C Boxplots showing average gene expression ( log10 ( FPKMs + 1 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 ) in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( left ) . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . showing expression profile ( log10 ( FPKMs + 1 ) ) of genes up - or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 mice ( mid ) . Genes are ranked by degree of expression . Numbers in colour scale show correspondence between gene expression values colours . Boxplots showing average TSS accessibility ( log10 ( ATAC reads + 1 ) , N = 3 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 versus WT mice hippocampus ( right ) . Exact P values are reported in Appendix Table S3 . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . D Average gene expression ( closed or open regions FPKMs / unchanged regions FPKMs ) for genes associated with differential accessible regions in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( adjusted P - value < 0 . 05 , N = 3 ) . Each point corresponds to value from an sample . Data is shown as mean ± S . E . M . * P < 0 . 05 as compared with WT mice ( two - unpaired Student ' s t ) . Exact P values are reported in Appendix Table S3 .

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

C Boxplots showing average gene expression (log10(FPKMs +1) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2) in R6/1 (N=9) versus WT (N=9) mice (left). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. Heatmap showing expression profile (log10 (FPKMs +1)) of genes up- or down-regulated (adjusted P-value<0.001, |FC|>2, N=9) in R6/1 mice (mid). Genes are ranked by the degree of expression. Numbers in colour scale show the correspondence between gene expression values and colours. Boxplots showing average TSS chromatin accessibility (log10(ATAC reads +1), N=3) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2, N=9) in R6/1 versus WT mice hippocampus (right). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. D Average gene expression (closed or open regions FPKMs/ unchanged regions FPKMs) for genes associated with differential accessible regions in R6/1 (N=9) versus WT (N=9) mice (adjusted P-value < 0.05, N=3). Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. *P < 0.05 as compared with WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.


C Boxplots showing average gene expression ( log10 ( FPKMs + 1 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 ) in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( left ) . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . showing expression profile ( log10 ( FPKMs + 1 ) ) of genes up - or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 mice ( mid ) . Genes are ranked by degree of expression . Numbers in colour scale show correspondence between gene expression values colours . Boxplots showing average TSS accessibility ( log10 ( ATAC reads + 1 ) , N = 3 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 versus WT mice hippocampus ( right ) . Exact P values are reported in Appendix Table S3 . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

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

C Boxplots showing average gene expression (log10(FPKMs +1) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2) in R6/1 (N=9) versus WT (N=9) mice (left). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. Heatmap showing expression profile (log10 (FPKMs +1)) of genes up- or down-regulated (adjusted P-value<0.001, |FC|>2, N=9) in R6/1 mice (mid). Genes are ranked by the degree of expression. Numbers in colour scale show the correspondence between gene expression values and colours. Boxplots showing average TSS chromatin accessibility (log10(ATAC reads +1), N=3) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2, N=9) in R6/1 versus WT mice hippocampus (right). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


A Boxplots of average expression ( log10 ( FPKMs + 1 ) ) for genes sublist 1 - 5 ( lowest to highest expression ) for wild - type ( WT , N = 3 ) mice ( left ) . Pie charts of gene distribution among generated sublists ( 1 - 5 ) for genes ( middle ) genes in LADs ( right ) in WT mice . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . B Average gene expression ( FPKMs ) of genes found in common wild - type ( WT , N = 3 ) specific LADs for WT ( N = 9 ) R6 / 1 ( N = 9 ) mice . Each point corresponds to value from an sample . Data is shown as mean ± S . E . M . * P < 0 . 05 as compared to WT mice ( two - unpaired Student ' s t ) . Exact P values are reported in Appendix Table S3 .

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

A Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. B Average gene expression (FPKMs) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=9) and R6/1 (N=9) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. * P < 0.05 as compared to WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.


A Boxplots of average expression ( log10 ( FPKMs + 1 ) ) for genes sublist 1 - 5 ( lowest to highest expression ) for wild - type ( WT , N = 3 ) mice ( left ) . Pie charts of gene distribution among generated sublists ( 1 - 5 ) for genes ( middle ) genes in LADs ( right ) in WT mice . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

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

A Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


C Average accessibility ( normalized read ) of genes found in common wild - type ( WT , N = 3 ) specific LADs for WT ( N = 3 ) R6 / 1 ( N = 3 ) mice . Each point corresponds to value from an sample . Data is shown as mean ± S . E . M . Wilcoxon Mann Whitney was used for statistical . Exact P values are reported in Appendix Table S3 .

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

C Average chromatin accessibility (normalized read counts) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=3) and R6/1 (N=3) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. Wilcoxon Mann Whitney test was used for statistical analysis. Exact P values are reported in Appendix Table S3.


D Venn diagram showing total number of genes found in common wild - type ( WT ) specific LADs regions ( left ) . Bar graph showing number of up - down - regulated genes found in common WT specific LADs regions filtering only according to adjusted P - value ( adjusted P - value < 0 . 001 ) or additionally with fold change ( adjusted P - value < 0 . 001 , | FC | > 2 ) obtained from deseq2 differential expression ( see methods ) .

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

D Venn diagram showing the total number of genes found in common and wild-type (WT) specific LADs regions (left). Bar graph showing the number of up- and down-regulated genes found in common and WT specific LADs regions filtering only according to adjusted P-value (adjusted P-value <0.001) or additionally with fold change (adjusted P-value <0.001, |FC| > 2) obtained from deseq2 differential expression analysis (see methods).


E UCSC genome browser capture of wild - type ( WT , N = 3 ) R6 / 1 ( N = 3 ) mice hippocampus ATAC - seq data , lamin B1 ChIP - seq ( log ( Lb1 ChIP / Input ) ) for WT ( N = 3 ) R6 / 1 ( N = 3 ) mice hippocampus , hippocampal H3K9ac , CTCF H3K9me3 for WT mice hippocampus in Fos locus ( left ) . Enhancer regions ( E1 - E5 ) are indicated with arrows . Boxplot of lamin B1 enrichment ( log10 ( Lb1 ChIP / Input ) ) for regions with unchanged , decreased ( closed in R6 / 1 ) increased ( opened in R6 / 1 ) accessibility in R6 / 1 ( N = 3 ) mice hippocampus . * P < 0 . 05 as compared to WT ( N = 3 ) mice . ( Wilcoxon Mann Whitney ) . Exact P values are reported in Appendix Table S3 . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

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

E UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, lamin B1 ChIP-seq (log(Lb1 ChIP/Input)) for WT (N=3) and R6/1 (N=3) mice hippocampus, hippocampal H3K9ac, CTCF and H3K9me3 for WT mice hippocampus in Fos locus (left). Enhancer regions (E1-E5) are indicated with arrows. Boxplot of lamin B1 enrichment (log10(Lb1 ChIP/Input)) for regions with unchanged, decreased (closed in R6/1) and increased (opened in R6/1) chromatin accessibility in R6/1 (N=3) mice hippocampus. * P < 0.05 as compared to WT (N=3) mice. (Wilcoxon Mann Whitney test). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


B , C Graphs show of time exploring each object with respect to total exploration time in ( B ) NOLT ( C ) NORT , 5 after treatment ( Veh , vehicle ; BA , betulinic acid ; WT , wild - type ) . * * * * P < 0 . 0001 compared to corresponding old location / object . WT veh N = 13 ; R6 / 1 veh N = 10 ; R6 / 1 + BA N = 10 .

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

B,C Graphs show the percentage of time exploring each object with respect to the total exploration time in the (B) NOLT and (C) NORT, 5 weeks after treatment (Veh, vehicle; BA, betulinic acid; WT, wild-type). ****P < 0.0001 compared to the corresponding old location/object. WT veh N = 13; R6/1 veh N = 10; R6/1+BA N = 10.


D Accelerating rotarod was assessed after 7 of treatment . * * P < 0 . 01 * * * P < 0 . 001 compared to vehicle - treated wild - type ( WT ) mice ; # P < 0 . 05 compared to vehicle - treated R6 / 1 mice . WT veh N = 13 ; R6 / 1 veh N = 9 ; R6 / 1 + BA N = 9 .

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

D Accelerating rotarod was assessed after 7 weeks of treatment. **P < 0.01 and ***P < 0.001 compared to vehicle-treated wild-type (WT) mice; #P < 0.05 compared to vehicle-treated R6/1 mice. WT veh N = 13; R6/1 veh N = 9; R6/1+BA N = 9.


E Lamin B1 were analysed by Western blot in striatum ( WT veh N = 10 ; R6 / 1 veh N = 6 ; R6 / 1 + BA N = 7 ) , hippocampus ( WT veh N = 12 ; R6 / 1 veh N = 8 ; R6 / 1 + BA N = 7 ) cortex ( WT veh N = 11 ; R6 / 1 veh N = 6 ; R6 / 1 + BA N = 7 ) after 12 of treatment . * P < 0 . 05 , * * P < 0 . 05 compared to vehicle - treated wild - type ( WT ) mice # P < 0 . 05 compared to vehicle - treated R6 / 1 mice . Representative immunoblots of lamin B1 α - tubulin ( as loading control ) for each treatment group are shown .

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

E Lamin B1 levels were analysed by Western blot in the striatum (WT veh N = 10; R6/1 veh N = 6; R6/1+BA N = 7), hippocampus (WT veh N = 12; R6/1 veh N = 8; R6/1+BA N = 7) and cortex (WT veh N = 11; R6/1 veh N = 6; R6/1+BA N = 7) after 12 weeks of treatment. *P < 0.05, **P < 0.05 compared to vehicle-treated wild-type (WT) mice and #P < 0.05 compared to vehicle-treated R6/1 mice. Representative immunoblots of lamin B1 and α-tubulin (as loading control) for each treatment group are shown.


F Lamin B1 were analysed by FANSI in hippocampal CA1 neurons nuclei from wild - type ( WT ) R6 / 1 mice after 12 of treatment . * P < 0 . 05 compared to vehicle - treated WT mice . * P < 0 . 05 as compared to vehicle - treated WT mice . Lamin B1 : WT veh N = 6 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 4 ; Lamin B1 circularity : WT veh N = 6 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 5 .

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

F Lamin B1 intensity and nuclear morphology were analysed by FANSI in hippocampal CA1 neurons nuclei from wild-type (WT) and R6/1 mice after 12 weeks of treatment. *P < 0.05 compared to vehicle-treated WT mice. *P < 0.05 as compared to vehicle-treated WT mice. Lamin B1 intensity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 4; Lamin B1 circularity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 5.


G permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 of treatment . * p < 0 . 05 compared to vehicle - treated wild - type ( WT ) mice . WT veh N = 4 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 6 .

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

G Nuclear permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 weeks of treatment. *p < 0.05 compared to vehicle-treated wild-type (WT) mice. WT veh N = 4; R6/1 veh N = 5; R6/1+BA N = 6.


( A ) Purification of Halo - tagged cohesin tetramer . Human cohesin tetramer tagged by Halo was purified from insect Alexa488 dye was conjugated to Halo - tag ( cohesinHalo488 ) .

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

(A) Purification of the Halo-tagged cohesin tetramer. Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo-tag (cohesinHalo488).


( B ) CohesinHalo488 loaded onto in presence of Scc2 - Scc4 was incubated in buffer with or without TEV protease . After high - salt washing , CohesinHalo488 intensities on were measured . Red bars denote median , lower , upper quartile values ( n ≥ 98 ; * p < 0 . 0001 , two - Mann - Whitney ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Kymograph of a single - interacting cohesinHalo488 particle . CohesinHalo488 is loaded onto in presence of Scc2 - Scc4 , after high - salt washing , single cohesin particles were observed in presence of ATP 100 mM KCl . Scale bar , 2 μm . ( D ) Histogram of net displacement ( xfinal - xinitial ) of cohesin particles treated as in ( C ) . magenta curve represents Gaussian fit .

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

(C) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit.


( E ) CohesinHalo488 was loaded washed in high salt as in ( C ) , cohesin particles were observed in presence of ATP 100 mM or 750 mM KCl . Kymograph of - associated cohesin particles under high - salt conditions ( 750 mM KCl , right ) mean square displacement ( MSD ) versus time ( left ) are shown . Dotted lines are regression lines used to estimate diffusion coefficient ( D ) . Scale bar , 2 μm .

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

(E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm.


( F ) MSD versus time for - bound cohesinWT - Halo488 or cohesinKA - Halo488 particles . CohesinWT - Halo488 or cohesinKA - Halo488 was loaded onto in presence of Scc2 - 4 ATP . After high - salt washing , cohesin particles on were observed in presence of 1 mM AMP - PCP or ATP 100 mM KCl . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Kymograph of CohesinHalo488 in presence or absence of Wapl - Pds5 . CohesinHalo488 was loaded onto in presence of Scc2 - Scc4 washed in high - salt buffer . - bound cohesin was further incubated with or without Wapl - Pds5 , then cohesinHalo488 particles were observed in presence of ATP 100 mM KCl . Scale bar , 1 μm . ( B ) MSD versus time of cohesin particles in ( A ) are shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( C ) Acetylated - or unacetylated - cohesinHalo488 was loaded onto in presence of Scc2 - Scc4 washed in high - salt buffer . - bound cohesin was further incubated with Wapl - Pds5 , then cohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( D ) Acetylated cohesinHalo488 was treated as in ( C ) , - bound cohesin was further incubated with buffer , Wapl - Pds5 , or Wapl - Pds5 plus Sororin , then cohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Acetylated cohesinHalo488 was loaded onto in presence of Scc2 - Scc4 washed in high - salt buffer . - bound cohesin was further incubated with Wapl - Pds5 plus Sororin , then treated with buffer , Plk1 , or Plk1 plus BI4834 . CohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( B ) Acetylated cohesinHalo488 was treated as in ( A ) , or treated with CDK1 or Aurora B rather than Plk1 . Sororin on was measured after immunofluorescence microscopy with anti - Sororin antibody ( n > 100 , * p < 0 . 0001 , two - Mann - Whitney ) .

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

(B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Acetylated cohesinHalo488 was treated as in ( A ) but treated with Aurora B rather than Plk1 . CohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Stretched was incubated in HSS in presence or absence of geminin . After washing HSS , endogenous Smc3 ( green ) Mcm2 ( magenta ) were detected by immunofluorescence . was counterstained with SYTOX ( gray ) . Scale bar , 2 µm . ( B ) Numbers of Smc3 Mcm2 signals were counted on 10 μm shown in ( A ) . ( 22 DNAs per condition , n = 3 , mean ± s . e . m . * p < 0 . 0001 , two - Mann - Whitney ) .

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

(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test).


( C ) formed in HSS in presence of geminin , cohesinHalo488 ( green ) was loaded onto with Scc2 - Scc4 . was detected by immunostaining of histone H3 ( magenta ) . was counterstained with SYTOX ( gray ) . distributions of cohesin particles histone H3 are shown as - scan plots ( bottom ) . Scale bar , 5 μm . ( D ) Cohesin particles observed in ( C ) were categorized as indicated ( > 37 particles per condition . n = 6 , mean ± s . e . m . ) . ( E ) Cohesin particle numbers for each condition . Red bars denote median , lower , upper quartile values ( n ≥ 12 , * p < 0 . 0004 , unpaired t - ) . ( F ) Colocalization of Mcm2 with Smc3 was quantified ( 50 Mcm2 particles per condition , n = 3 , mean ± s . e . m . )

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

(C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)


( A ) Stretched was incubated in HSS . Endogenous Smc3 ( green ) Mcm2 ( magenta ) were detected by immunofluorescence . was counterstained with SYTOX ( gray ) . Colocalization of Smc3 Mcm2 was quantified ( bottom , n = 3 , mean ± s . e . m . ) . Scale bar , 2 μm .

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

(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.


( B ) Fluorescently labeled Xenopus cohesins ( xCohesinHaloTMR ) were loaded onto in HSS , - bound cohesin particles were observed in HSS with or without 5 mM AMP - PNP supplementation . On kymograph , magenta lines indicate duration of binding of each cohesin particle . was counterstained with SYTOX . Scale bar , 5 μm .

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

(B) Fluorescently labeled Xenopus cohesins (xCohesinHaloTMR) were loaded onto DNA in the HSS, and DNA-bound cohesin particles were observed in the HSS with or without 5 mM AMP-PNP supplementation. On the kymograph, the magenta lines indicate the duration of DNA binding of each cohesin particle. DNA was counterstained with SYTOX. Scale bar, 5 μm.


( C ) Kymographs of - bound xCohesinHaloTMR ( green ) in HSS . After time - lapse imaging of xCohesinHaloTMR , nucleosomes were immunostained by anti - H3 antibody ( magenta ) . Result of H3 immunostaining was merged with kymograph . was counterstained with SYTOX . Scale bar , 5 μm .

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

(C) Kymographs of DNA-bound xCohesinHaloTMR (green) in HSS. After time-lapse imaging of xCohesinHaloTMR, nucleosomes were immunostained by anti-H3 antibody (magenta). Result of H3 immunostaining was merged with the kymograph. DNA was counterstained with SYTOX. Scale bar, 5 μm.


( D ) HSS were subjected to mock or XEco2 immunodepletion human Esco1 - His ( hEsco1 ) was added back to XEco2 - depleted HSS . Sperm was incubated in these extracts - bound were analyzed by immunoblotting .

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

(D) HSS were subjected to mock or XEco2 immunodepletion and human Esco1-His (hEsco1) was added back to XEco2-depleted HSS. Sperm chromatin was incubated in these extracts and chromatin-bound proteins were analyzed by immunoblotting.


( E ) Kymographs of - bound xCohesinHaloTMR in mock - , XEco2 - depleted , or hEsco1 - added XEco2 - depleted HSS . Scale bar , 2 μm . ( F ) MSD versus time of cohesin particle in experiment in ( E ) . D indicates diffusion coefficient ( n = 15 , mean ± s . e . m . ) .

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

(E) Kymographs of DNA-bound xCohesinHaloTMR in mock-, XEco2-depleted, or hEsco1-added XEco2-depleted HSS. Scale bar, 2 μm.(F) MSD versus time of cohesin particle in the experiment in (E). D indicates the diffusion coefficient (n = 15, mean ± s.e.m.).


( B ) Kymographs of examples for cohesin motion during replication . xCohesinHaloTMR ( magenta ) was loaded onto in HSS , NPE was introduced to allow replication . replication was monitored by xFen1D179A - PAGFP ( green ) . Thirthy - four particles were categorized into five groups . Arrowheads indicate time points when cohesin was dissociated from . Scale bar , 2μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymographs of examples for cohesin motion during DNA replication. xCohesinHaloTMR (magenta) was loaded onto DNA in the HSS, and NPE was introduced to allow DNA replication. DNA replication was monitored by xFen1D179A-PAGFP (green). Thirthy-four particles were categorized into five groups. Arrowheads indicate the time points when cohesin was dissociated from chromatin. Scale bar, 2μm.


A Hypocotyl lengths of wild - type suc2 seedlings grown under continuous at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . seedlings were grown on medium with or without 3 % sucrose . Representative seedlings are shown in upper panel . Different letters above each box indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ; n = 10 ) . Numbers indicate of hypocotyl lengths ( 28 ° C / 20 ° C ) . In box plots ( A - D ) , thick lines indicate median values , lower upper ends of boxes represent 25th 75th percentiles , ends of whiskers are set at 1 . 5 times interquartile range .

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

A Hypocotyl lengths of wild-type and suc2 seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium with or without 3% sucrose. Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). In the box plots (A-D), the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


B Hypocotyl lengths of wild - type tps1 - 2 ; GVG : : TPS1 seedlings grown under same growth conditions Representative seedlings are shown in upper panel . Different letters above each box indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ; n > 10 ) . Numbers indicate of hypocotyl lengths ( 28 ° C / 20 ° C ) .

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

B Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under the same growth conditions Representative seedlings are shown in the upper panel. Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 10). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C).


C Hypocotyl lengths of wild - type tps1 - 2 ; GVG : : TPS1 seedlings grown under continuous at 20 ° C for 4 days followed by 3 days at 28 ° C . seedlings were grown on medium containing either mock or 20 µM of dexamethasone ( DEX ) . ns , not significant ( Student ' s t - P ≥ 0 . 05 ; n > 10 ) . * * P < 0 . 01 ( Student ' s t - ) ; n > 10 . Number indicates of hypocotyl lengths ( Dex / Mock ) .

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

C Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown under continuous white light at 20 °C for 4 days followed by 3 days at 28 °C. The seedlings were grown on the medium containing either mock or 20 µM of dexamethasone (DEX). ns, not significant (Student's t-test P ≥ 0.05; n > 10). **P < 0.01 (Student's t-test); n > 10. Number indicates the ratio of hypocotyl lengths (Dex/Mock).


D Hypocotyl lengths of wild - type tps1 - 2 ; GVG : : TPS1 seedlings grown in at 20 ° C for 7 days . Representative seedlings are shown in left panel . ns , not significant ( Student ' s t - P ≥ 0 . 05 ; n > 10 ) .

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

D Hypocotyl lengths of wild-type and tps1-2;GVG::TPS1 seedlings grown in the dark at 20 °C for 7 days. Representative seedlings are shown in the left panel. ns, not significant (Student's t-test P ≥ 0.05; n > 10).


F - I qRT - PCR analyses of transcript of YUC8 , IAA19 , IAA29 , PIF4 . Transcript of each gene were normalized to those of PP2A presented as values relative to those of wild - type seedlings at 20 ° C . Error bars indicate s . d . ( n = 3 ) . Different letters above each bar indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ) . Numbers indicate of transcript in seedlings grown at two different temperatures ( 28 ° C / 20 ° C ) .

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

F-I The qRT-PCR analyses of the transcript levels of YUC8, IAA19, IAA29, and PIF4. Transcript levels of each gene were normalized to those of PP2A and presented as values relative to those of wild-type seedlings at 20 °C. Error bars indicate s.d. (n = 3). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05). Numbers indicate the ratio of transcript levels in seedlings grown at two different temperatures (28 °C/20 °C).


A Western blotting with anti - PIF4 antibody shows that PIF4 are decreased in tps1 - 2 ; GVG : : TPS1 mutants compared to wild - type . Seedlings were maintained under 12 - h / 12 - h cycles at 20 ° C for 4 days then transferred under continuous on 5th day . growth was increased to 28 ° C or kept at 20 ° C for 4 hours at ZT 20 - 24 before harvesting for total extraction . Equal loading of samples is shown by Ponceau S .

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

A Western blotting with anti-PIF4 antibody shows that PIF4 protein levels are decreased in the tps1-2;GVG::TPS1 mutants compared to wild-type plants. Seedlings were maintained under 12-h light/12-h dark cycles at 20 °C for 4 days and then transferred under continuous light on the 5th day. The growth temperature was increased to 28 °C or kept at 20°C for 4 hours at ZT 20-24 before harvesting for total protein extraction. Equal loading of samples is shown by Ponceau S staining.


B Western blotting showing of PIF4 - Myc in PIF4 - OX PIF4 - OX ; tps1 - 2 ; GVG : : TPS1 seedlings . Total was extracted from seedlings grown under continuous at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Western blotting was probed using an anti - Myc antibody . Ponceau S is shown for equal loading .

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

B Western blotting showing the levels of PIF4-Myc protein in PIF4-OX and PIF4-OX;tps1-2;GVG::TPS1 seedlings. Total protein was extracted from the seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Western blotting was probed using an anti-Myc antibody. Ponceau S staining is shown for equal loading.


C , D Hypocotyl lengths in seedlings of different genotypes . Seedlings were grown under continuous at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Representative seedlings are shown in ( C ) . Numbers in ( D ) indicate of hypocotyl lengths ( 28 ° C / 20 ° C ) . Different letters above each box indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ; n > 12 ) . In box plots , thick lines indicate median values , lower upper ends of boxes represent 25th 75th percentiles , ends of whiskers are set at 1 . 5 times interquartile range .

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

C, D Hypocotyl lengths in seedlings of different genotypes. Seedlings were grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (C). Numbers in (D) indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 12). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


A , B Hypocotyl lengths of wild - type KIN10 - OX seedlings grown under continuous at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Representative seedlings are shown in ( A ) . Numbers indicate of hypocotyl lengths ( 28 ° C / 20 ° C ) . Different letters above each box indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ; n > 15 ) . In box plots , thick lines indicate median values , lower upper ends of boxes represent 25th 75th percentiles , ends of whiskers are set at 1 . 5 times interquartile range .

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

A, B Hypocotyl lengths of wild-type and KIN10-OX seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (A). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 15). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


C Transient gene expression assays . reporter vector containing 35S : : Renilla luciferase IAA19p : : Firefly luciferase was co - transfected with 35S : : PIF4 35S : : KIN10 into Arabidopsis mesophyll protoplasts . activity of Firefly luciferase under control of IAA19 promoter was normalized to that of Renilla luciferase . Error bars indicate s . d . ( n = 4 ) . Different letters above each bar indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ) . T , 35S terminator .

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

C Transient gene expression assays. The reporter vector containing 35S::Renilla luciferase and IAA19p::Firefly luciferase was co-transfected with 35S::PIF4 and 35S::KIN10 into Arabidopsis mesophyll protoplasts. The activity of Firefly luciferase under the control of IAA19 promoter was normalized to that of Renilla luciferase. Error bars indicate s.d. (n = 4). Different letters above each bar indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05). T, 35S terminator.


D , E Hypocotyl lengths of seedlings grown under continuous at 20 ° C for 7 days or at 20 ° C for 4 days followed by 3 days at 28 ° C . Representative seedlings are shown in ( D ) . Numbers indicate of hypocotyl lengths ( 28 ° C / 20 ° C ) . Different letters above each box indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ; n > 15 ) . In box plots , thick lines indicate median values , lower upper ends of boxes represent 25th 75th percentiles , ends of whiskers are set at 1 . 5 times interquartile range .

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

D, E Hypocotyl lengths of seedlings grown under continuous white light at 20 °C for 7 days or at 20 °C for 4 days followed by 3 days at 28 °C. Representative seedlings are shown in (D). Numbers indicate the ratio of hypocotyl lengths (28 °C/20 °C). Different letters above each box indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n > 15). In the box plots, the thick lines indicate median values, the lower and upper ends of the boxes represent the 25th and 75th percentiles, and the ends of the whiskers are set at 1.5 times the interquartile range.


B , C Yeast two - hybrid assays showing direct interaction between PIF4 KIN10 . Yeast clones were grown on synthetic dropout medium with histidine ( + HIS ) or without histidine ( - HIS ) plus 25 mM of 3 - - 1 , 2 , 4 - triazole ( 3 - AT ) .

B, C Yeast two-hybrid assays showing the direct interaction between PIF4 and KIN10. Yeast clones were grown on the synthetic dropout medium with histidine (+HIS) or without histidine (-HIS) plus 25 mM of 3-amino-1,2,4-triazole (3-AT).


D Co - IP assays showing interaction between PIF4 KIN10 in vivo . extracts from protoplasts expressing PIF4 - Myc KIN10 - GFP ( or GFP ) were immunoprecipitated with anti - GFP antibody analyzed by immunoblotting with anti - GFP or anti - Myc antibody .

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

D Co-IP assays showing the interaction between PIF4 and KIN10 in vivo. Protein extracts from protoplasts expressing PIF4-Myc and KIN10-GFP (or GFP) were immunoprecipitated with anti-GFP antibody and analyzed by immunoblotting with anti-GFP or anti-Myc antibody.


E GRIK1 - activated KIN10 phosphorylates PIF4 . In vitro phosphorylation assays were performed at 30 ° C for 30 min using 1 μg of purified recombinant ( KIN10 , GRIK1 , or PIF4 ) . 32P - labeled were visualized by autoradiography . CBB , coomassie brilliant blue - stained gel .

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

E GRIK1-activated KIN10 phosphorylates PIF4. In vitro phosphorylation assays were performed at 30 °C for 30 min using 1 μg of purified recombinant proteins (KIN10, GRIK1, or PIF4). 32P-labeled proteins were visualized by autoradiography. CBB, coomassie brilliant blue-stained gel.


F T6P inhibits PIF4 phosphorylation by KIN10 . This was performed with 1 μg of each at 30 ° C for 30 min in presence of 1 mM T6P or sucrose ( Suc ) .

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

F T6P inhibits PIF4 phosphorylation by KIN10. This assay was performed with 1 μg of each protein at 30 °C for 30 min in the presence of 1 mM T6P or sucrose (Suc).


of PIF4 - Myc are negatively correlated with of KIN10 - GFP . Total were extracted from mesophyll protoplasts transfected with different amounts of KIN10 - GFP PIF4 - Myc analyzed by immunoblotting with anti - GFP or anti - Myc antibody . Ponceau S is shown for equal loading .

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

The levels of PIF4-Myc are negatively correlated with the levels of KIN10-GFP. Total proteins were extracted from mesophyll protoplasts transfected with different amounts of KIN10-GFP and PIF4-Myc and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal loading.


KIN10 reduces stability of PIF4 through 26S proteasome - mediated degradation . Transfected mesophyll protoplasts were pre - treated with or without 20 µM MG132 for 4 hours then incubated in presence of 100 µM cycloheximide ( CHX ) for indicated time . Total was extracted from protoplasts , analyzed by immunoblotting with anti - GFP or anti - Myc antibody . Ponceau S is shown for equal loading . Representative western blots are shown in ( B ) .

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

KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Ponceau S staining is shown for equal protein loading. Representative western blots are shown in (B).


KIN10 reduces stability of PIF4 through 26S proteasome - mediated degradation . Transfected mesophyll protoplasts were pre - treated with or without 20 µM MG132 for 4 hours then incubated in presence of 100 µM cycloheximide ( CHX ) for indicated time . Total was extracted from protoplasts , analyzed by immunoblotting with anti - GFP or anti - Myc antibody . of relative PIF4 - Myc is shown in ( C ) . PIF4 - Myc are presented as values relative to those at 0 h . In ( C ) , different letters above each point indicate statistically significant differences ( ANOVA Tukey ' s HSD ; P < 0 . 05 ; n = 3 ) . Error bars indicate s . d . ( n = 3 ) .

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

KIN10 reduces the stability of PIF4 through 26S proteasome-mediated degradation. Transfected mesophyll protoplasts were pre-treated with or without 20 µM MG132 for 4 hours and then incubated in the presence of 100 µM cycloheximide (CHX) for the indicated time. Total protein was extracted from the protoplasts, and analyzed by immunoblotting with anti-GFP or anti-Myc antibody. Quantification of relative PIF4-Myc levels is shown in (C). PIF4-Myc levels are presented as values relative to those at 0 h. In (C), different letters above each point indicate statistically significant differences (ANOVA and Tukey's HSD; P < 0.05; n = 3). Error bars indicate s.d. (n = 3).


Western blotting showing endogenous PIF4 in seedlings grown Total extracted from seedlings was analyzed by immunoblotting with anti - PIF4 antibody . Ponceau S shows equal loading .

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

Western blotting showing endogenous PIF4 protein levels in the seedlings grown Total protein extracted from the seedlings was analyzed by immunoblotting with anti-PIF4 antibody. Ponceau S staining shows equal protein loading.


( a - d ) Onset of rod visualized by for envelope LaminA ( a ) , caspase3 ( arrowheads , magenta red signal , b ) TUNEL ( arrows , brown signal , c , d ) . Blue in a b shows DAPI . A retinal flat mount with a view onto photoreceptor layer is shown in d .

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

(a-d) Onset of rod death visualized by staining for cleaved nuclear envelope protein LaminA (a), cleaved caspase3 (arrowheads, magenta and red signal, b) and TUNEL (arrows, brown signal, c, d). Blue in a and b shows nuclear DAPI staining. A retinal flat mount with a view onto the photoreceptor layer is shown in d.


( e - h ) Progression of rod determined by reduction of ONL was visualized by hematoxylin eosin .

(e-h) Progression of rod death determined by the reduction of the ONL was visualized by hematoxylin and eosin staining.


( i - q ) End phase of rod was assessed by section ( i - l ) or by retinal flat mounts ( m - q ) . In Rho − / − mouse , onset of rod was around PW5 ( a ) progressed up to PW25 ( l ) . By PW17 , ONL was reduced to one row of ( h , j ) remaining rods died in following 8 ( j - q ) , as seen by immunofluorescence with an antibody to Gnat1 on sections of progressively older ( j - l ) . Retinal flat mounts showing rods visualized by immunofluorescence microscopy with an antibody Gnat1 are shown in m - q . entire retina is shown in m , higher magnifications around optic nerve head are shown in n o , peripheral region is shown in p . No signal was seen at PW25 on flat mounts ( q ) or sections ( l ) . ( in PW ) is indicated in panels . Vertical bar in a - c e - l indicates thickness of ONL .

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

(i-q) End phase of rod death was assessed by section analysis (i-l) or by retinal flat mounts (m-q). In the Rho−/− mouse, the onset of rod death was around PW5 (a) and progressed up to PW25 (l). By PW17, the ONL was reduced to one row of cells (h, j) and the remaining rods died in the following 8 weeks (j-q), as seen by immunofluorescence with an antibody to Gnat1 on sections of progressively older animals (j-l). Retinal flat mounts showing rods visualized by immunofluorescence microscopy with an antibody Gnat1 are shown in m-q. The entire retina is shown in m, higher magnifications around the optic nerve head are shown in n and o, and the peripheral region is shown in p. No signal was seen at PW25 on flat mounts (q) or sections (l). Age (in PW) is indicated in the panels. Vertical bar in a-c and e-l indicates thickness of the ONL.


( a ) qRT - PCR for Opn1sw during cone degeneration .

(a) qRT-PCR analysis for Opn1sw during cone degeneration.


( b - o ) panels show retinal flat mounts except for i l . Green signal indicates PNA expression red signal indicates red / green or blue opsin . wild - type retina is shown at P35 in b - d . Red / green opsin ( b ) PNA ( c , d ) expression were detected both dorsally ventrally , whereas blue opsin ( c , d ) was detected only ventrally .

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

(b-o) All panels show retinal flat mounts except for i and l. Green signal indicates PNA expression and red signal indicates red/green or blue opsin. The wild-type retina is shown at P35 in b-d. Red/green opsin (b) and PNA (c, d) expression were detected both dorsally and ventrally, whereas blue opsin (c,d) was detected only ventrally.


( b - o ) panels show retinal flat mounts except for i l . Green signal indicates PNA expression red signal indicates red / green or blue opsin . wild - type retina is shown at P35 in b - d . Red / green opsin ( b ) PNA ( c , d ) expression were detected both dorsally ventrally , whereas blue opsin ( c , d ) was detected only ventrally . Pde6b − / − mouse is analyzed in e - g j - o . A central to peripheral gradient of PNA shortening of cone outer segments is shown in e - g . At P20 , there were fewer elongated outer segments in center ( e ) as compared with periphery . Higher magnifications of a central or peripheral outer segment from e are shown in f g , a wild - type outer segment is shown in h ( marks outer segment ) . A of outer segment at 3 is shown in i ( error bars represent s . d . of 15 measurements each ) . With shortening of outer segments during degeneration , red / green opsin was localized throughout of , PNA , which detects an extracellular ( s ) , was reduced to a small dot attached to residual outer segment ( j ) ( yellow shows red / green PNA overlap , arrow ) . A higher magnification of a cone showing red / green at is displayed in k ( arrow ) . A cross section showing red / green in is shown in l ( arrows ) ( P70 ; j - l ) . During degeneration , red / green opsin was detected mainly dorsally ( m ) , whereas PNA ( m , n ) or blue opsin ( o ) expression were not altered ( m n , P21 ; o , P49 ) . GCL , ganglion layer ; INL , inner layer .

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

(b-o) All panels show retinal flat mounts except for i and l. Green signal indicates PNA expression and red signal indicates red/green or blue opsin. The wild-type retina is shown at P35 in b-d. Red/green opsin (b) and PNA (c, d) expression were detected both dorsally and ventrally, whereas blue opsin (c,d) was detected only ventrally. The Pde6b−/− mouse is analyzed in e-g and j-o. A central to peripheral gradient of PNA and shortening of cone outer segments is shown in e-g. At P20, there were fewer elongated outer segments in the center (e) as compared with the periphery. Higher magnifications of a central or peripheral outer segment from e are shown in f and g, and a wild-type outer segment is shown in h (white line marks the outer segment). A quantification of outer segment length at 3 weeks is shown in i (error bars represent s.d. of 15 measurements each). With the shortening of outer segments during degeneration, red/green opsin was localized throughout the membrane of the cell body, and PNA, which detects an extracellular protein(s), was reduced to a small dot attached to the residual outer segment (j) (yellow shows red/green and PNA overlap, arrow). A higher magnification of a cone showing red/green at the membrane is displayed in k (arrow). A cross section showing red/green in cell body is shown in l (arrows) (P70; j-l). During degeneration, red/ green opsin was detected mainly dorsally (m), whereas PNA (m, n) or blue opsin (o) expression were not altered (m and n, P21; o, P49). GCL, ganglion cell layer; INL, inner nuclear layer.


( b ) Summary of rod cone kinetics . Time is indicated in postnatal days or . ( c ) Immunofluorescence microscopy on retinal flat mounts showing ventral reduction of red / green opsin expression found in four mutants during cone degeneration . time , in postnatal days , are indicated below each image ( for higher magnification of wild type , see Fig . 2b ) .

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

(b) Summary of rod and cone death kinetics. Time is indicated in postnatal days or weeks. (c) Immunofluorescence microscopy on retinal flat mounts showing the ventral reduction of red/green opsin expression found in the four mutants during cone degeneration. Strain and time, in postnatal days, are indicated below each image (for higher magnification of wild type, see Fig. 2b).


( a - c ) P - mTOR in wild - type retinae . Dorsal ( top ) enrichment of P - mTOR ( a ) . Higher magnifications of dorsal ventral regions are shown to right , with P - mTOR in red cone segments in green , as detected by PNA . Dorsal retinal sections stained for P - mTOR ( red ) PNA ( b ) or antibody to β - galactosidase ( green , c ) . Higher magnification ( insets ) suggest that P - mTOR signal is located in lower part of outer segment ( OS ) . IS , inner segment .

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

(a-c) P-mTOR levels in wild-type retinae. Dorsal (top) enrichment of P-mTOR (a). Higher magnifications of the dorsal and ventral regions are shown to the right, with P-mTOR in red and cone segments in green, as detected by PNA. Dorsal retinal sections stained for P-mTOR (red) and PNA (b) or antibody to β-galactosidase (green, c). Higher magnification (insets) images suggest that the P-mTOR signal is located in the lower part of the outer segment (OS). IS, inner segment.


( d - g ) Rapamycin treatment of wild - type mice led to downregulation of red / green opsin ventrally ( e ) but not dorsally ( red , d ) . Ventral blue opsin ( red , f ) remained unaffected , as did PNA ( d - g ) ( green ) P - mTOR itself ( red , g ) .

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

(d-g) Rapamycin treatment of wild-type mice led to downregulation of red/green opsin ventrally (e) but not dorsally (red, d). Ventral blue opsin (red, f) remained unaffected, as did PNA (d-g) (green) and P-mTOR itself (red, g).


( h - m ) Reduced of dorsal P - mTOR during photoreceptor degeneration ( red ) . wild - type control is shown in h Pde6b mutant is shown in i j . Reduction started during rod at P15 ( i ) as outer segments ( green , PNA ) detached from retinal pigmented epithelium . By P30 , only a few cones ( green signal : α - β - galactosidase ) showed high of P - mTOR ( red ) ( j ) . ( k - m ) Similar reduction was seen in dorsal cones of other three mutants ( cones marked in green by PNA ; Pde6g − / − is shown at P35 in k ; Rho − / − is shown at PW20 in l ; P23H is shown at PW70 in m ) . panels show immunofluorescence microscopy on retinal flat mounts ( photoreceptor side up ) with exception of b , c g , which show retinal sections . Blue indicates DAPI .

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

(h-m) Reduced levels of dorsal P-mTOR during photoreceptor degeneration (red). The wild-type control is shown in h and the Pde6b mutant is shown in i and j. Reduction started during rod death at P15 (i) as the outer segments (green, PNA) detached from the retinal pigmented epithelium. By P30, only a few cones (green signal: α-β-galactosidase) showed high levels of P-mTOR (red) (j). (k-m) Similar reduction was seen in dorsal cones of the other three mutants (cones marked in green by PNA; Pde6g−/− is shown at P35 in k; Rho−/− is shown at PW20 in l; P23H is shown at PW70 in m). All panels show immunofluorescence microscopy on retinal flat mounts (photoreceptor side up) with the exception of b, c and g, which show retinal sections. Blue indicates DAPI.


panels show immunofluorescent . Blue shows DAPI green shows cones marked with PNA . ( a - f ) for HIF - 1α ( red signal ) . ( a ) Wild type ( PW10 ) stained for HIF - 1α ( inset shows higher magnification ) . ( b , c ) Cross sections in wild type stained for HIF - 1α ( PW10 ) . DAPI overlap is shown in c . ( d - f ) During cone degeneration in Pde6b − / − ( PW10 ) mice , we found increased of HIF - 1α in cones ( inset in d shows higher magnification with DAPI overlap ) . Cross sections indicated that increase of Hif - 1α occurred mainly in cones ( arrows point to cones that are located in top layer of inner layer at this stage ; e ) . DAPI overlap is shown in f .

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

All panels show immunofluorescent staining. Blue shows nuclear DAPI staining and green shows cones marked with PNA. (a-f) Staining for HIF-1α (red signal). (a) Wild type (PW10) stained for HIF-1α (inset shows higher magnification). (b, c) Cross sections in wild type stained for HIF-1α (PW10). DAPI overlap is shown in c. (d-f) During cone degeneration in Pde6b−/− (PW10) mice, we found increased levels of HIF-1α in cones (inset in d shows higher magnification with DAPI overlap). Cross sections indicated that the increase of Hif-1α occurred mainly in cones (arrows point to cones that are located in the top layer of the inner nuclear layer at this stage; e). DAPI overlap is shown in f.


( g ) GLUT1 expression in wild type ( PW10 ) ( red ) . Most of signal in between cones reflects expression in rods . ( h - j ) Increased expression of GLUT1 in cones during degeneration seen in flat mounts ( h ) sections ( i , j ) . PNA overlap of j is shown in i . Retinal flat mounts are shown in a , d , g h . Retinal sections are shown in b , c , e , f , i j .

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

(g) GLUT1 expression in wild type (PW10) (red). Most of the signal in between the cones reflects expression in rods. (h-j) Increased expression of GLUT1 in cones during degeneration seen in flat mounts (h) and sections (i, j). PNA overlap of j is shown in i. Retinal flat mounts are shown in a, d, g and h. Retinal sections are shown in b, c, e, f, i and j.


( a - c ) Immunofluorescence microscopy on retinal flat mounts ( LAMP - 2 is shown in green , red / green opsin in red DAPI in blue ) . Insets show enlarged ( arrow ) . A wild - type retinae at PW5 showing a lysosome ( small green dots ) with normal LAMP - 2 distribution is imaged in a . Weak red / green opsin signal was detected at level of photoreceptornuclei , as it was mainly found in outer segments in wild type . Enlarged lysosomes ( dots ) resulting from an accumulation of LAMP - 2 at lysosomal were seen specifically in cones from Pde6b − / − micePW5 ( b ) . Confocal section of same field as in b taken at level of inner layer showed LAMP - 2 similar to those in wild type ( c ) .

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

(a-c) Immunofluorescence microscopy on retinal flat mounts (LAMP-2 is shown in green, red/green opsin in red and DAPI in blue). Insets show enlarged cells (arrow). A wild-type retinae at PW5 showing a lysosome (small green dots) with normal LAMP-2 distribution is imaged in a. Weak red/green opsin signal was detected at the level of the photoreceptornuclei, as it was mainly found in the outer segments in the wild type. Enlarged lysosomes (dots) resulting from an accumulation of LAMP-2 at the lysosomal membrane were seen specifically in cones from Pde6b−/− micePW5 (b). Confocal section of same field as in b taken at the level of the inner nuclear layer showed LAMP-2 levels similar to those in wild type (c).


( d ) qRT - PCR for three different LAMP - 2 splice forms showing relative ratios between Pde6b − / − mutant wild type . Error bars represent s . d . of three measurements .

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

(d) qRT-PCR for the three different LAMP-2 splice forms showing the relative concentration and the ratios between the Pde6b−/− mutant and wild type. Error bars represent s.d. of three measurements.


( a - c ) Retinal flat mounts of Pde6b − / − mutants at PW7 stained for LacZ to detect cones ( see Methods Supplementary Fig . 9 ) . An example of untreated control is shown in a , an example of a mouse injected with streptozotocin is shown in b an example of a mouse injected daily with insulin is shown in c . ( d ) of cone survival after four of treatment . Data represents an average of at least eight retinae . y axis represents of cone surface versus surface of entire retina ( see Supplementary Figs . 8 10 ) .

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

(a-c) Retinal flat mounts of Pde6b−/− mutants at PW7 stained for LacZ to detect cones (see Methods and Supplementary Fig. 9). An example of untreated control is shown in a, an example of a mouse injected with streptozotocin is shown in b and an example of a mouse injected daily with insulin is shown in c. (d) Quantification of cone survival after four weeks of treatment. Data represents an average of at least eight retinae. The y axis represents the percentage of the cone surface area versus the surface area of entire retina (see Supplementary Figs. 8 and 10).


( e , f ) Measurements of blood glucose ( e ) ( f ) performed weekly over time span of experiment .

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

(e, f) Measurements of blood glucose levels (e) and body weight (f) performed weekly over the time span of the experiment.


( g , h ) Immunofluorescence microscopy on retinal flat mounts for HIF - 1α ( red ) PNA ( green ) in untreated control Pde6b − / − ( g ) Pde6b − / − mice treated for 4 with insulin ( h ) . DAPI is shown in blue . Error bars in d - f represent s . d .

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

(g, h) Immunofluorescence microscopy staining on retinal flat mounts for HIF-1α (red) and PNA (green) in untreated control Pde6b−/− (g) and Pde6b−/− mice treated for 4 weeks with insulin (h). DAPI is shown in blue. Error bars in d-f represent s.d.


a ) Rapamycin ( Rap ) treatment or nutrient deprivation attenuated expression of Wnt reporter TopFlash in HEK - 293T . ( n = 3 ; two asterisks , P 0 . 01 ) . DMSO , dimethylsulphoxide .

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

a) Rapamycin (Rap) treatment or nutrient deprivation attenuated the expression of the Wnt reporter TopFlash in HEK-293T cells. (n = 3; two asterisks, P 0.01). DMSO, dimethylsulphoxide.


( b ) Expression of Wnt target genes by quantitative RT - PCR after 4 h of treatment with rapamycin . ( n = 3 ; asterisk , P 0 . 05 ; two asterisks , P 0 . 01 ) . Wnt3a CM , Wnt3a conditioned medium .

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

(b) Expression analysis of Wnt target genes by quantitative RT-PCR after 4 h of treatment with rapamycin. (n = 3; asterisk, P 0.05; two asterisks, P 0.01). Wnt3a CM, Wnt3a conditioned medium.


( c ) Knockdown of LC3 or Beclin 1 potentiated Wnt signalling . ( n = 3 ; asterisk , P 0 . 05 ) .

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

(c) Knockdown of LC3 or Beclin 1 potentiated Wnt signalling. (n = 3; asterisk, P 0.05).


( d ) Atg5 knockout enhanced Wnt signalling . TopFlash reporter activity induced by Wnt3a CM in Atg5 − / − or wild - type MEFs . Results are means ± s . d . after normalization to Renilla luciferase activity . ( n = 3 ; asterisk , P 0 . 05 ) .

