Figure 3A , Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells . The quantification is presented in the right panel ( mean ± SD or three biological replicates ) . D , MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D ) for the indicated times . TGFβ receptor inhibitor SB505124 ( SB , 5 μM ) was added when indicated in D . Cells were lysed and proteins analyzed by WB . Quantification of the protein levels from D is represented at the lower panel ( mean ± SD of three biological replicates ) E , GFP - labelled MSC were seeded in Matrigel - coated transwells with SW620 and HCT116 cells , and supplemented with SB when indicated . After 48 h non - invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed . GFP - labelled cells were counted in five different fields per filter by ImageJ software and represented ( bottom ) . A representative picture of SW620 cells in each condition is shown ( top ) . The bar corresponds to 500 μm . The figure shows the mean ± SD of three experiments .

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

Figure 3A, Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells. The quantification is presented in the right panel (mean ± SD or three biological replicates).D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates)E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.


Figure 4C , Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft - agar colony formation ( 21 days ) ( left ) . Representative micrographs of SW620 cells are shown in Appendix Fig S7B . Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments ( right ) . Cells were seeded for tumor - sphere formation in tumor organoid complete medium . In E , SW620 cells were supplemented with LGK ( 10 μM ) , starting the day of seeding . After four days , the wells were stained ( top ) and the number of colonies counted and represented ( bottom ) ( mean ± SD of three biological replicates ) . A more detailed micrograph of the colonies is shown in Appendix Fig S7D . F , RNA was isolated from SW620 control , stably - depleted of Ror2 , treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5 , ASCL2 , SMOC2 and the differentiation marker KRT20 was assessed by qRT - PCR . The figure shows the mean ± SD of at least three biological replicates ( performed in triplicate ) represented with respect to the corresponding control ( dashed line ) .

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

Figure 4C, Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft-agar colony formation (21 days) (left). Representative micrographs of SW620 cells are shown in Appendix Fig S7B. Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments (right).Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).


Figure 5B , C , Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin - embedded tumors obtained when tumors were initially detected ( days 18 or 22 ) . Staining with an anti - human antibody is also enclosed . The scale bar corresponds to 10 μm ( B ) . Fibronectin and Vimentin staining were quantified as described in Methods ( six tumors , three areas per tumor ) and represented in C ( mean ± SD ) . D , Representative images of the invading front of the tumors . The scale bar corresponds to 4 μM . E , Percentage of tumors showing muscle invasion .

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

Figure 5B, C, Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin-embedded tumors obtained when tumors were initially detected (days 18 or 22). Staining with an anti-human antibody is also enclosed. The scale bar corresponds to 10 μm (B). Fibronectin and Vimentin staining were quantified as described in Methods (six tumors, three areas per tumor) and represented in C (mean ± SD).D, Representative images of the invading front of the tumors. The scale bar corresponds to 4 μM. E, Percentage of tumors showing muscle invasion.


Figure 6F , G , In vivo tumor growth of MTO cells subcutaneously injected in NOD / SCID mice ( F ) ( 5x105 cells / flank ; eight tumors per condition ) and treated with WntC59 ( 10 mg / kg / day in the drinking water ) or vehicle . Tumor weight of NOD / SCID mice at day 26 is presented in G . In F and G , the mean ± SD of the eight tumors is shown . I , J , WB ( I ) and RNA analysis ( J ) of MTO cells infected with shRor2 or shCtl . Quantification of the WB is also presented ( I , right ) . Results are the mean ± SD of three biological replicates ( performed in triplicated in J ) . N , O , C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle . In N , kinetics of in vivo luminescence ( mean ± SD of the values in five mice ) ; in O , the percentage of mice showing in vivo luminescence .

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

Figure 6F, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.I, J, WB (I) and RNA analysis (J) of MTO cells infected with shRor2 or shCtl. Quantification of the WB is also presented (I, right). Results are the mean ± SD of three biological replicates (performed in triplicated in J).N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.


Figure 7A - C , Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h . In B , cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid . The figure shows the mean ± SD of the results of three experiments . D , Cells were treated with cisplatin for the indicated times . Total extracts were lysed and analyzed by WB . I , MTO cell viability was determined as above ( mean ± SD of the results of three - four experiments ) .

