Figure 4Cdc48 occupancy at the Flp - bound FRT site . Levels of Cdc48 - TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in asynchronous cells before and after Flp - cc induction with galactose . The graph shows enrichment of qPCR signal over the unrelated intergenic region . Data are presented as means ± SDs of 3 to 4 independent biological replicates . p - values for + 200 bp were defined by 2 ‑ way ANOVA using Tukey ' s multiple comparisons test ( n . s . , non - significant ; * * * * p < 0 . 0001 ) . In addition to the indicated mutations , all strains are Cdc48 - TAP . No FRT strain was used as a negative control . Ubiquitination of the FRT locus . Asynchronous cultures were grown in 2 % raffinose ( raf ) or additionally supplemented with 3 % galactose ( gal ) to induce flp - H305L expression . Cells were also collected during glucose repression of flp - H305L expression at time points 30 min ( glu 30 ' ) and 120 min ( glu 120 ' ) . Ubiquitin antibody was used for ChIP - qPCR analysis following formaldehyde crosslinking . Data are shown as the mean ± SDs of three to six independent replicates . p - values for + 200 bp were defined by 2 ‑ way ANOVA using Tukey ' s multiple comparisons test ( n . s . , non - significant ; * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . In addition to all indicated mutations , all strains are bar1 ∆ Ddi1 - TAP .

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 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

Figure 4Cdc48 occupancy at the Flp-bound FRT site. Levels of Cdc48-TAP were monitored by ChIP and qPCR following formaldehyde crosslinking in asynchronous cells before and after Flp-cc induction with galactose. The graph shows enrichment of qPCR signal over the unrelated intergenic region. Data are presented as means ± SDs of 3 to 4 independent biological replicates. p-values for +200 bp were defined by 2‑way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; **** p < 0.0001). In addition to the indicated mutations, all strains are Cdc48-TAP. No FRT strain was used as a negative control.Ubiquitination of the FRT locus. Asynchronous cultures were grown in 2% raffinose (raf) or additionally supplemented with 3% galactose (gal) to induce flp-H305L expression. Cells were also collected during glucose repression of flp-H305L expression at time points 30 min (glu 30') and 120 min (glu 120'). Ubiquitin antibody was used for ChIP-qPCR analysis following formaldehyde crosslinking. Data are shown as the mean ± SDs of three to six independent replicates. p-values for +200 bp were defined by 2‑way ANOVA using Tukey's multiple comparisons test (n.s., non-significant; *p < 0.05; *** p < 0.001; **** p < 0.0001). In addition to all indicated mutations, all strains are bar1∆ Ddi1-TAP.


Figure 5ubx5 ∆ restores wss1 ∆ resistance to HU and FA but not upon additional deletion of DDI1 . Strains were grown in YEPD and either treated with 40 mM FA for 15 min or directly spotted on plates containing 100 mM of HU or 5 μg / mL CPT . Plates were incubated for 2 days at 30 ° C . Endogenous HA - Ddi1 levels are still elevated upon additional ubx5 ∆ mutation . Levels in total cell extracts were compared by immunoblotting . For quantification , anti - HA signal was normalized to Pgk1 and then normalized to WT ( see ( D ) ) . Quantification of HA - Ddi1 levels shown in ( C ) . The anti - HA signal was compared to Pgk1 and normalized to WT . Quantifications of 5 biological replicates are presented as means ± SDs . Significance was defined by ordinary one - way ANOVA using Dunnett ' s multiple comparisons test and WT as a control ( * p < 0 . 05 ; * * p < 0 . 01 ) . Catalytic activity of the protease Ddi1 is essential for survival of the ubx5 ∆ wss1 ∆ yeast mutant . Mutants carrying wild - type ( WT ) Ddi1 , D220A catalytic mutant or ddi1 ∆ in combination with ubx5 ∆ , wss1 ∆ or double ubx5 ∆ wss1 ∆ were grown in YEPD . Cells were treated with 40 mM formaldehyde ( FA ) for 15 min or directly spotted on plates supplemented with 100 mM hydroxyurea ( HU ) . Plates were incubated for 2 days at 30 ° C .

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 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 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

Figure 5ubx5∆ restores wss1∆ resistance to HU and FA but not upon additional deletion of DDI1. Strains were grown in YEPD and either treated with 40 mM FA for 15 min or directly spotted on plates containing 100 mM of HU or 5 μg/mL CPT. Plates were incubated for 2 days at 30°C.Endogenous HA-Ddi1 levels are still elevated upon additional ubx5∆ mutation. Levels in total cell extracts were compared by immunoblotting. For quantification, anti-HA signal was normalized to Pgk1 and then normalized to WT (see (D)). Quantification of HA-Ddi1 levels shown in (C). The anti-HA signal was compared to Pgk1 and normalized to WT. Quantifications of 5 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparisons test and WT as a control (*p < 0.05; ** p < 0.01).Catalytic activity of the protease Ddi1 is essential for survival of the ubx5∆ wss1∆ yeast mutant. Mutants carrying wild-type (WT) Ddi1, D220A catalytic mutant or ddi1∆ in combination with ubx5∆, wss1∆ or double ubx5∆ wss1∆ were grown in YEPD. Cells were treated with 40 mM formaldehyde (FA) for 15 min or directly spotted on plates supplemented with 100 mM hydroxyurea (HU). Plates were incubated for 2 days at 30°C.


Figure 6Ubx5 loss restores HU - induced Rpb1 degradation in wss1 ∆ and requires Ddi1 . Rpb1 levels were defined in cells growing in the presence of 200 mM HU and 100 μg / mL cycloheximide ( CHX ) at the indicated time points . Rpb1 and Pgk1 levels in total cell extracts were probed by immunoblotting and quantified using fluorescent secondary antibodies . See ( B ) for quantifications . Images show immunoblotting using chemiluminescence . Rpb1 chromatin enrichment following HU treatment . ( C ) Chromatin fractions were isolated from cells treated for 2 h with 200 mM HU and 100 μg / mL CHX . Pgk1 and H3 were used as controls to monitor the fractionation . Quantifications of 8 biological replicates are presented as means ± SDs . Significance was defined by ordinary one - way ANOVA using Dunnett ' s multiple comparison test and WT as a control ( n . s . , non - significant ; * * p < 0 . 01 ) . Rpb1 chromatin enrichment following HU treatment . ( D ) Quantifications of Rpb1 and H3 levels in chromatin fractions . Relative Rpb1 to H3 levels were set to 1 in the respective WT samples . Quantifications of 8 biological replicates are presented as means ± SDs . Significance was defined by ordinary one - way ANOVA using Dunnett ' s multiple comparison test and WT as a control ( n . s . , non - significant ; * * p < 0 . 01 ) .

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 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

Figure 6Ubx5 loss restores HU-induced Rpb1 degradation in wss1∆ and requires Ddi1. Rpb1 levels were defined in cells growing in the presence of 200 mM HU and 100 μg/mL cycloheximide (CHX) at the indicated time points. Rpb1 and Pgk1 levels in total cell extracts were probed by immunoblotting and quantified using fluorescent secondary antibodies. See (B) for quantifications. Images show immunoblotting using chemiluminescence.Rpb1 chromatin enrichment following HU treatment. (C) Chromatin fractions were isolated from cells treated for 2 h with 200 mM HU and 100 μg/mL CHX. Pgk1 and H3 were used as controls to monitor the fractionation. Quantifications of 8 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparison test and WT as a control (n.s., non-significant; ** p < 0.01).Rpb1 chromatin enrichment following HU treatment. (D) Quantifications of Rpb1 and H3 levels in chromatin fractions. Relative Rpb1 to H3 levels were set to 1 in the respective WT samples. Quantifications of 8 biological replicates are presented as means ± SDs. Significance was defined by ordinary one-way ANOVA using Dunnett's multiple comparison test and WT as a control (n.s., non-significant; ** p < 0.01).


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 .

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 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

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 .

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 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

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 1Colocalization of AcGFP - Siwi , AcGFP - BmAgo3 and mCherry - BmVasa in BmN4 cells . Line scans ( white line in the enlarged region ) show that Siwi - D670A mutant but not BmAgo3 - D697A mutant dissociates from BmVasa foci . Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel . Scale bar , 8 μm . Colocalization of AcGFP - Siwi and mCherry - BmDcp2 in BmN4 cells . Line scans ( white line in the enlarged region ) show that Siwi - D670A mutant but not BmAgo3 - D697A mutant overlaps with BmDcp2 foci . Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel . Scale bar , 8 μm .

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 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

Figure 1Colocalization of AcGFP-Siwi, AcGFP-BmAgo3 and mCherry-BmVasa in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant dissociates from BmVasa foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.Colocalization of AcGFP-Siwi and mCherry-BmDcp2 in BmN4 cells. Line scans (white line in the enlarged region) show that Siwi-D670A mutant but not BmAgo3-D697A mutant overlaps with BmDcp2 foci. Line scans of fluorescence intensity were normalized to the highest value and depicted at the right panel. Scale bar, 8 μm.


Figure 2 ( Left ) Effect of 1 , 6 - Hexanediol treatment on AcGFP - Siwi and AcGFP - Siwi - D670A foci . Z - stacks were taken prior to the addition of 1 , 6 - Hexanediol or medium and after 30 minutes incubation at RT . Z - projections of the middle 6 μm stacks are then normalized and pseudo - colored with Fire LUT to visualize the Siwi foci . Scale bar 10 μm . Scale bars represent pixel intensity in arbitrary units ( A . U . ) ( Right ) High granule / cell intensity ratio of Siwi - D670A persists despite 1 , 6 - Hexanediol treatment . Average intensity ratio from each of the independent Z - stacks ( n = 30 per set ) is quantified ( see Materials and Methods ) and depicted as box plots . Representative data from N = 3 independent experiments are shown . P - values were calculated by Asymptotic Wilcoxon rank sum test . Co - expression of Siwi - D670A increases granule - to - cell intensity ratio of BmSpnE and BmQin foci . ( Upper ) Representative Z - projections ( maximum intensity ) of mCherry - BmSpnE and mCherry - BmQin foci . AcGFP signal from FITC channel is not shown . Scale bar 8 μm . ( Bottom ) Average intensity ratio from each of the independent Z - stacks ( n = 6 per set ) is quantified ( see Materials and Methods ) and depicted as box plots . Representative data from N ≥ 3 independent experiments are shown . P - values were calculated by Asymptotic Wilcoxon rank sum test . Depletion of BmSpnE or BmQin results in segregation of Siwi - D670A from BmDcp2 - containing P - bodies . ( Top - left ) Representative Z - projections ( Maximum intensity ) . Scale bar 8 μm . ( Top - right ) Enlarged area from the white box . Scale bar 2 μm . ( Bottom ) Box plot showing that depletion of BmQin ( n = 32 cells ) or BmSpnE ( n = 32 cells ) reduces colocalization ratio between Siwi - D670A and BmDcp2 , compared to control ( dsRLuc , n = 32 cells ) . Representative data from N = 3 independent experiments are shown . Bonferroni - corrected P - values were calculated by Asymptotic Wilcoxon rank sum test . See also Figure EV3 .

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 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

Figure 2(Left) Effect of 1,6-Hexanediol treatment on AcGFP-Siwi and AcGFP-Siwi-D670A foci. Z-stacks were taken prior to the addition of 1,6-Hexanediol or medium and after 30 minutes incubation at RT. Z-projections of the middle 6 μm stacks are then normalized and pseudo-colored with Fire LUT to visualize the Siwi foci. Scale bar 10 μm. Scale bars represent pixel intensity in arbitrary units (A.U.) (Right) High granule/cell intensity ratio of Siwi-D670A persists despite 1,6-Hexanediol treatment. Average intensity ratio from each of the independent Z-stacks (n=30 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N = 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.Co-expression of Siwi-D670A increases granule-to-cell intensity ratio of BmSpnE and BmQin foci. (Upper) Representative Z-projections (maximum intensity) of mCherry-BmSpnE and mCherry-BmQin foci. AcGFP signal from FITC channel is not shown. Scale bar 8 μm. (Bottom) Average intensity ratio from each of the independent Z-stacks (n=6 per set) is quantified (see Materials and Methods) and depicted as box plots. Representative data from N ≥ 3 independent experiments are shown. P-values were calculated by Asymptotic Wilcoxon rank sum test.Depletion of BmSpnE or BmQin results in segregation of Siwi-D670A from BmDcp2-containing P-bodies. (Top-left) Representative Z-projections (Maximum intensity). Scale bar 8 μm. (Top-right) Enlarged area from the white box. Scale bar 2 μm. (Bottom) Box plot showing that depletion of BmQin (n=32 cells) or BmSpnE (n=32 cells) reduces colocalization ratio between Siwi-D670A and BmDcp2, compared to control (dsRLuc, n=32 cells). Representative data from N = 3 independent experiments are shown. Bonferroni-corrected P-values were calculated by Asymptotic Wilcoxon rank sum test. See also Figure EV3.


Figure 3Depletion of Siwi results in segregation of BmQin from BmDcp2 - containing P - bodies . Box plot showing that depletion of Siwi ( dsSiwi , n = 47 cells ) reduces colocalization ratio between BmQin and BmDcp2 , compared to control ( dsRLuc , n = 55 cells ) . Representative data from N = 4 independent experiments are shown . Depletion of Siwi results in segregation of BmSpnE from BmDcp2 - containing P - bodies . Box plot showing that depletion of Siwi ( dsSiwi , n = 55 cells ) reduces colocalization ratio between BmSpnE and BmDcp2 , compared to control ( dsRLuc , n = 55 cells ) . Representative data from N = 6 independent experiments are shown . Depletion of Siwi results in colocalization of BmSpnE and BmVasa . Box plot showing that depletion of Siwi ( dsSiwi , n = 18 cells ) significantly up - regulates colocalization ratio between BmSpnE and BmVasa , compared to control ( dsRLuc , n = 18 cells ) . Representative data from N = 3 independent experiments are shown . Depletion of BmVasa results in segregation of Siwi from BmAgo3 foci . Box plot showing that depletion of BmVasa ( dsBmVasa , n = 12 cells ) down - regulates colocalization ratio between Siwi and BmAgo3 , compared to control ( dsRLuc , n = 12 cells ) . Data points collected from N = 2 independent experiments are shown ( statistical test is not performed for N < 3 data ) . Depletion of BmVasa results in entanglement of Siwi at piP - bodies . Interaction interfaces are indicated by white arrows . Box plot showing that depletion of BmVasa ( dsBmVasa , n = 45 cells ) mildly up - regulates colocalization ratio between Siwi and BmDcp2 , compared to control ( dsRLuc , n = 49 cells ) . Representative data from N = 5 independent experiments are shown .

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 O O O O O O O O O O O

Figure 3Depletion of Siwi results in segregation of BmQin from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=47 cells) reduces colocalization ratio between BmQin and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 4 independent experiments are shown. Depletion of Siwi results in segregation of BmSpnE from BmDcp2-containing P-bodies. Box plot showing that depletion of Siwi (dsSiwi, n=55 cells) reduces colocalization ratio between BmSpnE and BmDcp2, compared to control (dsRLuc, n=55 cells). Representative data from N = 6 independent experiments are shown. Depletion of Siwi results in colocalization of BmSpnE and BmVasa. Box plot showing that depletion of Siwi (dsSiwi, n=18 cells) significantly up-regulates colocalization ratio between BmSpnE and BmVasa, compared to control (dsRLuc, n=18 cells). Representative data from N = 3 independent experiments are shown. Depletion of BmVasa results in segregation of Siwi from BmAgo3 foci. Box plot showing that depletion of BmVasa (dsBmVasa, n=12 cells) down-regulates colocalization ratio between Siwi and BmAgo3, compared to control (dsRLuc, n=12 cells). Data points collected from N = 2 independent experiments are shown (statistical test is not performed for N < 3 data). Depletion of BmVasa results in entanglement of Siwi at piP-bodies. Interaction interfaces are indicated by white arrows. Box plot showing that depletion of BmVasa (dsBmVasa, n=45 cells) mildly up-regulates colocalization ratio between Siwi and BmDcp2, compared to control (dsRLuc, n=49 cells). Representative data from N = 5 independent experiments are shown.


Figure 4Co - expression of BmVasa - E339Q / Siwi - D670A results in aggregates with larger sizes . Representative Z - projections ( Maximum intensity ) of mutant aggregates . Scale bar 1 μm .

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Figure 4Co-expression of BmVasa-E339Q/Siwi-D670A results in aggregates with larger sizes. Representative Z-projections (Maximum intensity) of mutant aggregates. Scale bar 1 μm.


Figure 5Split violin plots of piRNA expression fold change between AcGFP ( control ) and piRNA factor ( s ) overexpressed libraries . Overexpression of Siwi - D670A , BmVasa - E339Q and both mutants increase non - TE - derived piRNA ( red , 626 genes ) while TE - derived piRNA ( blue , 818 genes ) does not change . Bonferroni - corrected P - values and the effect sizes ( r ) were calculated by Asymptotic Wilcoxon rank sum 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

Figure 5Split violin plots of piRNA expression fold change between AcGFP (control) and piRNA factor(s) overexpressed libraries. Overexpression of Siwi-D670A, BmVasa-E339Q and both mutants increase non-TE-derived piRNA (red, 626 genes) while TE-derived piRNA (blue, 818 genes) does not change. Bonferroni-corrected P-values and the effect sizes (r) were calculated by Asymptotic Wilcoxon rank sum test.


Figure 2C . ) Live epifluorescence microscopy of PfMev parasites that are expressing api - SFG ( green ) and the P . falciparum DPCK - mCherry protein ( red ) with DAPI marking the nuclei ( blue ) . The borders of the red blood cell and parasite are marked with dotted blue and white lines , respectively . Microscopy images represent fields that are 10µm long by 10µm wide .

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Figure 2C.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and the P. falciparum DPCK-mCherry protein (red) with DAPI marking the nuclei (blue). The borders of the red blood cell and parasite are marked with dotted blue and white lines, respectively. Microscopy images represent fields that are 10µm long by 10µm wide.


Figure 3B . ) Live epifluorescence microscopy of PfMev parasites that are expressing api - SFG ( green ) and api - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . Data information : All microscopy images represent fields that are 10µm long by 10µm wide . D . ) Live epifluorescence microscopy of the PfMev api - EcDPCK - mCherry Δdpck parasite line that is expressing api - SFG ( green ) and api - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . E . ) Live epifluorescence microscopy of the PfMev api - EcDPCK - mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate . This parasite line expresses api - SFG ( green ) and api - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . Data information : All microscopy images represent fields that are 10µm long by 10µm wide . F . ) Attempted PCR detection of the ldh , sufB , and cox1 genes from the nuclear ( N ) , apicoplast ( A ) , and mitochondrial ( M ) genomes , respectively . We failed to amplify sufB from the azithromycin - treated ( + Azith ) PfMev api - EcDPCK - mCherry Δdpck parasites , indicating the loss of the apicoplast organellar genome .

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 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 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

Figure 3B.) Live epifluorescence microscopy of PfMev parasites that are expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.D.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck parasite line that is expressing api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). E.) Live epifluorescence microscopy of the PfMev api-EcDPCK-mCherry Δdpck line after treatment with 1x azithromycin for 7 days under supplementation with 50μM mevalonate. This parasite line expresses api-SFG (green) and api-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.F.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We failed to amplify sufB from the azithromycin-treated (+ Azith) PfMev api-EcDPCK-mCherry Δdpck parasites, indicating the loss of the apicoplast organellar genome.


Figure 4B . ) Live epifluorescence microscopy of the PfMev EcDPCK - mCherry line . This parasite line expresses api - SFG ( green ) and EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . D . ) Live epifluorescence microscopy of the PfMev EcDPCK - mCherry Δdpck line . This parasite line expresses api - SFG ( green ) and EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . Data information : All microscopy images represent fields that are 10µm long by 10µm wide . E . ) Attempted PCR detection of the ldh , sufB , and cox1 genes from the nuclear ( N ) , apicoplast ( A ) , and mitochondrial ( M ) genomes , respectively . We were successful in amplifying sufB from the PfMev EcDPCK - mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate , indicating retention of the apicoplast organellar genome .

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

Figure 4B.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). D.) Live epifluorescence microscopy of the PfMev EcDPCK-mCherry Δdpck line. This parasite line expresses api-SFG (green) and EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). Data information: All microscopy images represent fields that are 10µm long by 10µm wide.E.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were successful in amplifying sufB from the PfMev EcDPCK-mCherry Δdpck parasite line grown under continuous supplementation with 50μM mevalonate, indicating retention of the apicoplast organellar genome.


Figure 5C . ) Live epifluorescence microscopy of the PfMev CLD - EcDPCK - mCherry - apt Δdpck line . This parasite line expresses api - SFG ( green ) and CLD - EcDPCK - mCherry ( red ) with DAPI marking the nuclei ( blue ) . This parasite line was grown in the presence of 0 . 5μM aTc . D . ) The PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown in the absence of aTc for 48 hours . The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite . E . ) The PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown in the presence of 0 . 5μM aTc , and in the presence of 0 . 5μM Shield1 ( Shld ) for 48 hours . F . ) The PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown in the absence of aTc , and in the presence of 0 . 5μM Shield1 ( Shld ) for 48 hours . The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite . Data information : All microscopy images represent fields that are 10µm long by 10µm wide . G . ) Growth curve of the PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line grown either in the presence of 0 . 5μM aTc and the absence 0 . 5μM Shield1 ( permissive condition , blue ) , or the absence of 0 . 5μM aTc and the presence of 0 . 5μM Shield1 ( non - permissive condition , red ) . Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1 : 5 on day 4 . Treatment under non - permissive conditions resulted in reduced parasite growth beginning at day 1 ( two - way ANOVA , followed by Bonferroni ' s correction ; * , P < 0 . 05 ) . Error bars represent the standard error of the mean from two independent experiments , each conducted in quadruplicate .

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 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

Figure 5C.) Live epifluorescence microscopy of the PfMev CLD-EcDPCK-mCherry-apt Δdpck line. This parasite line expresses api-SFG (green) and CLD-EcDPCK-mCherry (red) with DAPI marking the nuclei (blue). This parasite line was grown in the presence of 0.5μM aTc. D.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. E.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the presence of 0.5μM aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. F.) The PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown in the absence of aTc, and in the presence of 0.5μM Shield1 (Shld) for 48 hours. The brightness of the red fluorescence image was increased to demonstrate the lack of mCherry signal associated with the parasite. Data information: All microscopy images represent fields that are 10µm long by 10µm wide.G.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate was supplied at 50nM in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 1 (two-way ANOVA, followed by Bonferroni's correction; *, P < 0.05). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.


Figure 6A . ) Attempted PCR detection of the ldh , sufB , and cox1 genes from the nuclear ( N ) , apicoplast ( A ) , and mitochondrial ( M ) genomes , respectively . We were unsuccessful in amplifying sufB from the PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line after treatment with 100nM azithromycin ( Azith ) in the presence of 50μM mevalonate , indicating the disruption of the apicoplast organelle . B . ) Live epifluorescence microscopy of the apicoplast - negative PfMev CLD - EcDPCK - mCherry - apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate . This parasite line expresses api - SFG ( green ) and the CLD - EcDPCK - mCherry construct ( red ) with DAPI marking the nuclei ( blue ) . Images represent fields that are 10µm long by 10µm wide . C . ) Growth curve of the PfMev CLD - EcDPCK - mCherry - apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0 . 5μM aTc and the absence 0 . 5μM Shield1 ( permissive condition , blue ) , or the absence of 0 . 5μM aTc and the presence of 0 . 5μM Shield1 ( non - permissive condition , red ) . Pantothenate ( 50nM ) and mevalonate ( 50μM ) were supplied in both conditions and the cultures were diluted 1 : 5 on day 4 . Treatment under non - permissive conditions resulted in reduced parasite growth beginning at day 2 ( two - way ANOVA , followed by Bonferroni ' s correction ; * * , P < 0 . 01 ) . Error bars represent the standard error of the mean from two independent experiments , each conducted in quadruplicate .

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 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 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

Figure 6A.) Attempted PCR detection of the ldh, sufB, and cox1 genes from the nuclear (N), apicoplast (A), and mitochondrial (M) genomes, respectively. We were unsuccessful in amplifying sufB from the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line after treatment with 100nM azithromycin (Azith) in the presence of 50μM mevalonate, indicating the disruption of the apicoplast organelle.B.) Live epifluorescence microscopy of the apicoplast-negative PfMev CLD-EcDPCK-mCherry-apt Δdpck line post treatment with 100nM azithromycin in the presence of 50μM mevalonate. This parasite line expresses api-SFG (green) and the CLD-EcDPCK-mCherry construct (red) with DAPI marking the nuclei (blue). Images represent fields that are 10µm long by 10µm wide.C.) Growth curve of the PfMev CLD-EcDPCK-mCherry-apt Δdpck parasite line containing a disrupted apicoplast grown either in the presence of 0.5μM aTc and the absence 0.5μM Shield1 (permissive condition, blue), or the absence of 0.5μM aTc and the presence of 0.5μM Shield1 (non-permissive condition, red). Pantothenate (50nM) and mevalonate (50μM) were supplied in both conditions and the cultures were diluted 1:5 on day 4. Treatment under non-permissive conditions resulted in reduced parasite growth beginning at day 2 (two-way ANOVA, followed by Bonferroni's correction; **, P < 0.01). Error bars represent the standard error of the mean from two independent experiments, each conducted in quadruplicate.


Figure 1A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag - USP19 ( top ) or endogenous USP19 in HeLa cells ( bottom ) treated with USP19 - specific siRNA or scrambled ( Scr ) siRNA . B Representative images of GFP - LC3B puncta in HeLa - GFP - LC3B cells transfected with USP19 - specific siRNA or scrambled siRNA during growth in normal medium ( DMEM ) or EBSS medium for 3 h . Arrows denote representative autophagosomes . Scale bar , 200 μm . C Quantification of GFP - LC3B puncta in HeLa - GFP - LC3B cells transfected with USP19 - specific siRNA or scrambled siRNA . Bars represented mean ± standard error of the mean ( SEM ) of triplicate samples ( 20 cells per sample ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . D Immunoblot detection of the relative accumulation of LC3 I and LC3 II in HeLa cells transfected with USP19 - specific siRNA or scrambled siRNA with rapamycin ( 250 nM ) treatment for 12 h . E HeLa cells were transfected with USP19 - specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h , p62 levels were detected by immunoblot . F Human peripheral blood mononuclear cells ( PBMCs ) transfected with control or USP19 - specific siRNA were treated with rapamycin ( 250 nM ) for 18 h , the lysates were analyzed with each antibody . G Flag - USP19 stably expressing 293T cells were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine ( CQ ) . Cells were then assayed for the relative accumulation of LC3 - I and LC3 - II by immunoblot analysis using anti - LC3 antibody . H Flag - USP19 inducible HeLa cells were treated with 200 ng / ml doxycycline ( Doxy ) for overnight to induce the expression of Flag - USP19 . The protein levels of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations . I GFP - LC3B stably expressing HeLa cells were transfected with pcDNA3 . 1 empty vector or plasmid expressing the wild - type and the mutant forms of USP19 ( CS or CS / HA ) . Pictures in ( B ) and ( I ) were taken using Leica DMI3000 B microscope with a × 100 oil - immersion objective . Scale bar , 200 μm . J Average GFP - LC3B puncta per cell were calculated . Bars represented mean ± SEM of triplicate samples ( 20 cells per sample ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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 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 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 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 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 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 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 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 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

Figure 1A Immunoblot analysis of the knockdown of exogenous USP19 in 293T cells expressing Flag-USP19 (top) or endogenous USP19 in HeLa cells (bottom) treated with USP19-specific siRNA or scrambled (Scr) siRNA.B Representative images of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA during growth in normal medium (DMEM) or EBSS medium for 3 h. Arrows denote representative autophagosomes. Scale bar, 200 μm.C Quantification of GFP-LC3B puncta in HeLa-GFP-LC3B cells transfected with USP19-specific siRNA or scrambled siRNA. Bars represented mean ± standard error of the mean (SEM) of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.D Immunoblot detection of the relative accumulation of LC3 I and LC3 II in HeLa cells transfected with USP19-specific siRNA or scrambled siRNA with rapamycin (250 nM) treatment for 12 h.E HeLa cells were transfected with USP19-specific siRNA or scrambled siRNA and treated with indicated concentration of rapamycin for 12 h, p62 levels were detected by immunoblot.F Human peripheral blood mononuclear cells (PBMCs) transfected with control or USP19-specific siRNA were treated with rapamycin (250 nM) for 18 h, the lysates were analyzed with each antibody.G Flag-USP19 stably expressing 293T cells were incubated with DMEM or EBSS medium for 3 h in the absence or presence of 50 μM of chloroquine (CQ). Cells were then assayed for the relative accumulation of LC3-I and LC3-II by immunoblot analysis using anti-LC3 antibody.H Flag-USP19 inducible HeLa cells were treated with 200 ng/ml doxycycline (Doxy) for overnight to induce the expression of Flag-USP19. The protein levels of p62 were analyzed after rapamycin treatment for 12 h at indicated concentrations.I GFP-LC3B stably expressing HeLa cells were transfected with pcDNA3.1 empty vector or plasmid expressing the wild-type and the mutant forms of USP19 (CS or CS/HA). Pictures in (B) and (I) were taken using Leica DMI3000 B microscope with a×100 oil-immersion objective. Scale bar, 200 μm.J Average GFP-LC3B puncta per cell were calculated. Bars represented mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.


Figure 2A 293T cells were transfected with plasmids encoding HA - USP19 and Flag - tagged Beclin - 1 complex members ( Flag - ATG14L , Flag - VPS15 , Flag - Beclin - 1 , Flag - VPS34 , Flag - UVRAG and Flag - AMBRA1 ) , followed by immunoprecipitation ( IP ) with anti - Flag beads and immunoblot analysis with anti - HA . Throughout was the immunoblot analysis of whole - cell lysates ( WCL ) without immunoprecipitation . B , C Extracts of A549 cells ( B ) or human PBMCs ( C ) incubated with EBSS for various time points ( above lanes ) were subjected to immunoprecipitation with anti - Beclin - 1 and immunoblot analysis with indicated antibodies ( shown on the left ) . D HeLa cells were incubated in EBSS medium for 1 h after co - transfection eGFP - USP19 and dsRed - Beclin - 1 for 48 h . Cells were imaged for eGFP and dsRed . Scale bar , 200 μm . E Quantitative data are mean ± SEM of triplicate samples ( 20 cells per sample ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . F Co - immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of USP19 plasmid along with vector encoding Flag - Beclin - 1 . G Co - immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of Beclin - 1 plasmid along with vector encoding HA - USP19 .

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 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 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 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 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

Figure 2A 293T cells were transfected with plasmids encoding HA-USP19 and Flag-tagged Beclin-1 complex members (Flag-ATG14L, Flag-VPS15, Flag-Beclin-1, Flag-VPS34, Flag-UVRAG and Flag-AMBRA1), followed by immunoprecipitation (IP) with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates (WCL) without immunoprecipitation.B, C Extracts of A549 cells (B) or human PBMCs (C) incubated with EBSS for various time points (above lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with indicated antibodies (shown on the left).D HeLa cells were incubated in EBSS medium for 1 h after co-transfection eGFP-USP19 and dsRed-Beclin-1 for 48 h. Cells were imaged for eGFP and dsRed. Scale bar, 200 μm.E Quantitative data are mean ± SEM of triplicate samples (20 cells per sample). *p < 0.05, **p < 0.01 and ***p < 0.001.F Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of USP19 plasmid along with vector encoding Flag-Beclin-1.G Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with deletion mutants of Beclin-1 plasmid along with vector encoding HA-USP19.


Figure 3A Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag - Beclin - 1 and increasing doses of expression vector for HA - USP19 ( wedge ) . B HeLa cell line harboring inducible Flag - USP19 was induced with indicated concentration of Doxy for overnight , and the protein was harvested for immunoblot analysis . C BECN1 mRNA levels of the same sample ( B ) were detected by real - time PCR . Bars represented mean ± SEM of triplicate samples . D Human PBMCs were transfected with USP19 - specific or scramble siRNA and treated with rapamycin ( 250 nM ) for 18 h . The extracts were analyzed by immunoblot with indicated antibodies . E Wild - type ( WT ) and USP19 KO A549 cells were cultured in EBSS for indicated time points , and the protein expression levels of Beclin - 1 and ULK1 were detected by immunoblot . F USP13 KO HEK293T cells were transfected with plasmids encoding Myc - USP5 , Myc - USP19 and Myc - USP38 and the cellular extracts were analyzed by immunoblot . G 293T cells transfected with plasmid expressing Flag - Beclin - 1 and empty vector or Myc - tagged USP19 ( WT , CS , CS / HA or ΔTMD ) , the lysates were analyzed by immunoblot . H 293T cells were transfected with plasmid encoding Flag - Beclin - 1 and treated with CHX ( 100 μg / ml ) alone or with MG132 ( 10 μM ) , 3MA ( 10 mM ) , CQ ( 50 μM ) as combination for 6 h or transfected with the HA - USP19 plasmid . The cell lysates were analyzed by immunoblot .

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Figure 3A Immunoblot analysis of extracts of 293T cells transfected with expression vector for Flag-Beclin-1 and increasing doses of expression vector for HA-USP19 (wedge).B HeLa cell line harboring inducible Flag-USP19 was induced with indicated concentration of Doxy for overnight, and the protein was harvested for immunoblot analysis.C BECN1 mRNA levels of the same sample (B) were detected by real-time PCR. Bars represented mean ± SEM of triplicate samples.D Human PBMCs were transfected with USP19-specific or scramble siRNA and treated with rapamycin (250 nM) for 18 h. The extracts were analyzed by immunoblot with indicated antibodies.E Wild-type (WT) and USP19 KO A549 cells were cultured in EBSS for indicated time points, and the protein expression levels of Beclin-1 and ULK1 were detected by immunoblot.F USP13 KO HEK293T cells were transfected with plasmids encoding Myc-USP5, Myc-USP19 and Myc-USP38 and the cellular extracts were analyzed by immunoblot.G 293T cells transfected with plasmid expressing Flag-Beclin-1 and empty vector or Myc-tagged USP19 (WT, CS, CS/HA or ΔTMD), the lysates were analyzed by immunoblot.H 293T cells were transfected with plasmid encoding Flag-Beclin-1 and treated with CHX (100 μg/ml) alone or with MG132 (10 μM), 3MA (10 mM), CQ (50 μM) as combination for 6 h or transfected with the HA-USP19 plasmid. The cell lysates were analyzed by immunoblot.


Figure 4A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 ( 10 μM ) treatment for 3 h . Protein extracts were immunoprecipitated using anti - Beclin - 1 antibody or with IgG as a negative control and analyzed by immunoblot using anti - ubiquitin and anti - Beclin - 1 antibodies . B Lysates of 293T cells transfected with plasmid expressing Flag - Beclin - 1 and HA - tagged ubiquitin ( HA - Ub ( wild type ) , K6 - linked - Ub , K11 - linked - Ub , K27 - linked - Ub , K29 - linked - Ub , K33 - linked - Ub , K48 - linked - Ub or K63 - linked - Ub ) , together with the empty vector or expression vector of Myc - USP19 and treated with MG132 ( 10 μM ) for 3 h were immunoprecipitated with anti - Flag and immunoblotted with anti - HA . C HeLa cells were transfected with USP19 - specific siRNA and plasmid encoding Flag - Beclin - 1 , plus K11 - linked - Ub . Protein was harvested after MG132 ( 10 μM ) treatment for 3 h and immunoprecipitated with anti - Flag and immunoblotted with anti - HA . D 293T cells were transfected with plasmid for Flag - Beclin - 1 and HA - K11 - Ub , together with the empty vector or expression vector for Myc - USP19 ( WT , CS or CS / HA ) and treated with MG132 ( 10 μM ) . Cell lysates were subjected to immunoprecipitation with anti - Flag and immunoblot analysis with anti - HA . E Purified ubiquitinated Beclin - 1 was incubated with immunopurified Flag - USP19 in vitro in deubiquitinating buffer . The immunoblot was probed with anti - HA .

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 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 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 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 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

Figure 4A HeLa cells were transfected with USP19 siRNA or scrambled siRNA and protein extracts were harvested after MG132 (10 μM) treatment for 3 h. Protein extracts were immunoprecipitated using anti-Beclin-1 antibody or with IgG as a negative control and analyzed by immunoblot using anti-ubiquitin and anti-Beclin-1 antibodies.B Lysates of 293T cells transfected with plasmid expressing Flag-Beclin-1 and HA-tagged ubiquitin (HA-Ub (wild type), K6-linked-Ub, K11-linked-Ub, K27-linked-Ub, K29-linked-Ub, K33-linked-Ub, K48-linked-Ub or K63-linked-Ub), together with the empty vector or expression vector of Myc-USP19 and treated with MG132 (10 μM) for 3 h were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.C HeLa cells were transfected with USP19-specific siRNA and plasmid encoding Flag-Beclin-1, plus K11-linked-Ub. Protein was harvested after MG132 (10 μM) treatment for 3 h and immunoprecipitated with anti-Flag and immunoblotted with anti-HA.D 293T cells were transfected with plasmid for Flag-Beclin-1 and HA-K11-Ub, together with the empty vector or expression vector for Myc-USP19 (WT, CS or CS/HA) and treated with MG132 (10 μM). Cell lysates were subjected to immunoprecipitation with anti-Flag and immunoblot analysis with anti-HA.E Purified ubiquitinated Beclin-1 was incubated with immunopurified Flag-USP19 in vitro in deubiquitinating buffer. The immunoblot was probed with anti-HA.


Figure 5A Lysates of 293T cells transfected with plasmid for Flag - Beclin - 1 and HA - K11 - linked - Ub , together with empty vector or expression vector for Myc - USP5 or Myc - USP19 were immunoprecipitated with anti - Flag and immunoblotted with anti - HA . B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag - Beclin - 1 and HA - K11 - linked ubiquitin . C 293T cells were transfected with Flag - Beclin - 1 ( WT or K437R ) and treated with CHX ( 100 μg / ml ) for indicated time points . The expression levels of WT and K437R Flag - Beclin - 1 were analyzed by immunoblot . D Quantification of the expression levels of WT and K437R Flag - Beclin - 1 . E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA - USP19 , together with plasmid expressing WT or K427R , K430R or K437R mutant of Flag - Beclin - 1 . F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag - Beclin - 1 were stained by anti - LC3 antibody . Scale bar , 200 μm . G Quantification of LC3 puncta shown in ( F ) . Bars represented mean ± SEM of triplicate samples . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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 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 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 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 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

Figure 5A Lysates of 293T cells transfected with plasmid for Flag-Beclin-1 and HA-K11-linked-Ub, together with empty vector or expression vector for Myc-USP5 or Myc-USP19 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA.B Immunoprecipitation and immunoblot analysis of 293T cells transfected with vectors expressing Flag-Beclin-1 and HA-K11-linked ubiquitin.C 293T cells were transfected with Flag-Beclin-1 (WT or K437R) and treated with CHX (100 μg/ml) for indicated time points. The expression levels of WT and K437R Flag-Beclin-1 were analyzed by immunoblot.D Quantification of the expression levels of WT and K437R Flag-Beclin-1.E Immunoblot analysis of protein extracts of 293T cells transfected with empty vector or vector for HA-USP19, together with plasmid expressing WT or K427R, K430R or K437R mutant of Flag-Beclin-1.F HeLa cells transfected with BECN1 sgRNA and restored with WT and K437R Flag-Beclin-1 were stained by anti-LC3 antibody. Scale bar, 200 μm.G Quantification of LC3 puncta shown in (F). Bars represented mean ± SEM of triplicate samples. *p < 0.05, **p < 0.01 and ***p < 0.001.


Figure 6A Luciferase activity in 293T cells transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter and together with plasmid encoding RIG - I ( N ) or cGAS and STING . B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies . C RT - PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points . D Phase - contrast microscopy ( PH ) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV - eGFP at an MOI of 0 . 01 for the indicated time length . Scale bar , 50 μm . E Human PBMCs were transfected with control or USP19 - specific siRNA , followed by treatment with influenza A / Puerto Rico / 8 / 34 ( H1N1 ) ( PR8 ) ( MOI = 5 ) at different time points , the lysates were analyzed with each antibody . F Human PBMCs were transfected with control or USP19 - specific siRNA were infected with H1N1 virus ( MOI = 5 ) at different time points . Relative expression levels of IFNB , ISG15 , ISG54 , ISG56 mRNA and H1N1 nucleoprotein ( NP ) RNA were measured by real - time PCR . G Relative expression levels of IFNB mRNA and interferon - stimulated genes , including ISG54 and ISG56 in WT , BECN1 , ATG5 and SQSTM1 KO cells with or without USP19 . H , I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE ( H ) or IFN - β ( I ) promoter - driven luciferase reporter after infection with SeV for indicated time points .

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 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 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 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 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 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 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

Figure 6A Luciferase activity in 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter and together with plasmid encoding RIG-I (N) or cGAS and STING.B Protein lysates of WT and USP19 KO A549 cells infected with SeV at indicated time points were immunoblotted with indicated antibodies.C RT-PCR analysis of gene transcription in WT and USP19 KO A549 cells after infection with SeV at indicated time points.D Phase-contrast microscopy (PH) and fluorescence microscopy images of WT and USP19 KO A549 cells infected with VSV-eGFP at an MOI of 0.01 for the indicated time length. Scale bar, 50 μm.E Human PBMCs were transfected with control or USP19-specific siRNA, followed by treatment with influenza A/Puerto Rico/8/34 (H1N1) (PR8) (MOI = 5) at different time points, the lysates were analyzed with each antibody.F Human PBMCs were transfected with control or USP19-specific siRNA were infected with H1N1 virus (MOI = 5) at different time points. Relative expression levels of IFNB, ISG15, ISG54, ISG56 mRNA and H1N1 nucleoprotein (NP) RNA were measured by real-time PCR.G Relative expression levels of IFNB mRNA and interferon-stimulated genes, including ISG54 and ISG56 in WT, BECN1, ATG5 and SQSTM1 KO cells with or without USP19.H, I Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (H) or IFN-β (I) promoter-driven luciferase reporter after infection with SeV for indicated time points.


Figure 7A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE ( left ) or IFN - β ( right ) promoter - driven luciferase reporter after infection with SeV for 12 h . B WT and BECN1 KO 293T cells were infected with SeV for indicated time points , then protein extracts were analyzed by immunoblot using the indicated antibodies . C Protein extracts of THP - 1 cells infected with SeV for various time points ( upper lanes ) were subjected to immunoprecipitation with anti - Beclin - 1 and immunoblot analysis with antibodies shown on the left . D Co - immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV . E 293T cells were transfected with vectors encoding HA - Beclin - 1 and Flag - MAVS or Flag - MAVSΔCARD , followed by IP with anti - Flag beads and immunoblot analysis with anti - HA . F Co - immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA - MAVS plasmid along with vector encoding Flag - RIG - I ( N ) in the absence or presence of USP19 . G Protein extracts of scramble or USP19 - specific siRNA treated 293T cells transfected with HA - MAVS plasmid along with vector encoding for Flag - RIG - I ( N ) in the absence or presence of Beclin - 1 were subjected to immunoprecipitation and immunoblot analysis . H Luciferase activity in 293T cells transfected with scramble or USP19 - specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 . I Luciferase activity in 293T cells transfected with scramble or USP19 - specific siRNA and IFN - β luciferase reporter after SeV infection for indicated time points with or without Beclin - 1 .

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 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 B-PANEL_START 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 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 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 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 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

Figure 7A Luciferase activity in WT and BECN1 KO 293T cells transfected with an ISRE (left) or IFN-β (right) promoter-driven luciferase reporter after infection with SeV for 12 h.B WT and BECN1 KO 293T cells were infected with SeV for indicated time points, then protein extracts were analyzed by immunoblot using the indicated antibodies.C Protein extracts of THP-1 cells infected with SeV for various time points (upper lanes) were subjected to immunoprecipitation with anti-Beclin-1 and immunoblot analysis with antibodies shown on the left.D Co-immunoprecipitation and immunoblot analysis of protein lysates of A549 cells infected with SeV.E 293T cells were transfected with vectors encoding HA-Beclin-1 and Flag-MAVS or Flag-MAVSΔCARD, followed by IP with anti-Flag beads and immunoblot analysis with anti-HA.F Co-immunoprecipitation and immunoblot analysis of WT and BECN1 KO 293T cells transfected with HA-MAVS plasmid along with vector encoding Flag-RIG-I (N) in the absence or presence of USP19.G Protein extracts of scramble or USP19-specific siRNA treated 293T cells transfected with HA-MAVS plasmid along with vector encoding for Flag-RIG-I (N) in the absence or presence of Beclin-1 were subjected to immunoprecipitation and immunoblot analysis.H Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and ISRE luciferase reporter after SeV infection for indicated time points with or without Beclin-1.I Luciferase activity in 293T cells transfected with scramble or USP19-specific siRNA and IFN-β luciferase reporter after SeV infection for indicated time points with or without Beclin-1.


Figure 1 ( A ) Expression patterns of Hoxa5 and Hoxc8 in MNs ( demarcated by white dashed lines ) along the rostrocaudal axis of the spinal cord ( cervical C4 to thoracic T1 ) from mouse embryos from E9 . 5 to E12 . 5 . Pink scale bars : 12 . 5 μm for E9 . 5 ; 12 . 5 μm for E10 . 5 ; 25 μm for E11 . 5 ; and 50 μm for E12 . 5 .

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Figure 1(A) Expression patterns of Hoxa5 and Hoxc8 in MNs (demarcated by white dashed lines) along the rostrocaudal axis of the spinal cord (cervical C4 to thoracic T1) from mouse embryos from E9.5 to E12.5. Pink scale bars: 12.5 μm for E9.5; 12.5 μm for E10.5; 25 μm for E11.5; and 50 μm for E12.5.


Figure 3Expression patterns of Hoxa5 and Hoxc8 mRNAs , as revealed by in situ hybridization , aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs ( demarcated with pink / white dashed lines ) along the cervical spinal cord ( cervical C5 to C7 ) in mouse embryos at E12 . 5 . Pink scale bar , 50 μm . White and pink dash lines demarcate the postmitotic MN region . Dot - plots showing expression of genes ( rows ) that distinguish MN subtypes ( columns ) . The height of each colored shape is proportional to the percentage of cells expressing the marker ( ≥ 1 UMI ) , and its width reflects the average transcript count within expressing cells .

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 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

Figure 3Expression patterns of Hoxa5 and Hoxc8 mRNAs, as revealed by in situ hybridization, aligned with corresponding sections showing Hoxa5 and Hoxc8 immunostainings in MNs (demarcated with pink/white dashed lines) along the cervical spinal cord (cervical C5 to C7) in mouse embryos at E12.5. Pink scale bar, 50 μm. White and pink dash lines demarcate the postmitotic MN region.Dot-plots showing expression of genes (rows) that distinguish MN subtypes (columns). The height of each colored shape is proportional to the percentage of cells expressing the marker (≥1 UMI), and its width reflects the average transcript count within expressing cells.


Figure 6Immunostaining ( C ) of EBs 96 h after ESC differentiation , and quantification ( D ) of Hoxa5on in MNs ( Hb9on ) . Hoxa5 expression upon miR - 27 knockdown , with exposure to RAhi for 72 h , or with exposure to RAhi for 48 h , and then switching to RAlow plus RAR inhibitor ( 1 µM ) for a further 24 h , or with exposure to RAmed for 72 h .

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Figure 6Immunostaining (C) of EBs 96 h after ESC differentiation, and quantification (D) of Hoxa5on in MNs (Hb9on).Hoxa5 expression upon miR-27 knockdown, with exposure to RAhi for 72 h, or with exposure to RAhi for 48 h, and then switching to RAlow plus RAR inhibitor (1 µM) for a further 24 h, or with exposure to RAmed for 72 h.


Figure 8 ( C ) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6 / 7 segments of miR - 23 ~ 27 ~ 24 double KO ( DKO ) mice . miR - 23a ~ 27a ~ 24 - 2 + / - ; miR - 23b ~ 27b ~ 24 - 1 - / - mice were used as a control ( ctrl ) . ( E ) Conversely , Hoxc8 is shifted into the MNs of cervical C5 segments of both miR - 196a2 / b DKO and miR - 196a1 / a2 / b triple knockout ( TKO ) embryos . Age - matched wild type embryos were used as controls . Scale bar represents 50 μm . White dash lines demarcate the postmitotoic MN domain ( D , F ) Quantifications of Hoxa5on , Hoxc8on , and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos ( mean ± SD , N = 3 embryos , * p < 0 . 01 , Student ' s t - tests ) .

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

Figure 8(C) Immunostaining at cervical spinal cord sections reveals Hoxa5 expansion into the MNs of cervical C6/7 segments of miR-23~27~24 double KO (DKO) mice. miR-23a~27a~24-2+/-;miR-23b~27b~24-1-/- mice were used as a control (ctrl). (E) Conversely, Hoxc8 is shifted into the MNs of cervical C5 segments of both miR-196a2/b DKO and miR-196a1/a2/b triple knockout (TKO) embryos. Age-matched wild type embryos were used as controls. Scale bar represents 50 μm. White dash lines demarcate the postmitotoic MN domain (D, F) Quantifications of Hoxa5on, Hoxc8on, and Hoxa5on Hoxc8on cells in the cervical spinal cord of control and knockout embryos (mean ± SD, N=3 embryos, *p < 0.01, Student's t-tests ).


Figure 1G . Relative expression levels of shade - regulated lncRNAs by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 20 µE , B = 20 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) .

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

Figure 1G. Relative expression levels of shade-regulated lncRNAs by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 20 µE, B = 20 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).


Figure 2Relative expression levels of PUAR under WL , SH , and dark ( DK ) conditions determined by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . The seedlings were grown under DK for 4 d or transferred to WL for 6 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) . Relative expression levels of PUAR in Col - 0 , phyB - 9 , phyA - 211 , and pif7 - 1 by RT - qPCR under Red ( R = 100 µE ) , Far - red ( FR = 10 µE ) , or SH conditions . For R and FR conditions , the seedlings were grown under R or FR for 4 d . For SH conditions , the seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 1 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) . Hypocotyl phenotypes of Col - 0 , PUARox , PUAR - S1ox , The seedlings were grown under white light for 4 d , and then maintained under white light or transferred to shade for 3 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . The scale bar represents 2 mm . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) . Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions . The scale bar represents 2 mm . P - values indicate statistically significant differences ( * P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) . Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions . The scale bar represents 2 mm . P - values indicate statistically significant differences ( * P - value < 0 . 05 ) by t - test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) . J . Hypocotyl phenotypes of Col - 0 , PHYAox , and amiR - puar . The seedlings were grown under white light for 3 d , and then maintained under white light or transferred to shade for 4 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . The scale bar represents 2 mm . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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 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 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 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 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

Figure 2Relative expression levels of PUAR under WL, SH, and dark (DK) conditions determined by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. The seedlings were grown under DK for 4 d or transferred to WL for 6 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by t-test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).Relative expression levels of PUAR in Col-0, phyB-9, phyA-211, and pif7-1 by RT-qPCR under Red (R = 100 µE), Far-red (FR = 10 µE), or SH conditions. For R and FR conditions, the seedlings were grown under R or FR for 4 d. For SH conditions, the seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 1 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by t-test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).Hypocotyl phenotypes of Col-0, PUARox, PUAR-S1ox, The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions. The scale bar represents 2 mm. P-values indicate statistically significant differences (*P-value < 0.05) by t-test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).Relative hypocotyl lengths under FR compared with those of seedlings grown under DK conditions. The scale bar represents 2 mm. P-values indicate statistically significant differences (*P-value < 0.05) by t-test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates). J. Hypocotyl phenotypes of Col-0, PHYAox, and amiR-puar. The seedlings were grown under white light for 3 d, and then maintained under white light or transferred to shade for 4 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). The scale bar represents 2 mm. Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).


Figure 3Relative expression levels of PHYA in Col - 0 , pif7 - 1 , PUARox , PUAR - S1ox , by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . The left Y - axis is relative expression level of PUAR , and the right Y - axis is the ratio of relative expression level under shade conditions to that under white light conditions . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) . PHYA protein levels in Col - 0 , pif7 - 1 , PUARox , PUAR - S1ox , by western - blot . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Tubulin indicates the loading control . Relative expression levels of PHYA in Col - 0 TATA - mut , and amiR - puar by RT - qPCR . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . The left Y - axis is relative expression level of PUAR , and the right Y - axis is the ratio of relative expression level under shade conditions to that under white light conditions . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n = 3 , n refers to biological replicates ) . PHYA protein levels in Col - 0 , TATA - mut , and amiR - puar by western - blot . The seedlings were grown under white light for 6 d , and then maintained under white light or transferred to shade for 4 h . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Tubulin indicates the loading control . G - H . Hypocotyl phenotypes of Col - 0 , phyA - 211 , PUARox , PUAR - S1ox , PUARox / phyA - 211 , GFPox , PHYAox , PUAR - S1ox , and PHYAox * PUAR - S1ox . The seedlings were grown under white light for 4 d , and then maintained under white light or transferred to shade for 3 d . WL , white light ( R = 20 µE , B = 20 µE , FR = 5 µE ) . SH , shade ( R = 10 µE , B = 5 µE , FR = 60 µE ) . Different letters indicate statistically significant differences ( P - value < 0 . 05 ) by one - way ANOVA with Tukey ' s HSD test . The data shown are the mean ± SDs ( n ≥ 16 , n refers to biological replicates ) .

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 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 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 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 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

Figure 3Relative expression levels of PHYA in Col-0, pif7-1, PUARox, PUAR-S1ox, by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. The left Y-axis is relative expression level of PUAR, and the right Y-axis is the ratio of relative expression level under shade conditions to that under white light conditions. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).PHYA protein levels in Col-0, pif7-1, PUARox, PUAR-S1ox, by western-blot. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Tubulin indicates the loading control.Relative expression levels of PHYA in Col-0 TATA-mut, and amiR-puar by RT-qPCR. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. The left Y-axis is relative expression level of PUAR, and the right Y-axis is the ratio of relative expression level under shade conditions to that under white light conditions. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n = 3, n refers to biological replicates).PHYA protein levels in Col-0, TATA-mut, and amiR-puar by western-blot. The seedlings were grown under white light for 6 d, and then maintained under white light or transferred to shade for 4 h. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Tubulin indicates the loading control.G-H. Hypocotyl phenotypes of Col-0, phyA-211, PUARox, PUAR-S1ox, PUARox/phyA-211, GFPox, PHYAox, PUAR-S1ox, and PHYAox*PUAR-S1ox. The seedlings were grown under white light for 4 d, and then maintained under white light or transferred to shade for 3 d. WL, white light (R = 20 µE, B = 20 µE, FR = 5 µE). SH, shade (R = 10 µE, B = 5 µE, FR = 60 µE). Different letters indicate statistically significant differences (P-value < 0.05) by one-way ANOVA with Tukey's HSD test. The data shown are the mean ± SDs (n ≥ 16, n refers to biological replicates).