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

(d) Atg5 knockout enhanced Wnt signalling. TopFlash reporter activity induced by Wnt3a CM in Atg5−/− or wild-type MEFs. Results are means ± s.d. after normalization to Renilla luciferase activity. (n = 3; asterisk, P 0.05).


( a ) Dvl2 underwent autophagy - mediated degradation . E - cadherin was used as a loading control for fraction , tubulin as a loading control for . asterisk indicates phosphorylated form of Dvl2 .

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

(a) Dvl2 underwent autophagy-mediated degradation. E-cadherin was used as a loading control for membrane fraction, and tubulin as a loading control for cytosolic proteins. The asterisk indicates the phosphorylated form of Dvl2.


( b ) half - life of Dvl2 was prolonged in Atg5 − / − . MEFs were subjected to starvation ( starv . ) for indicated durations harvested for immunoblotting with indicated antibodies . Dvl2 were quantified ( right panel ) . Autophagy induction is indicated by of LC3 II p62 degradation .

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

(b) The half-life of Dvl2 was prolonged in Atg5−/− cells. MEFs were subjected to starvation (starv.) for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel). Autophagy induction is indicated by the levels of LC3 II and p62 degradation.


( c ) Reintroduction of Atg5 in Atg5 − / − MEFs restored starvation - induced Dvl2 degradation . MEFs infected with recombinant adenovirus expressing GFP or Atg5 were starved for indicated durations , lysates were subjected to immunoblotting with indicated antibodies .

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

(c) Reintroduction of Atg5 in Atg5−/− MEFs restored starvation-induced Dvl2 degradation. MEFs infected with recombinant adenovirus expressing GFP or Atg5 were starved for the indicated durations, and the cell lysates were subjected to immunoblotting with the indicated antibodies.


( d ) Dvl2 degradation was slowed down in Atg7 − / − MEFs . MEFs were subjected to starvation for indicated durations harvested for immunoblotting with indicated antibodies . Dvl2 were quantified ( right panel ) .

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

(d) Dvl2 degradation was slowed down in Atg7−/− MEFs. MEFs were subjected to starvation for the indicated durations and harvested for immunoblotting with the indicated antibodies. Dvl2 levels were quantified (right panel).


( e ) continued degradation of Dvl2 in Atg5 − / − is proteasome - dependent . MEFs were starved for 2 , 4 or 8 h with MG132 ( 1 μM ) or BFA1 ( 0 . 1 μM ) for different durations , followed by anti - Dvl2 immunoblotting to detect endogenous Dvl2 . Tubulin served as a loading control . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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

(e) The continued degradation of Dvl2 in Atg5−/− cells is proteasome-dependent. MEFs were starved for 2, 4 or 8 h with MG132 (1 μM) or BFA1 (0.1 μM) for different durations, followed by anti-Dvl2 immunoblotting to detect endogenous Dvl2 protein levels. Tubulin served as a loading control. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Immunoelectron microscopy of HeLa transfected with Flag - Dvl2 under nutrient - rich or starvation conditions . arrowheads indicate Flag - Dvl2 . Scale bars , 1 μm ( left ) ; 250 nm ( right ) .

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

(a) Immunoelectron microscopy analysis of HeLa cells transfected with Flag-Dvl2 under nutrient-rich or starvation conditions. The arrowheads indicate Flag-Dvl2. Scale bars, 1 μm (left); 250 nm (right).


( b ) Localization of endogenous Dvl ( red ) GFP - LC3 ( green ) in GFP - LC3 stable HeLa in nutrient - rich medium or under starvation ( starv . ) conditions with BFA1 for 4 h . right panel shows co - localization between Dvls GFP - LC3 - positive dots . Scale bar , 5 μm .

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

(b) Localization of endogenous Dvl proteins (red) and GFP-LC3 (green) in GFP-LC3 stable HeLa cells in nutrient-rich medium or under starvation (starv.) conditions with BFA1 for 4 h. The right panel shows the percentage co-localization between Dvls and GFP-LC3-positive dots. Scale bar, 5 μm.


( c ) Immunoelectron microscopy of HeLa transfected with Flag - Dvl2 GFP - LC3 under starvation conditions . Arrows indicate Flag - Dvl2 ; arrowheads indicate GFP - LC3 . Scale bar , 250 nm .

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

(c) Immunoelectron microscopy analysis of HeLa cells transfected with Flag-Dvl2 and GFP-LC3 under starvation conditions. Arrows indicate Flag-Dvl2; arrowheads indicate GFP-LC3. Scale bar, 250 nm.


( d ) Dvl2 - LC3 interaction in vivo . IB , immunoblotting ; IP , immunoprecipitation ; S , starvation ; WCL , whole - lysate ; N , nutrient - rich .

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

(d) Dvl2-LC3 interaction in vivo. IB, immunoblotting; IP, immunoprecipitation; S, starvation; WCL, whole-cell lysate; N, nutrient-rich.


( e , f ) Dvl2 interacts directly with LC3 ( e ) GABARAP ( f ) but not with GATE - 16 in vitro . GST pulldown using in vitro - translated Flag - Dvl2 immobilized GST or indicated GST fusions . Precipitated were detected with anti - Flag antibody . Coomassie of GST GST - LC3 is shown .

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

(e, f) Dvl2 interacts directly with LC3 (e) and GABARAP (f) but not with GATE-16 in vitro. GST pulldown assay using in vitro-translated Flag-Dvl2 and immobilized GST or the indicated GST fusions. Precipitated proteins were detected with anti-Flag antibody. Coomassie staining of GST and GST-LC3 protein is shown.


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


( g ) LC - 3 - binding motif is important for Dvl2 - LC3 interaction . GST - LC3 - immobilized beads were incubated with lysates from expressing wild - type ( WT ) Dvl2 or W444A , I447A mutant ( MUT ) ; pulldowns were then subjected to anti - haemagglutinin ( anti - HA ) immunoblotting . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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

(g) The LC-3-binding motif is important for the Dvl2-LC3 interaction. GST-LC3-immobilized beads were incubated with the lysates from the cells expressing wild-type (WT) Dvl2 or the W444A, I447A mutant (MUT); the pulldowns were then subjected to anti-haemagglutinin (anti-HA) immunoblotting. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Interaction between LC3 wild - type Dvl2 or its point mutants .

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

(a) Interaction between LC3 and wild-type Dvl2 or its point mutants.


( b ) Ubiquitylation of wild - type Dvl2 its variants . Transfected HEK - 293T were treated with BFA1 ( 0 . 2 μM ) for 6 h . Ubiquitylated were precipitated with Ni - nitrilotriacetate ( Ni - NTA ) beads , followed by anti - HA immunoblotting . Ub , ubiquitin .

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

(b) Ubiquitylation of wild-type Dvl2 and its variants. Transfected HEK-293T cells were treated with BFA1 (0.2 μM) for 6 h. Ubiquitylated proteins were precipitated with Ni-nitrilotriacetate (Ni-NTA) beads, followed by anti-HA immunoblotting. Ub, ubiquitin.


( c ) Degradation kinetics of wild - type Dvl2 its variants under starvation conditions . Lower panel : of immunoblotting results .

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

(c) Degradation kinetics of wild-type Dvl2 and its variants under starvation conditions. Lower panel: quantification of the immunoblotting results.


( d ) Starvation promotes co - localization of Dvl , ubiquitin p62 . Scale bar , 10 μm .

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

(d) Starvation promotes co-localization of Dvl, ubiquitin and p62. Scale bar, 10 μm.


( e ) Starvation induces formation of high - molecular - mass Dvl2 .

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

(e) Starvation induces the formation of high-molecular-mass Dvl2 aggregates.


( f ) p62 knockdown alters Dvl localization . Scale bar , 10 μm . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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

(f) p62 knockdown alters Dvl localization. Scale bar, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Knockdown of p62 expression attenuates LC3 - Dvl2 interaction . shRNA , short hairpin .

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

(a) Knockdown of p62 expression attenuates the LC3-Dvl2 interaction. shRNA, short hairpin RNA.


( b ) Co - localization of GFP - LC3 , p62 Dvl in HeLa GFP - LC3 stable on starvation . visualized by immunofluorescence with anti - GFP ( green ) , anti - p62 ( blue ) anti - Dvl ( red ) antibodies . Scale bar , 10 μm .

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

(b) Co-localization of GFP-LC3, p62 and Dvl in HeLa GFP-LC3 stable cells on starvation. Proteins visualized by immunofluorescence with anti-GFP (green), anti-p62 (blue) and anti-Dvl (red) antibodies. Scale bar, 10 μm.


( c ) p62 knockdown slows degradation of Dvl2 on starvation . HEK - 293T were transfected with p62 siRNA . At 48 h after transfection , were subjected to starvation for various times . lysates were subjected to immunoblotting with antibodies indicated . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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

(c) p62 knockdown slows the degradation of Dvl2 on starvation. HEK-293T cells were transfected with p62 siRNA. At 48 h after transfection, cells were subjected to starvation for various times. The cell lysates were subjected to immunoblotting with the antibodies indicated. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a ) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL .

(a) Ubiquitylation of Dvl2 enhanced by ectopically expressed pVHL.


( b ) In vitro ubiquitylation of Dvl2 by pVHL E3 ligase including pVHL , Elongin B , Elongin C , Rbx1 Cul2 . In vitro - translated Flag - Dvl2 was incubated with or without purified E1 , E2 E3 ligase , Elongin C pVHL . reaction was subjected to anti - Flag immunoprecipitation . Dvl2 ubiquitylation was examined by using anti - ubiquitin immunoblotting .

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

(b) In vitro ubiquitylation of Dvl2 by the pVHL E3 ligase complex including pVHL, Elongin B, Elongin C, Rbx1 and Cul2. In vitro-translated Flag-Dvl2 was incubated with or without purified E1, E2 and E3 ligase complex, Elongin C and pVHL. The reaction was subjected to anti-Flag immunoprecipitation. Dvl2 ubiquitylation was examined by using anti-ubiquitin immunoblotting.


( c ) Ubiquitylation of Dvl2 is decreased by pVHL knockdown .

(c) Ubiquitylation of Dvl2 is decreased by pVHL knockdown.


( d ) Starvation elevates Dvl2 - Cul2 interaction . After co - transfection with Myc - Cul2 Flag - Dvl2 for 36 h , HEK - 293T were cultured in nutrient - rich medium or starved with BFA1 ( 0 . 1 μM ) for 4 h . were harvested for anti - Myc immunoprecipitation followed by anti - Flag immunoblotting . Total expression was confirmed by immunoblotting with whole - lysates .

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

(d) Starvation elevates Dvl2-Cul2 interaction. After co-transfection with Myc-Cul2 and Flag-Dvl2 for 36 h, HEK-293T cells were cultured in nutrient-rich medium or starved with BFA1 (0.1 μM) for 4 h. Cells were harvested for anti-Myc immunoprecipitation followed by anti-Flag immunoblotting. Total protein expression was confirmed by immunoblotting with whole-cell lysates.


( e ) pVHL knockdown or UbKOGA blocks Dvl2 binding with p62 under starvation conditions .

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

(e) pVHL knockdown or UbKOGA blocks Dvl2 binding with p62 under starvation conditions.


( f ) Dvl2 ubiquitylation is required for its direct interaction with p62 . Dvl2 was ubiquitylated by pVHL E3 ligase in vitro , followed by in vitro binding with recombinant p62 .

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

(f) Dvl2 ubiquitylation is required for its direct interaction with p62. Dvl2 was ubiquitylated by the pVHL E3 ligase complex in vitro, followed by in vitro binding assay with recombinant p62.


( g ) pVHL knockdown decreases Dvl2 degradation under starvation conditions .

(g) pVHL knockdown decreases Dvl2 degradation under starvation conditions.


( h ) Knockdown of p62 or LC3 attenuates pVHL - promoted Dvl2 degradation under starvation conditions . Uncropped of blots are shown in Supplementary Information , Fig . S8 .

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

(h) Knockdown of p62 or LC3 attenuates the pVHL-promoted Dvl2 degradation under starvation conditions. Uncropped images of blots are shown in Supplementary Information, Fig. S8.


( a , b ) Depletion of Beclin 1 by shRNA promotes growth in soft agar . SW480 stably expressing Beclin 1 shRNA or control shRNA were seeded in soft agar treated with or without Dkk ( a ) ; colonies were then scored after 10 days ( b ) . Numbers in bars indicate numbers of colonies . Results are means ± s . d . ( n = 4 ) . ( Asterisk , P 0 . 05 ) . Scale bar , 200 μm .

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

(a, b) Depletion of Beclin 1 by shRNA promotes growth in soft agar. SW480 cells stably expressing Beclin 1 shRNA or control shRNA were seeded in soft agar and treated with or without Dkk (a); the colonies were then scored after 10 days (b). Numbers in bars indicate the numbers of colonies. Results are means ± s.d. (n = 4). (Asterisk, P 0.05). Scale bar, 200 μm.


( c ) Representative from colon cancer tissues by immunohistochemistry . Negative correlation between Beclin1 Dvl was observed in late stages of tumours , whereas positive correlation between p62 Dvl in late stages of tumours . Scale bar , 25 μm .

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

(c) Representative images from colon cancer tissues by immunohistochemistry staining. Negative correlation between Beclin1 and Dvl was observed in late stages of tumours, whereas positive correlation between p62 and Dvl in late stages of tumours. Scale bar, 25 μm.


( d ) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies . N , normal tissue ; T , tumour .

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

(d) Immunoblotting of human colon tumour samples versus normal tissues using indicated antibodies. N, normal tissue; T, tumour.


( D ) U2OS ( EGFP - BIR - 5085 ) expressing shRNAs for RAD51 , POLD3 or shRNA vector ( Ctrl ) were infected by lentiviruses encoding endonuclease I - SceI . of EGFP positive was assayed by FACS 4 days later ( left ) . Expression of RAD51 POLD3 is shown by Western blot ( right ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 , * * * * P < 0 . 0001 n . s . ( not significant ) P > 0 . 05 .

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

(D) U2OS (EGFP-BIR-5085) cells expressing shRNAs for RAD51, POLD3 or shRNA vector (Ctrl) were infected by lentiviruses encoding endonuclease I-SceI. The percentage of EGFP positive cells was assayed by FACS analysis 4 days later (left). Expression of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ****P < 0.0001 and n.s. (not significant) P>0.05.


( B ) U2OS WT or PIF1 - KO were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr then IdU for another 30 min . Labeled were processed for fiber . Representative of stalled or restarted forks forks with new origin firing were shown ( left ) . of restarted forks was quantified by analyzing of 110 - 130 for each experiment ( right ) . Experiments were repeated four times for each sample . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 , * * * P < 0 . 001 .

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

(B) U2OS WT or PIF1-KO cells were labeled with CldU for 30 min followed by incubation with 2 mM HU for 2 hr and then IdU for another 30 min. Labeled cells were processed for DNA fiber analysis. Representative images of stalled or restarted forks and forks with new origin firing were shown (left). The percentage of restarted forks was quantified by analyzing of 110-130 fibers for each experiment (right). Experiments were repeated four times for each sample. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.


( D ) U2OS ( EGFP - BIR - 5085 ) lines carrying Dox - inducible Cas9 / sgRNA - 5085 ( Dox - Cas9 ) or Cas9n / sgRNA - 5085 ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) assayed by FACS 2 days later ( left ) . U2OS ( EGFP - BIR - 5085 , Dox - Cas9 or Dox - Cas9n ) expressing shRNAs RAD51 , POLD3 PIF1 or shRNA vector ( Ctrl ) were incubated with 5 µg / ml Dox , FACS was performed after 2 days ( right ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 , * * * P < 0 . 001 .

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

(D) U2OS (EGFP-BIR-5085) cell lines carrying Dox-inducible Cas9/sgRNA-5085 (Dox-Cas9) or Cas9n/sgRNA-5085 (Dox-Cas9n) were incubated with or without Dox (5 µg/ml) and assayed by FACS analysis 2 days later (left). U2OS (EGFP-BIR-5085, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with 5 µg/ml Dox, and FACS analysis was performed after 2 days (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01, ***P < 0.001.