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

Figure 7A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.I, MTO cell viability was determined as above (mean ± SD of the results of three-four experiments).


Figure 1A Quantification and representative immunoblots of lamin B1 in the striatum ( 8w , 12w and 20w ; N = 6 for both genotypes ; 30w ; N = 4 and 5 for WT and R6 / 1 mice , respectively ) , hippocampus ( 8w ; N = 8 and 6 for WT and R6 / 1 mice , respectively ; 12w ; N = 7 and 6 for WT and R6 / 1 mice , respectively ; 20w N = 5 and 6 for WT and R6 / 1 mice , respectively ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) and cortex ( 8w ; N = 5 and 4 for WT and R6 / 1 mice , respectively ; 12w and 20w ; N = 6 for both genotypes ; 30w ; N = 6 and 5 for WT and R6 / 1 mice , respectively ) . Data information : Data is expressed as a percentage of controls . Each point corresponds to the value from an individual sample . Bars represent the mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 as compared with WT mice ( two - tailed unpaired Student ' s t test ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 . B Quantification and representative immunoblots of lamin B2 in the striatum ( 8w , 12w and 20w ; N = 6 for both genotypes , 30w ; N = 5 for both genotypes ) , hippocampus ( 8w ; N = 8 and 6 for WT and R6 / 1 mice , respectively ; 12w ; N = 6 and 7 for WT and R6 / 1 mice , respectively ; 20w ; N = 5 for both genotypes ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) and cortex ( 8w and 12w ; N = 6 for both genotypes ; 20w ; N = 5 for both genotypes ; 30w ; N = 8 and 5 for WT and R6 / 1 mice , respectively ) . Data information : Data is expressed as a percentage of controls . Each point corresponds to the value from an individual sample . Bars represent the mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 as compared with WT mice ( two - tailed unpaired Student ' s t test ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 .

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Figure 1A Quantification and representative immunoblots of lamin B1 in the striatum (8w, 12w and 20w; N = 6 for both genotypes; 30w; N = 4 and 5 for WT and R6/1 mice, respectively), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 7 and 6 for WT and R6/1 mice, respectively; 20w N = 5 and 6 for WT and R6/1 mice, respectively; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w; N = 5 and 4 for WT and R6/1 mice, respectively; 12w and 20w; N = 6 for both genotypes; 30w; N = 6 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3. B Quantification and representative immunoblots of lamin B2 in the striatum (8w, 12w and 20w; N = 6 for both genotypes, 30w; N = 5 for both genotypes), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 6 and 7 for WT and R6/1 mice, respectively; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w and 12w; N = 6 for both genotypes; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3.


Figure 2A Mouse brain tissue was processed for immunohistochemistry by combining anti - lamin B1 ( red ) and anti - NeuN ( green ) antibodies . Representative images ( maximal Z - projections ) showing the distribution of lamin B1 in the striatum of 30 - week - old wild - type ( WT ) and R6 / 1 mice . Yellow arrowheads show co - localization between lamin B1 and NeuN , and white arrowheads show NeuN negative lamin B1 positive nuclei . Scale bar 50 μm and 25 μm for low and high magnification , respectively . B Lamin B1 distribution in human putamen was analysed by immunohistochemistry . Antibody against lamin B1 ( red ) was combined with DAPI - Fluoromount G ( blue ) to label nuclei . Representative images show the distribution of lamin B1 in the putamen of non - affected individuals ( CTL ) and HD patients at different stages of the disease ( VS II - IV : Vonsattel grades ) . Yellow and white arrowheads indicate MSNs and glial cells , respectively . Scale bar 50 μm and 20 μm for low and high magnification , respectively . C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry . Anti - lamin B1 antibody ( red ) was combined with anti - GFAP antibody ( green ) and nuclei were labelled with DAPI - Fluoromount G ( blue ) . Representative images show the distribution of lamin B1 at Vonsattel grade III . Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP - positive cells . Scale bar 10 μm .

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

Figure 2A Mouse brain tissue was processed for immunohistochemistry by combining anti-lamin B1 (red) and anti-NeuN (green) antibodies. Representative images (maximal Z-projections) showing the distribution of lamin B1 in the striatum of 30-week-old wild-type (WT) and R6/1 mice. Yellow arrowheads show co-localization between lamin B1 and NeuN, and white arrowheads show NeuN negative lamin B1 positive nuclei. Scale bar 50 μm and 25 μm for low and high magnification, respectively.B Lamin B1 distribution in human putamen was analysed by immunohistochemistry. Antibody against lamin B1 (red) was combined with DAPI-Fluoromount G (blue) to label nuclei. Representative images show the distribution of lamin B1 in the putamen of non-affected individuals (CTL) and HD patients at different stages of the disease (VS II-IV: Vonsattel grades). Yellow and white arrowheads indicate MSNs and glial cells, respectively. Scale bar 50 μm and 20 μm for low and high magnification, respectively. C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry. Anti-lamin B1 antibody (red) was combined with anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue). Representative images show the distribution of lamin B1 at Vonsattel grade III. Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP-positive cells. Scale bar 10 μm.


Figure 3A Representative images showing the distribution of lamin B1 . On the left , images showing maximal Z - projection . Scale bar 150 μm . Small images correspond to CA1 and DG magnified images showing independent DAPI , GFAP and Lamin B1 channels , and merge , of representative confocal Z - stack images . Scale bar 50 μm . B Left , representative images showing the distribution of lamin B1 in the nuclei of CA - 1 hippocampal neurons from WT and R6 / 1 mice along different Z axis planes ( 1 - 6 ) . On the right , a 3D reconstruction of the Z - stack confocal images generated using ImageJ . Scale bar 20 μm .

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Figure 3A Representative images showing the distribution of lamin B1. On the left, images showing maximal Z-projection. Scale bar 150 μm. Small images correspond to CA1 and DG magnified images showing independent DAPI, GFAP and Lamin B1 channels, and merge, of representative confocal Z-stack images. Scale bar 50 μm. B Left, representative images showing the distribution of lamin B1 in the nuclei of CA-1 hippocampal neurons from WT and R6/1 mice along different Z axis planes (1-6). On the right, a 3D reconstruction of the Z-stack confocal images generated using ImageJ. Scale bar 20 μm.


Figure 4A Graphs show the quantification of different parameters in mice striatal MSNs nuclei ( Ctip2 + / NeuN + ) . N = 4 for both genotypes . An average of 5 , 000 nuclei was analysed for each sample . Representative images are shown . Scale bar 7 μm . B Graphs show the quantification of different parameters in mice striatal interneurons ( Ctip2 - / NeuN + ) . N = 4 for both genotypes . An average of 5 , 000 nuclei was analysed for each sample . Representative images are shown . Scale bar 7 μm . C Graphs show the quantification of lamin B1 intensity and circularity in 30 - week - old R6 / 1 mouse striatal neurons nuclei with ( mHtt + ) or without ( mHtt - ) nuclear inclusions . N = 6 . EM48 antibody was used to label mHtt inclusions . Representative images are shown . Scale bar 7 μm . D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease ( VS : Vonsattel grade ) and corresponding controls ( CTL : non - affected individuals ) . N = 6 for CTL , N = 3 for VS I - II and N = 4 for VS III - IV . An average of 50 nuclei was examined for each sample . Representative images ( maximal Z - projections ) are shown . Scale bar 7μm . E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2 + / Prox1 - ) and DG ( Ctip2 + / Prox1 + ) neurons nuclei . N = 4 for each genotype . Representative images are shown . Scale bar 7 μm .

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

Figure 4A Graphs show the quantification of different parameters in mice striatal MSNs nuclei (Ctip2+/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.B Graphs show the quantification of different parameters in mice striatal interneurons (Ctip2-/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.C Graphs show the quantification of lamin B1 intensity and circularity in 30-week-old R6/1 mouse striatal neurons nuclei with (mHtt +) or without (mHtt -) nuclear inclusions. N = 6. EM48 antibody was used to label mHtt inclusions. Representative images are shown. Scale bar 7 μm. D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease (VS: Vonsattel grade) and corresponding controls (CTL: non-affected individuals). N = 6 for CTL, N = 3 for VS I-II and N = 4 for VS III-IV. An average of 50 nuclei was examined for each sample. Representative images (maximal Z-projections) are shown. Scale bar 7μm. E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2+/Prox1-) and DG (Ctip2+/Prox1+) neurons nuclei. N = 4 for each genotype. Representative images are shown. Scale bar 7 μm.


