH expression is shown as an internal control . E Quantification of siRNA knockdown in cultured astrocytes ( n = 3 independent experiments , one - way ANOVA with repeated measures ) . Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes . Knockdown efficiency of Axl and Mertk was quantified from western blots . Results are normalized to Gapdh or GAPDH , and are shown as ratios to the value of non - transfected astrocytes . F , G Flow cytometric analysis of microglial debris uptake by cultured astrocytes . After knockdown of TAM family members , astrocytes were co - cultured with fluorescence - labelled apoptotic microglia . Fluorescence of internalized microglial debris in GLAST + astrocytes was quantified by flow cytometry ( n = 4 independent experiments , one - way ANOVA with repeated measures ) . Results are shown as ratios to the value of non - transfected astrocytes .

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Figure 6Cultured astrocytes engulf microglial debris. CAG-EGFP mouse-derived primary astrocytes were co-cultured with or without fluorescence-labelled apoptotic microglia. (A) Fluorescence images of GFP (green), microglial debris (red) and GFAP immunoreactivity (blue). Images were acquired using the same laser power and sensitivity, and image processing was the same in the two images (red). Scale bar, 50 µm.(B) Fluorescence images of GFP (green), microglial debris (red) and LAMP-1 immunoreactivity (cyan). Scale bar, 30 µm and 5 µm (inset).C Flow cytometric analysis of uptake of microglial debris by cultured astrocytes. Fluorescence of internalized microglial debris was measured in GLAST+ astrocytes co-cultured with or without fluorescence-labelled apoptotic microglia.D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes. Tyro3, Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting. Arrows indicate the corresponding protein band(s). The secondary antibody against goat IgG generates a ~115 kDa non-specific band. The ~115 kDa band in the Axl image is the Axl-specific band overlapping the non-specific band. The ~115 kDa band in the Mertk image is the non-specific band. Axl and Mertk expression was knocked down by two siRNAs. GAPDH expression is shown as an internal control. E Quantification of siRNA knockdown in cultured astrocytes (n = 3 independent experiments, one-way ANOVA with repeated measures). Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes. Knockdown efficiency of Axl and Mertk was quantified from western blots. Results are normalized to Gapdh or GAPDH, and are shown as ratios to the value of non-transfected astrocytes.F, G Flow cytometric analysis of microglial debris uptake by cultured astrocytes. After knockdown of TAM family members, astrocytes were co-cultured with fluorescence-labelled apoptotic microglia. Fluorescence of internalized microglial debris in GLAST+ astrocytes was quantified by flow cytometry (n = 4 independent experiments, one-way ANOVA with repeated measures). Results are shown as ratios to the value of non-transfected astrocytes.


Figure 7B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w . Sections were stained with anti - Iba1 or anti - CD11b antibodies ( red ) . Scale bar , 100 µm . C Quantification of microglial number ( n = 5 animals per group , one - way ANOVA ) . Note that Irf8 - / - mice were analyzed by CD11b immunostaining , while other strains were analyzed by Iba1 staining . D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) and anti - cleaved caspase - 3 ( blue ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Arrows indicate cleaved caspase - 3 + apoptotic cells . Insets are higher magnification images around apoptotic cells . Scale bar , 20 µm and 5 µm ( insets ) . E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice . 3D images reconstructed from confocal images were analyzed ( n = 36 - 50 cells from five animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice ( n = 36 - 50 cells from five animals per group , one - way ANOVA with post hoc Tukey ' s test ) .

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Figure 7B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w. Sections were stained with anti-Iba1 or anti-CD11b antibodies (red). Scale bar, 100 µm. C Quantification of microglial number (n = 5 animals per group, one-way ANOVA). Note that Irf8-/- mice were analyzed by CD11b immunostaining, while other strains were analyzed by Iba1 staining.  D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets). E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice. 3D images reconstructed from confocal images were analyzed (n = 36-50 cells from five animals per group, Kruskal-Wallis test with post hoc Dunn's test). F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice (n = 36-50 cells from five animals per group, one-way ANOVA with post hoc Tukey's test).


Figure 1Representative images of HeLa ( B ) or HCT - 8 ( C ) cells infected with Shigella WT pre - treated or not with arsenite , analyzed at the indicated times post - infection Cfu quantification of intracellular bacteria in HeLa and HCT - 8 cells pre - treated or not with arsenite and infected with ShigellaRepresentative images ( E ) and cfu quantification ( F ) of intracellular bacteria in HeLa cells infected with Shigella WT after pre - treatment with TNF - α , H2O2 , anisomycin , hypoxia and corresponding controls , analyzed at 0 . 5 hpiRepresentative images ( G ) and cfu quantification ( H ) of intracellular Shigella in HeLa cells pre - treated with arsenite , anisomycin , stressors plus NAC , and corresponding

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Figure 1Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with ShigellaRepresentative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia and corresponding controls, analyzed at 0.5 hpiRepresentative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls