( C ) Degree of conformational changes in CM induced by 60b linear ssDNA . Blue bar , Arm / Smc5 - Nse2 ( wild - type ) ; grey bar , Arm / Smc5 - Nse2 ( K333E / K344E ) ; green bar , Arm / Smc5 - Nse2 ( K764E ) ; pink bar , Arm / Smc5 - Nse2 ( K333E / K344E / K764E ) ; red bar , Arm / Smc5 - Nse2 ( K743E / K745E ) . Reactions were performed at least in three different independent experiments . Data values are mean ± s . d . n = 3 technical replicates . Significance was measured by a two - unpaired t - relative to wild - type . * * * * P < 0 . 0001

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(C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001


( B ) Growth of wild - type , nse2 - CH indicated smc5 - KE mutants ; 10 - fold serial dilutions of liquid cultures were spotted in YPD pictures taken after 48 hours

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(B) Growth test analysis of wild-type, nse2-CH and the indicated smc5-KE mutants; 10-fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours


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(C) Same as in (B) but using the nse2-CH, smc5-3KE, smc5-K743,745E, smc5-K743,745R, smc5-7KE and smc5-7KR mutants


( A ) extracts from exponentially growing wild - type , smc5 - 3KE , smc5 - K743 , 745E , smc5 - 4KE smc5 - K743 , 745R were prepared under denaturing conditions ; 6xHis - Flag tagged SUMO ( HF - SUMO ) was pulled down from extracts to purify sumoylated species analyzed by western blot . A with no HF - tag was used as control ; arrow points to unmodified form of , vertical bar to sumoylated forms

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(A) Protein extracts from exponentially growing wild-type, smc5-3KE, smc5-K743,745E, smc5-4KE and smc5-K743,745R cells were prepared under denaturing conditions; 6xHis-Flag tagged SUMO (HF-SUMO) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot. A strain with no HF-tag was used as control; arrow points to unmodified form of the protein, vertical bar to sumoylated forms


( B ) Left , same as in ( A ) , but using wild - type two independent clones of smc5 - 7KE ; right , same as in ( A ) , but using wild - type , smc5 - 7KE smc5 - 7KR mutant

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(B) Left, same as in (A), but using wild-type and two independent clones of smc5-7KE; right, same as in (A), but using wild-type, smc5-7KE and smc5-7KR mutant cells


( C ) of Smc5 sumoylated species in pull downs from indicated smc5 - KE mutants , relative to wild - type controls from at least three independent experiments . Boxes , 25 - 75 % data range ; whiskers , total data range ; black bar , median ; grey cross , mean . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 . 1 - way ANOVA , n = number of samples analyzed

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(C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed


( D ) Co - immunoprecipitation of Smc5 - Nse2 interactions . NSE2 - 6HA were transformed with centromeric plasmids expressing indicated SMC5 alleles , extracts from exponentially growing subjected to anti - HA immunoprecipitation . A with no HA tag was used as control

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(D) Co-immunoprecipitation analysis of the Smc5-Nse2 interactions. NSE2-6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles, and protein extracts from exponentially growing cells subjected to anti-HA immunoprecipitation. A strain with no HA tag was used as control


( E ) Same as in ( D ) , but using SMC6 - 6HA

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(E) Same as in (D), but using SMC6-6HA cells


( F ) of Smc5 - 6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of indicated genotypes . mean value on wild - type spreads was arbitrarily set to 1 ; each dot represents one nucleus ; red , median . 1 - way ANOVA , n = number of nuclei analyzed . * * * * P < 0 . 0001

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(F) Quantification of Smc5-6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes. The mean value on wild-type spreads was arbitrarily set to 1; each dot represents one nucleus; red line, median. 1-way ANOVA, n=number of nuclei analyzed. ****P < 0.0001


( b ) RNAscope of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript ( N = 3 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars =

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(b) RNAscope analysis of Lgr5 mRNA in DSS colitis demonstrating loss of Lgr5 transcript (N=3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm


( c ) Hematoxylin Eosin of colonic epithelium following DSS induced damaged . Crypts are dramatically injured by d5 . At d8 through d12 , regenerating crypts are observed . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars =

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(c) Hematoxylin and Eosin staining of the colonic epithelium following DSS induced damaged. Crypts are dramatically injured by d5. At d8 through d12, regenerating crypts are observed. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm


( d - e ) Compared to untreated controls , DSS treatment ablates Lgr5 - GFP + results in functional loss of tdTomato + lineage tracing as early as d5 . Lgr5 - GFP + reappear by d12 ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars = 50µm Data are represented as mean ± SD ( e ) analyzed using Student ' s t - . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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(d-e) Compared to untreated controls, DSS treatment ablates Lgr5-GFP+ stem cells and results in functional loss of tdTomato+ lineage tracing as early as d5. Lgr5-GFP+ stem cells reappear by d12 (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm Data are represented as mean ± SD (e) analyzed using Student's t-test. *P≤0.05, ** P≤0.01.


( f ) Effects of DSS colitis injury on expression of ( Lgr5 Krt19 ) progenitor ( Atoh1 Notch1 ) markers . Lgr5 mRNA expression is significantly decreased after exposure to DSS ( N = 6 - 7 per condition ) . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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(f) Effects of DSS colitis injury on RNA expression levels of stem (Lgr5 and Krt19) and progenitor (Atoh1 and Notch1) cell markers. Lgr5 mRNA expression is significantly decreased after exposure to DSS (N=6-7 per condition). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.


Mice ( h ) are comparable between three treatments : DSS + DT ( N = 12 ) , only DSS ( N = 8 ) or only DT ( N = 3 ) . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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Mice body weight (h) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.


Mice survival ( i ) are comparable between three treatments : DSS + DT ( N = 12 ) , only DSS ( N = 8 ) or only DT ( N = 3 ) . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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Mice survival (i) are comparable between three treatments: DSS+DT (N=12), only DSS (N=8) or only DT (N=3). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.


( j ) Hematoxylin Eosin of colonic epithelium shows no morphological difference between DSS alone DSS + DT treated mice . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . scale bar ( j ) = 500µm .

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(j) Hematoxylin and Eosin staining of the colonic epithelium shows no morphological difference between DSS alone and DSS+DT treated mice. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bar (j) =500µm.


( k ) extent of epithelial damage is similar in mice treated with DSS alone versus DSS + DT ( to ablate Lgr5 + ) ( N = 3 DSS ; N = 6 DSS + DT ) . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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(k) The extent of epithelial damage is similar in mice treated with DSS alone versus DSS+DT (to ablate Lgr5+ stem cells) (N=3 DSS; N=6 DSS+DT). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05, ** P≤0.01.


( l ) RNAscope for Lgr5 in Lgr5DTR - GFP mice showing loss of Lgr5 transcripts after DT treatment ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars =

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(l) RNAscope for Lgr5 in Lgr5DTR-GFP mice showing loss of Lgr5 transcripts after DT treatment (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50µm


( b - c ) Krt19 + lineage trace entire colonic crypts during both normal homeostasis injury ( N = 3 - 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines outline basement . Scale bars ( b ) = 45µm Data are represented as mean ± * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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(b-c) Krt19+ cells lineage trace entire colonic crypts during both normal homeostasis and injury (N=3-4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. Scale bars (b) =45µm Data are represented as mean ± SEM *P≤0.05, **P≤0.01.


DT ablation of Krt19 + ( N ≥ 3 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines outline basement . scale bar ( e ) =

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DT ablation of Krt19+ cells (N≥3 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (e) =30µm


DT ablation of Krt19 + significantly reduced number of lineage labeled crypts ( N ≥ 3 per condition ) . Data are represented as mean ± using Student ' s t - * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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DT ablation of Krt19+ cells significantly reduced the number of lineage labeled crypts (N≥3 per condition). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.


( g ) Mice survival remain unchanged between DSS DSS + DT treated groups ( N = 6 control ; N = 7 DT ) . Data are represented as mean ± using Student ' s t - P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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(g) Mice body weight and survival remain unchanged between DSS and DSS+DT treated groups (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test P≤0.05, **P≤0.01.


Hematoxylin Eosin of colonic epithelium after DSS versus DSS + DT showing more damage is seen in mice treated with both DSS + DT treatment ( N = 6 control ; N = 7 DT ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines outline basement . scale bar ( h ) = 1mm .

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Hematoxylin and Eosin staining of the colonic epithelium after DSS versus DSS+DT showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines outline the basement membrane. scale bar (h) =1mm.


of damaged epithelial ( i ) , showing more damage is seen in mice treated with both DSS + DT treatment ( N = 6 control ; N = 7 DT ) . Data are represented as mean ± using Student ' s t - * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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quantification of damaged epithelial area (i), showing more damage is seen in mice treated with both DSS+DT treatment (N=6 control; N=7 DT). Data are represented as mean ± SEM using Student's t-test *P≤0.05, **P≤0.01.


( b - c ) Notch1 + progenitors are able to generate Lgr5 + contribute to epithelial renewal in homeostasis ; however , do not contribute to regeneration after colitis ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars = 50μm . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons . * * * * P ≤ 0 . 0001 .

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(b-c) Notch1+ progenitors are able to generate Lgr5+ stem cells and contribute to epithelial renewal in homeostasis; however, do not contribute to regeneration after colitis (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test. ****P≤0.0001.


of whole mount showing Notch1 lineage labeled crypts . No partially labeled crypts were found after Lgr5 + ablation . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars = 50μm .

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Images of whole mount showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.


of tissue sections Notch1CreERT2 ; ROSA26tdTomato ; Lgr5DTR - GFP mice ( f ) showing Notch1 lineage labeled crypts . No partially labeled crypts were found after Lgr5 + ablation . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars = 50μm .

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Images of tissue sections Notch1CreERT2; ROSA26tdTomato;Lgr5DTR-GFP mice (f) showing Notch1 lineage labeled crypts. No partially labeled crypts were found after Lgr5+ stem cell ablation. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars=50μm.


( a - b ) Atoh1 + progenitors show capacity during normal homeostasis regeneration . At early stages of recovery , d10 - 12 , Atoh1 + lineage trace colonic crypt ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars = 50μm Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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(a-b) Atoh1+ progenitors show stem cell capacity during normal homeostasis and regeneration. At early stages of recovery, d10-12, Atoh1+ cells lineage trace the colonic crypt (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.


( c - d ) By d30 after DSS , Atoh1 derived continue to lineage trace complete colonic crypts ( N = 4 per condition ) . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars first two panels of c = 50μm ; scale bars ( last panel of c = 100μm . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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(c-d) By d30 after DSS, Atoh1 derived stem cells continue to lineage trace complete colonic crypts (N=4 per condition). Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars first two panels of c =50μm; scale bars (last panel of c =100μm. Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test *P≤0.05, ***P≤0.001.


Whole mount ( f ) of Atoh1 lineage labeled crypts . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars =

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Whole mount (f) images of Atoh1 lineage labeled crypts. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm


section ( g ) of Atoh1 lineage labeled crypts . Majority of crypts are labeled after Lgr5 + ablation remain label with appearance of Lgr5 + ( inset , arrowheads ) . Atoh1 lineage labeled crypts remain by d30 . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . scale bars = 100μm .

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section (g) images of Atoh1 lineage labeled crypts. Majority of crypts are labeled after Lgr5+ stem cell ablation and remain label with the appearance of Lgr5+ stem cells (inset, arrowheads). Atoh1 lineage labeled crypts remain by d30. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. scale bars =100μm.


( h ) Significantly increased labeling of crypt base was observed at d8 in DT treated mice ( N = 4 per condition ) . Data are represented as mean ± SD analyzed using Student ' s t - with Welch ' s correction * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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(h) Significantly increased labeling of crypt base was observed at d8 in DT treated mice (N=4 per condition). Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.


( j - k ) Scattered labeling of Atoh1 + in control conditions , while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5 + . Data information : Nuclei are counterstained with Dapi ( blue ) ; dashed lines indicate basement . Scale bars = 50μm Data are represented as mean ± SD analyzed using Student ' s t - with Welch ' s correction * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

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(j-k) Scattered labeling of Atoh1+ cells in control conditions, while DSS injury lead to Atoh1 lineage labeled crypts lacking Lgr5+ stem cells. Data information: Nuclei are counterstained with Dapi (blue); white dashed lines indicate basement membrane. Scale bars =50μm Data are represented as mean ± SD analyzed using Student's t-test with Welch's correction *P≤0.05, ***P≤0.001.


( b - c ) Sporadic labeling of Atoh1 + after 4 - OHT induction . Following irradiation , entire colonic organoids exhibit full lineage labeling of Atoh1 + , characteristic of multipotent ( N = 3 per condition ) . Data information : Scale bars = 100μm Data are represented as mean ± SD analyzed using Student ' s t - ( c )

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(b-c) Sporadic labeling of Atoh1+ cells after 4-OHT induction. Following irradiation, entire colonic organoids exhibit full lineage labeling of Atoh1+ cells, characteristic of multipotent stem cells (N=3 per condition). Data information: Scale bars =100μm Data are represented as mean ± SD analyzed using Student's t-test (c)


( d - e ) Flow cytometry for tdTomato + in crypt epithelium from DT treated Atoh1CreERT2 ; Lgr5DTR - GFP ; ROSA26tdTomato mice .

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(d-e) Flow cytometry for tdTomato+ cells in crypt epithelium from DT treated Atoh1CreERT2;Lgr5DTR-GFP;ROSA26tdTomato mice.


( f ) tdTomato + colonic organoids from single Atoh1 - tdTomato + / Lgr5 - negative ( N = 1 ; n = 3 technical replicates per condition ) . Data information : Scale bars =

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(f) tdTomato+ colonic organoids from single Atoh1-tdTomato+/Lgr5-negative cells (N=1; n=3 technical replicates per condition). Data information: Scale bars =100μm


( g ) Post - passage colonic organoids from RFP + of control or DT treated mice continue growing . tdTomato - negative of control or DT treated mice also form colonic organoids after passaging . Data information scale bars ( g ) = 50μm .

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(g) Post-passage colonic organoids from RFP+ cells of control or DT treated mice continue growing. tdTomato-negative cells of control or DT treated mice also form colonic organoids after passaging. Data information scale bars (g) =50μm.


( h ) tdTomato + colonic organoids established from mice in which Lgr5 + have been ablated show higher growth efficiency compared to their colonic organoid counterparts ( N = 1 ; n = 3 technical replicates per condition ) . Data are represented as mean ± SD analyzed using two - way ANOVA with Sidak ' s multiple comparisons ( h ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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(h) tdTomato+ colonic organoids established from mice in which Lgr5+ stem cells have been ablated show higher growth efficiency compared to their colonic organoid counterparts (N=1; n=3 technical replicates per condition). Data are represented as mean ± SD analyzed using two-way ANOVA with Sidak's multiple comparisons test (h). *P≤0.05, **P≤0.01, ***P≤0.001.


( b ) Atoh1 + lineage labelling post - DSS DSS + DT . Data information : Scale bars ( b ) =

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(b) Atoh1+ lineage labelling post-DSS and DSS+DT. Data information: Scale bars (b)=200μm


( c ) DSS + DT treated - mice exhibit significant loss lower survival compared to DSS alone ( N = 4 - 7 control ; N = 3 - 4 DSS ) . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 .

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(c) DSS+DT treated-mice exhibit significant weight loss and lower survival compared to DSS alone (N=4-7 control; N=3-4 DSS). Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.


( d - e ) Hematoxylin Eosin of colonic epithelium after DSS versus DSS + DT ( d ) . Extensive epithelial damage was observed ( d , arrow ) quantified of in DSS + DT treated mice ( N = 7 control ; N = 4 DSS ) . Data information scale bars ( d ) = 400μ Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 .

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(d-e) Hematoxylin and Eosin staining of colonic epithelium after DSS versus DSS+DT (d). Extensive epithelial damage was observed (d, arrow) and quantified of in DSS+DT treated mice (N=7 control; N=4 DSS). Data information scale bars (d)=400μ Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.


( g - h ) DT treatment effectively ablates Atoh1 + shown by reduced number of tdTomato + per organoid . Data information scale bars = 100μm . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 .

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(g-h) DT treatment effectively ablates Atoh1+ cells shown by reduced number of tdTomato+ cells per organoid. Data information scale bars =100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.


( i - j ) Concurrent irradiation injury Atoh1 + ablation results in significantly reduced organoid survival ( N = 3 ; n = 3 technical replicates per condition ) . Data information : ; scale bars = 100μm . Data are represented as mean ± analyzed using Student ' s t - . * P ≤ 0 . 05 .

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(i-j) Concurrent irradiation injury and Atoh1+ cell ablation results in significantly reduced organoid survival (N=3; n=3 technical replicates per condition). Data information: ; scale bars =100μm. Data are represented as mean ± SEM analyzed using Student's t-test. *P≤0.05.


B : Double - of coronal brain sections ( 40 μm ) of a littermate ( LM ) a NexCre cTKO mouse ( : 5 months ) stained for Ctip2 ( green , expressed in layer V / VI ) Calbindin ( red , expressed in layer II / III to some extent in layer V ) . Note that no gross abnormalities in cortical lamination were observed . Layers of DAPI stained nuclei are denoted on right ( I - VI ) , scale bar : 100 µm .

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B: Double-IHC of coronal brain sections (40 μm) of a littermate (LM) and a NexCre cTKO mouse (age: 5 months) stained for Ctip2 (green, expressed in layer V/VI) and Calbindin (red, expressed in layer II/III and to some extent in layer V). Note that no gross abnormalities in cortical lamination were observed. Layers of DAPI stained cell nuclei are denoted on the right (I-VI), scale bar: 100 µm.