Figure 5 A Graphs show the quantification of lamin B1 intensity and circularity . N = 3 . An average of 12 nuclei was examined in each culture . Data are expressed as a percentage of controls . Bars represent the mean ± S . E . M . * P < 0 . 05 and * * P < 0 . 01 as compared to mApple - C1 control neurons ( two - tailed unpaired Student ' s t test ) . Exact P values are reported in Appendix Table S3 . B Representative images showing primary striatal neurons transfected with mApple - C1 or with mApple - LB1 , both in red . Neuronal nuclei were stained with DAPI - Fluoromount G ( blue ) . Lamin B1 is shown in white . Scale bar 10 μm .

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Figure 5 A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3. B Representative images showing primary striatal neurons transfected with mApple-C1 or with mApple-LB1, both in red. Neuronal nuclei were stained with DAPI-Fluoromount G (blue). Lamin B1 is shown in white. Scale bar 10 μm.


Figure 6 B - D Nuclear permeability in 30 - week - old wild - type ( WT ) and R6 / 1 mice striatal MSNs , CA1 and DG neurons nuclei , respectively . ( B and C ) N = 4 for each condition ; ( D ) N = 5 for each condition . An average of 25 nuclei were analysed for each sample

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Figure 6 B-D Nuclear permeability in 30-week-old wild-type (WT) and R6/1 mice striatal MSNs, CA1 and DG neurons nuclei, respectively.(B and C) N = 4 for each condition; (D) N = 5 for each condition. An average of 25 nuclei were analysed for each sample


Figure 7A UCSC genome browser capture of lamin B1 ChIP - seq signal ( log ( LB1 / Input ) ) and LADs discovered in wild - type ( WT LAD ) and R6 / 1 ( R6 / 1 LADs ) mice in combination with NPC lamin B1 DamID ( top ) . Black arrows highlight differences in identified LADs between WT and R6 / 1 mice ChIP - seq data . Boxplots of LADs size ( log10 ( LADs size + 1 ) ) and of LADs score ( log10 ( LADs score + 1 ) ) obtained by EDD ( bottom ) from WT ( N = 3 ) and R6 / 1 ( N = 3 ) lamin B1 ChIP - seq data . Hipp , hippocampus . 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 UCSC genome browser capture of lamin B1 ChIP - seq signal ( log ( LB1 / Input ) ) LADs discovered in wild - type ( WT LADs ) and R6 / 1 ( R6 / 1 LADs ) mice ; and common ( green ) , wild - type ( WT ) specific ( WT spec . LADs , red ) and R6 / 1 specific ( R6 / 1 spec . LADs , blue ) LADs identified by EDD ( top , WT ( N = 3 ) , R6 / 1 ( N = 3 ) ) . Black arrows highlight WT specific LADs not identified in R6 / 1 mice . Boxplot of average size ( log10 ( LADs size + 1 ) ) for common , WT specific and R6 / 1 specific LADs ( right ) . 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 Lamin B1 enrichment in common , wild - type ( WT , N = 3 ) specific ( spec ) and R6 / 1 ( N = 3 ) specific LADs ( log10 ( LB1 / Input reads + 1 ) in hippocampus ( Hipp ; left ) . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes within common and WT specific LADs ( right ) . Gene - term enrichment was estimated by DAVID using a modified Fisher ' s exact test and Benjamini multiple correction test . Bars represents the - log10 ( Benjamini ' s adjusted P - value ) . Exact P values are reported in Appendix Table S3 . E Representaive immunoblot showing lamin B1 levels in the nucleoplasm ( Nucleop ) and chromatin ( Chrom ) in the hippocampus of 30 - week - old wild - type ( WT , N = 7 ) and R6 / 1 ( N = 7 ) mice . TBP , TATA binding protein .

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

Figure 7A UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) and LADs discovered in wild-type (WT LAD) and R6/1 (R6/1 LADs) mice in combination with NPC lamin B1 DamID (top). Black arrows highlight differences in identified LADs between WT and R6/1 mice ChIP-seq data. Boxplots of LADs size (log10(LADs size + 1)) and of LADs score (log10(LADs score + 1)) obtained by EDD (bottom) from WT (N=3) and R6/1 (N=3) lamin B1 ChIP-seq data. Hipp, hippocampus. 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 UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) LADs discovered in wild-type (WT LADs) and R6/1 (R6/1 LADs) mice; and common (green), wild-type (WT) specific (WT spec. LADs, red) and R6/1 specific (R6/1 spec. LADs, blue) LADs identified by EDD (top, WT (N=3), R6/1 (N=3)). Black arrows highlight WT specific LADs not identified in R6/1 mice. Boxplot of average size (log10(LADs size + 1)) for common, WT specific and R6/1 specific LADs (right). 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 Lamin B1 enrichment in common, wild-type (WT, N=3) specific (spec) and R6/1 (N=3) specific LADs (log10(LB1/Input reads +1) in hippocampus (Hipp; left). Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes within common and WT specific LADs (right). Gene-term enrichment was estimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3. E Representaive immunoblot showing lamin B1 levels in the nucleoplasm (Nucleop) and chromatin (Chrom) in the hippocampus of 30-week-old wild-type (WT, N = 7) and R6/1 (N = 7) mice. TBP, TATA binding protein.


Figure 8 B UCSC genome browser capture of wild - type ( WT , N = 3 ) and R6 / 1 ( N = 3 ) mice hippocampus ATAC - seq data , hippocampal H3K9ac and unchanged , closed in R6 / 1 and open in R6 / 1 accessible detected regions ( left ) in Tuba1α , Gabra2α and Dlx2 gene locus . Arrows in blue ( closed in R6 / 1 ) and red ( open in R6 / 1 ) indicate differential accessible regions . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes associated with decreased ( right top ) or increased ( right bottom ) chromatin accessibility regions . Gene - term enrichment westimated by DAVID using a modified Fisher ' s exact test and Benjamini multiple correction test . Bars represents the - log10 ( Benjamini ' s adjusted P - value ) . 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 ) . 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 .

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

Figure 8 B UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, hippocampal H3K9ac and unchanged, closed in R6/1 and open in R6/1 accessible detected regions (left) in Tuba1α, Gabra2α and Dlx2 gene locus. Arrows in blue (closed in R6/1) and red (open in R6/1) indicate differential accessible regions. Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes associated with decreased (right top) or increased (right bottom) chromatin accessibility regions. Gene-term enrichment westimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). 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). 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.


Figure 9 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 . 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 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 ) 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 .

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

Figure 9 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.  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 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) 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.


Figure 10B , 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 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 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 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 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 .

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

Figure 10B,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 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 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 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 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.


Figure 1 ( 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 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 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 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 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 . ) .

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

Figure 1(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 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 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 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 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.).


Figure 2 ( 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 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 ) , 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 . ) .

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

Figure 2(A) 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 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), 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.).


Figure 3 ( 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 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 the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

Figure 3(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 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 the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


Figure 4 ( 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 ) 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 . )

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

Figure 4(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) 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.)


Figure 5 ( 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 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 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 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 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 . ) .

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

Figure 5(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 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 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 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 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.).


Figure 6 ( 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 .

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

Figure 6(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.


Figure 1A 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 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 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 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 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 ) .

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

Figure 1A 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 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 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 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 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).


Figure 2A 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 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 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 .

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

Figure 2A 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 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 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.


Figure 3A , 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 . 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 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 .

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

Figure 3A, 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. 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 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.


Figure 4B , 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 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 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 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 ) .

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

Figure 4B, 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 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 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 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).


Figure 5The 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 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 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 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 . 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 .

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

Figure 5The 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 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 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 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.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.


figf1 ( 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 death determined by the reduction of the ONL was visualized by hematoxylin and eosin staining . ( 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 .

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

figf1(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 death determined by the reduction of the ONL was visualized by hematoxylin and eosin staining.(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.


figf2 ( a ) qRT - PCR analysis for Opn1sw during cone degeneration . ( 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 ) 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 .

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

figf2(a) qRT-PCR analysis for Opn1sw during cone degeneration.(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) 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.


figf3 ( 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 ) .

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figf3(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).


figf5 ( 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 ) and P - mTOR itself ( red , g ) . ( 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 .

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

figf5(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) and P-mTOR itself (red, g).(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.