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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


( E ) U2OS ( EGFP - Flex1 - BIR - 5086 ) expressing shRNAs for RAD51 , POLD3 PIF1 or shRNA vector ( Ctrl ) were infected by retroviruses expressing RAS , of EGFP positive was quantified by FACS 4 days after retrovirus infection ( top ) . Expression level of RAD51 POLD3 is shown by Western blot ( bottom ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 .

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(E) U2OS (EGFP-Flex1-BIR-5086) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days after retrovirus infection (top). Expression level of RAD51 and POLD3 is shown by Western blot analysis (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( F ) U2OS WT or PIF1 - KO were infected by retrovirus expressing RAS or empty vector . profiling was plotted by counting numbers every 24 hr , normalized to number on first day . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 .

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(F) U2OS WT or PIF1-KO cells were infected by retrovirus expressing RAS or empty vector. Cell profiling was plotted by counting cell numbers every 24 hr, and normalized to the cell number on the first day. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( C ) U2OS ( EGFP - STGC - 1731 ) lines carrying Dox - inducible Cas9 / sgRNA - 1731 ( Dox - Cas9 ) or Cas9n / sgRNA ( Dox - Cas9n ) were incubated with or without Dox ( 5 µg / ml ) , of EGFP positive was quantified by FACS 2 days later ( top ) . U2OS ( EGFP - STGC - 1731 , Dox - Cas9 or Dox - Cas9n ) expressing shRNAs for RAD51 , POLD3 PIF1 or shRNA control ( Ctrl ) were incubated with Dox ( 5 µg / ml ) , of EGFP positive was quantified by FACS after 2 days ( bottom ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(C) U2OS (EGFP-STGC-1731) cell lines carrying Dox-inducible Cas9/sgRNA-1731 (Dox-Cas9) or Cas9n/sgRNA (Dox-Cas9n) were incubated with or without Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis 2 days later (top). U2OS (EGFP-STGC-1731, Dox-Cas9 or Dox-Cas9n) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA control (Ctrl) were incubated with Dox (5 µg/ml), and the percentage of EGFP positive cells was quantified by FACS analysis after 2 days (bottom). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) were treated with or without 2 mM HU for 24 hr , of EGFP positive by HU induction was quantified by FACS 4 days after removal of HU ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) expressing shRNAs for RAD51 , POLD3 PIF1 or shRNA vector ( Ctrl ) were treated with 2 mM HU for 24 hr , of EGFP positive was quantified by FACS 4 days later ( right ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(E) U2OS (EGFP-Flex1-STGC-1541) cells were treated with or without 2 mM HU for 24 hr, and the percentage of EGFP positive cells by HU induction was quantified by FACS analysis 4 days after removal of HU (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 or shRNA vector (Ctrl) were treated with 2 mM HU for 24 hr, and the percentage of EGFP positive cells was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( F ) U2OS ( EGFP - Flex1 - STGC - 1541 ) were infected by retroviruses expressing RAS or empty vector , of EGFP positive was assayed by FACS 5 days following infection ( left ) . U2OS ( EGFP - Flex1 - STGC - 1541 ) expressing shRNAs for RAD51 , POLD3 PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS , of EGFP positive was assayed by FACS 5 days after infection ( middle ) . Expression level of RAD51 POLD3 is shown by Western blot ( right ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(F) U2OS (EGFP-Flex1-STGC-1541) cells were infected by retroviruses expressing RAS or empty vector, and the percentage of EGFP positive cells was assayed by FACS 5 days following infection (left). U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RAD51, POLD3 and PIF1 shRNAs or shRNA vector were infected by retroviruses expressing RAS, and the percentage of EGFP positive cells was assayed by FACS 5 days after infection (middle). Expression level of RAD51 and POLD3 is shown by Western blot analysis (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( D ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) expressing shRNAs for POLD3 PIF1 or shRNA vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . of EGFP or mCherry positive after induction was quantified by FACS 2 days later to determine of EGFP or mCherry positive . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(D) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for POLD3 and PIF1 or shRNA vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later to determine the percentage of EGFP or mCherry positive cells. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - Flex1 - STGC - 1541 ) expressing shRNAs for RFC1 PCNA or vector ( Ctrl ) were treated with 2 mM HU for 24 hr . of EGFP positive after HU treatment was quantified by FACS 3 days after HU removal ( left ) . U2OS ( EGFP - STGC - 1731 ) expressing shRNAs for RFC1 PCNA or vector ( Ctrl ) were infected by lentivirus expressing I - SceI . of EGFP positive by I - SceI induction was quantified by FACS 4 days later ( right ) . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(E) U2OS (EGFP-Flex1-STGC-1541) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were treated with 2 mM HU for 24 hr. The percentage of EGFP positive cells after HU treatment was quantified by FACS analysis 3 days after HU removal (left). U2OS (EGFP-STGC-1731) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were infected by lentivirus expressing I-SceI. The percentage of EGFP positive cells by I-SceI induction was quantified by FACS analysis 4 days later (right). Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( F ) U2OS ( EGFP / STGC - mCherry / LTGC - 5034 , Dox - Cas9 ( left ) or Dox - Cas9n ( right ) ) expressing shRNAs for RFC1 PCNA or vector ( Ctrl ) were incubated with Dox ( 5 µg / ml ) . of EGFP or mCherry positive after induction was quantified by FACS 2 days later . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(F) U2OS (EGFP/STGC-mCherry/LTGC-5034, Dox-Cas9 (left) or Dox-Cas9n (right)) cells expressing shRNAs for RFC1 and PCNA or vector (Ctrl) were incubated with Dox (5 µg/ml). The percentage of EGFP or mCherry positive cells after induction was quantified by FACS analysis 2 days later. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( G ) FLAG - PIF1 was stably expressed in U2OS ( EGFP - Flex1 - STGC - 1541 ) by lentiviral infection . Enrichment of FLAG - PIF1 at Flex1 site or GAPDH site with or without HU ( 2 mM , 10 hr ) treatment was calculated by anti - FLAG ChIP ( left ) . When PCNA was depleted by shRNA using vector as a control ( Ctrl ) , enrichment of FLAG - PIF1 at Flex1 site was calculated by anti - FLAG ChIP ( right ) . ChIP value in without HU treatment is set up as 1 for normalization . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 n . s . ( not significant ) P > 0 . 05 .

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

(G) FLAG-PIF1 was stably expressed in U2OS (EGFP-Flex1-STGC-1541) cells by lentiviral infection. Enrichment of FLAG-PIF1 at Flex1 site or GAPDH site with or without HU (2 mM, 10 hr) treatment was calculated by anti-FLAG ChIP (left). When PCNA was depleted by shRNA using vector as a control (Ctrl), enrichment of FLAG-PIF1 at Flex1 site was calculated by anti-FLAG ChIP (right). ChIP value in cells without HU treatment is set up as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01 and n.s. (not significant) P>0.05.


( D ) Growth curves of WT or FANCM - KO HCT116 were plotted after expressing POLD3 ( left ) or PIF1 ( right ) shRNA or shRNA vector ( Ctrl ) . number was normalized to that on day one . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * * P < 0 . 01 .

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

(D) Growth curves of WT or FANCM-KO HCT116 cells were plotted after expressing POLD3 (left) or PIF1 (right) shRNA or shRNA vector (Ctrl). Cell number was normalized to that on day one. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as **P < 0.01.


( C ) Anti - γH2AX ChIP at FRA3B , FRA16D GAPDH genomic loci was performed in U2OS WT PIF1 - KO before after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D GAPDH loci in WT PIF1 KO was calculated using ChIP value in WT as 1 for normalization . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 n . s . ( not significant ) P > 0 . 05 .

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

(C) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci in WT and PIF1 KO cells was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


( E ) U2OS ( EGFP - BIR - 5085 ) expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I - SceI . of EGFP positive was quantified by FACS 5 days after I - SceI infection . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 n . s . ( not significant ) P > 0 . 05 .

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

(E) U2OS (EGFP-BIR-5085) cells expressing PIF1 WT or L319P mutant with endogenous PIF1 silenced by shRNA were infected by lentiviruses to express I-SceI. The percentage of EGFP positive cells was quantified by FACS analysis 5 days after I-SceI infection. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


( I ) Anti - γH2AX ChIP at FRA3B , FRA16D GAPDH genomic loci was performed in U2OS WT PIF1 - KO , or PIF1 - KO expressing PIF1 - WT , E307Q L319P mutants before after APH treatment ( 0 . 4 μM , 24 hr ) . Enrichment of γH2AX at FRA3B , FRA16D GAPDH loci was calculated using ChIP value in WT as 1 for normalization . Data information : Error bars represent standard deviation ( SD ) of at least three independent experiments . Signiﬁcance of differences was assayed by Two - non - paired parameters were applied in Student ' s t - . P value is indicated as * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 n . s . ( not significant ) P > 0 . 05 .

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

(I) Anti-γH2AX ChIP analysis at FRA3B, FRA16D and GAPDH genomic loci was performed in U2OS WT and PIF1-KO cells, or PIF1-KO cells expressing PIF1-WT, and E307Q and L319P mutants before and after APH treatment (0.4 μM, 24 hr). Enrichment of γH2AX at FRA3B, FRA16D and GAPDH loci was calculated using ChIP value in WT cells as 1 for normalization. Data information: Error bars represent the standard deviation (SD) of at least three independent experiments. Signiﬁcance of the differences was assayed by Two-tailed non-paired parameters were applied in Student's t-test. The P value is indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and n.s. (not significant) P>0.05.


Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2 - month - old mice . ( A ) In vivo cSLO examinations of mouse retina at 1 - 2 - post injection ( WPI ) of 2E9 total vg ( in 1 µl ) of sc - CMV - eGFP packaged with parental serotype AAV2 , state - of - art AAV2 . 7m8 , novel AAV2 . GL AAV2 . NN capsid variants .

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Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1- and 2-weeks post injection (WPI) of 2E9 total vg (in 1 µl) of sc-CMV-eGFP packaged with parental serotype AAV2 , state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.


( B ) Confocal scans of retinal cross sections immunolabelled for eGFP DAPI . Acquisition settings were kept constant for samples . Scale bar : 100 μm . ONL , outer layer ; INL , inner layer .

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(B) Confocal scans of retinal cross sections immunolabelled for eGFP and DAPI. Acquisition settings were kept constant for all samples. Scale bar: 100 μm. ONL, outer nuclear layer; INL, inner nuclear layer.


( C ) Confocal tile - scans of retinal cross sections immunolabelled for eGFP Hoechst . Scale bar : 500

(C) Confocal tile-scans of retinal cross sections immunolabelled for eGFP and Hoechst. Scale bar: 500 μm


( D - E ) Immunolabelled cross sections of mouse retina infected with AAV2 . GL AAV2 . NN respectively , with focus on ONL INL , stained for eGFP , recoverin DAPI . Scale bar : 50 μm ONL , outer layer ; INL , inner layer .

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(D-E) Immunolabelled cross sections of mouse retina infected with AAV2.GL and AAV2.NN respectively, with focus on the ONL and INL, stained for eGFP, recoverin and DAPI. Scale bar: 50 μm ONL, outer nuclear layer; INL, inner nuclear layer.


( F ) qRT - PCR data comparing fold change in eGFP transcript from retinal samples infected with capsid variants AAV2 . 7m8 , AAV2 . GL AAV2 . NN compared to AAV2 .

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(F) qRT-PCR data comparing fold change in eGFP transcript levels from retinal samples infected with capsid variants AAV2.7m8, AAV2.GL and AAV2.NN compared to AAV2.


Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2 - month - old mice . ( A ) In vivo cSLO examinations of mouse retina at 1 - , 2 - 3 - post injection ( WPI ) of 8E9 total vg of ss - hRho - eGFP packaged with parental serotype AAV2 , state - of - art AAV2 . 7m8 , novel AAV2 . GL AAV2 . NN capsid variants .

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

Vector mediated eGFP expression after intravitreal injection of novel capsid variants compared to controls in 2-month-old mice. (A) In vivo cSLO examinations of the mouse retina at 1-, 2- and 3- weeks post injection (WPI) of 8E9 total vg of ss-hRho-eGFP packaged with parental serotype AAV2, state-of-art AAV2.7m8, novel AAV2.GL and AAV2.NN capsid variants.


( B ) qRT - PCR data comparing fold change in eGFP transcript relative to ITR2 amplicons from retinal samples infected with four capsid variants .

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(B) qRT-PCR data comparing fold change in eGFP transcript levels relative to ITR2 amplicons from retinal samples infected with all four capsid variants.


( C ) Confocal scans of retinal cross sections , with focus on ONL , immunolabelled for DAPI , eGFP recoverin . upper panel shows only DAPI eGFP signal in ONL lower panel shows same ( noted with asterisk * ) with merged DAPI , eGFP recoverin signal . Acquisition settings were kept constant for samples . Scale bar : 50 μm . ONL , outer layer .

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(C) Confocal scans of retinal cross sections, with focus on the ONL, immunolabelled for DAPI, eGFP and recoverin. The upper panel shows only the DAPI and eGFP signal in the ONL and the lower panel shows the same images (noted with asterisk *) with merged DAPI, eGFP and recoverin signal. Acquisition settings were kept constant for all samples. Scale bar: 50 μm. ONL, outer nuclear layer.


Vector mediated eGFP expression after intravitreal injection in 10 - month - old dog eyes . ( A ) In vivo cSLO examinations of dog retina at four , five six post intravitreal injection of 2E11 total vg ( in 200 µl ) of sc - CMV - eGFP packaged with wildtype AAV2 ( right eye ) , AAV2 . GL ( right eye ) AAV2 . NN ( left eye ) capsid variants .

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Vector mediated eGFP expression after intravitreal injection in 10-month-old dog eyes. (A) In vivo cSLO examinations of the dog retina at four, five and six weeks post intravitreal injection of 2E11 total vg (in 200 µl) of sc-CMV-eGFP packaged with wildtype AAV2 (right eye), AAV2.GL (right eye) and AAV2.NN (left eye) capsid variants.


( B - C ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) stained for GFP DAPI at 6 WPI time point . : DAPI was used a marker . GFP = eGFP ; RPE , retinal pigmented epithelium ; OS , outer segments ; ONL , outer layer ; INL , inner layer ; GCL , ganglion layer .

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(B-C) Confocal scans of immunolabelled retinal cross sections (14 μm) stained for GFP and DAPI at 6 WPI time point. : DAPI was used a nuclear marker. GFP=eGFP; RPE, retinal pigmented epithelium; OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.


( D - E ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) at 6 WPI time point , focusing on photoreceptors , using antibodies against eGFP , Rhodopsin ( Rho ) M - opsin ( MO ) . OS , outer segments ONL , outer

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(D-E) Confocal scans of immunolabelled retinal cross sections (14 μm) at 6 WPI time point, focusing on the photoreceptors, using antibodies against eGFP, Rhodopsin (Rho) and M-opsin (MO). OS, outer segments ONL, outer nuclear layer


Vector mediated expression after intravitreal injection in ~ 3 - year old cynomolgus macaque eyes . ( A - B ) In vivo cSLO examination collaged to provide NHP fundus overview at 8 WPI of E12 total vg ( in 38 . 9 - 100 µl ) of sc - CMV - eGFP packaged with AAV2 . GL AAV2 . NN capsid variants , respectively .

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Vector mediated expression after intravitreal injection in ~3-year old cynomolgus macaque eyes. (A-B) In vivo cSLO examination images collaged to provide NHP fundus overview at 8 WPI of E12 total vg (in 38.9-100 µl) of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants, respectively.


( C - D ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) of fovea centralis from AAV2 . GL AAV2 . NN treated , respectively .

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(C-D) Confocal scans of immunolabelled retinal cross sections (14 μm) of the fovea centralis from AAV2.GL and AAV2.NN treated animals, respectively.


( E ) of eGFP + photoreceptors in ONL of foveal cross sections . AAV2 . GL technical repeats n = 4 , AAV2 . NN technical repeats n = 3 .

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(E) Percentage quantification of eGFP+ photoreceptors in the ONL of foveal cross sections. AAV2.GL technical repeats n=4, AAV2.NN technical repeats n=3.


( F - G ) Confocal scans of immunolabelled retinal cross sections ( 14 μm ) of periphery from AAV2 . GL ( F ) AAV2 . NN ( G ) treated retinae . ( Η - Ι ) Higher magnification confocal scans of immunolabelled peripheral retina . ONL , outer layer ; INL , inner

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(F-G) Confocal scans of immunolabelled retinal cross sections (14 μm) of the periphery from AAV2.GL (F) and AAV2.NN (G) treated retinae. (Η-Ι) Higher magnification confocal scans of immunolabelled peripheral retina. ONL, outer nuclear layer; INL, inner nuclear layer


Vector mediated eGFP expression in human retinal explants . ( B ) EVOS epifluorescence at 2x magnification of human retinal explants at three , six nine days in vitro ( DIV ) after transduction with 1E11 total vg of sc - CMV - eGFP packaged with AAV2 . GL AAV2 . NN capsid variants . signal indicates eGFP .

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Vector mediated eGFP expression in human retinal explants. (B) EVOS epifluorescence images at 2x magnification of the human retinal explants at three, six and nine days in vitro (DIV) after transduction with 1E11 total vg of sc-CMV-eGFP packaged with AAV2.GL and AAV2.NN capsid variants. White signal indicates eGFP.


( C ) Confocal scans of day 10 tissue sections immunolabelled for eGFP Hoechst . Scale bar : 50 μm .

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(C) Confocal scans of day 10 tissue sections immunolabelled for eGFP and Hoechst. Scale bar: 50 μm.


( A ) Representative photopic ERG flicker series at 1 , 5 10 Hz with 3 cd . s / m2 illumination at 10 WPI . Arrowheads indicate stimulus .

(A) Representative photopic ERG flicker series at 1, 5 and 10 Hz with 3 cd.s/m2 illumination at 10 WPI. Arrowheads indicate light stimulus.


( B ) Representative photopic single flash ERG signal with 1 , 3 10 cd . s / m2 illumination . vertical dotted indicates stimulus . ( C ) of photopic B - wave amplitude from single flash responses to 1 , 3 10 cd . s / m2 illumination of wildtype C57BL6 / J x 129 / sv eyes ( WT , n = 3 ) compared to treated eyes ( IVT / GL , n ≥ 3 ) untreated eyes ( UN , n ≥ 3 ) at 10 post injection .

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(B) Representative photopic single flash ERG signal with 1, 3 and 10 cd.s/m2 illumination. The vertical dotted line indicates light stimulus. (C) Quantification of photopic B-wave amplitude from single flash responses to 1, 3 and 10 cd.s/m2 illumination of wildtype C57BL6/J x 129/sv eyes (WT, n = 3) compared to treated eyes (IVT/GL, n ≥ 3) and untreated eyes (UN, n ≥ 3) at 10 weeks post injection.