C : Giemsa - stained , glycolmethacrylate - embedded coronal brain sections ( bregma - 1 . 9 ) displaying hippocampus adjacent callosal fiber tracts of a wildtype ( WT , top ) , littermate control ( LM , middle ) a NexCre cTKO ( cTKO , bottom ; : 5 - 6 months ) . Note agenesis of corpus callosum ( no CC ) in NexCre cTKO mice , which should always be present at this bregma in sections in which medial habenula ( mHb ) is present ( CC in WT LM ) . While WTs LMs show a compact layer of CA1 , NexCre cTKOs display a bilaminar cytoarchitecture ( boxed regions marking higher magnification to right ) with scattered ectopic in stratum radiatum ( arrowheads ) . In addition , CA3 appears less compact ( red arrowheads ) . Layer identity visualized via Calbindin ( right panel , scale bar : 50 µm ) .

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C: Giemsa-stained, glycolmethacrylate-embedded coronal brain sections (bregma -1.9) displaying the hippocampus and adjacent callosal fiber tracts of a wildtype (WT, top), littermate control (LM, middle) and a NexCre cTKO (cTKO, bottom; age: 5-6 months). Note the agenesis of the corpus callosum (no CC) in NexCre cTKO mice, which should always be present at this bregma and in sections in which the medial habenula (mHb) is present (CC in WT and LM). While WTs and LMs show a compact layer of CA1 cells, NexCre cTKOs display a bilaminar cytoarchitecture (boxed regions marking higher magnification to the right) with scattered ectopic cells in the stratum radiatum (white arrowheads). In addition, the CA3 appears less compact (red arrowheads). Layer identity visualized via Calbindin staining (right panel, scale bar: 50 µm).


C : Representative 3D reconstructions of CA1 pyramidal neurons from deep ( left , blue ) superficial ( right , blue ) radial layers .

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C: Representative 3D reconstructions of CA1 pyramidal neurons from deep (left, dark blue) and superficial (right, light blue) radial layers.


D : Sholl reveals no genotype effect on basal dendritic segments of deep CA1 pyramidal neurons ( dCA1 ; LM : n = 21 , N = 7 ; cTKO : n = 21 / N = 6 ) . E : Compared to littermate controls , dCA1 neurons of NexCre cTKO show no significant reduction in total dendritic in basal dendrites ( unpaired Student ' s t - , nsp = 0 . 4979 ) . F : number of basal dendrites is significantly reduced in NexCre cTKO compared to LM in dCA1 ( Mann - Whitney , * * p = 0 . 0032 ) . G :

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D: Sholl analysis reveals no genotype effect on basal dendritic segments of deep CA1 pyramidal neurons (dCA1; LM: n=21, N=7; cTKO: n=21/N=6). E: Compared to littermate controls, dCA1 neurons of NexCre cTKO animals show no significant reduction in total dendritic length in basal dendrites (unpaired Student's t-test, nsp = 0.4979). F: The number of primary basal dendrites is significantly reduced in NexCre cTKO animals compared to LM in dCA1 (Mann-Whitney test, **p = 0.0032). G:


A , B : Spine density of deep CA1 pyramidal is significantly reduced in basal dendritic segments ( A ; unpaired Student ' s t - , * * p = 0 . 0067 ) , while it is unchanged in midapical dendritic segments ( B ; unpaired Student ' s t - , nsp = 0 . 4949 ) . C : Representative of basal midapical dendritic segments from both radial layers . Brightness contrast were adjusted to ensure a uniform appearance . Scale bar : 5 µm . D

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A, B: Spine density of deep CA1 pyramidal cells is significantly reduced in basal dendritic segments (A; unpaired Student's t-test, **p = 0.0067), while it is unchanged in midapical dendritic segments (B; unpaired Student's t-test, nsp = 0.4949). C: Representative images of basal and midapical dendritic segments from both radial layers. Brightness and contrast were adjusted to ensure a uniform appearance. Scale bar: 5 µm. D


A : mEPSC mIPSC sample traces of recordings from deep CA1 pyramidal . B - E : Bar graphs of mEPSC mIPSC frequencies amplitudes in deep CA1 pyramidal . mEPSC frequency was not different in deep CA1 pyramidal neurons of adult ( 4 months ) NexCre cTKO versus littermate controls ( B , p = 0 . 6415 ) . mEPSC amplitude was increased in deep CA1 pyramidal of NexCre cTKO compared to LM control ( C , * p = 0 . 0363 ) . mIPSC frequency was not different in deep CA1 pyramidal neurons of adult ( 4 months ) NexCre cTKO versus littermate controls ( D , p = 0 . 0564 ) mIPSC amplitude was increased in deep CA1 pyramidal of NexCre cTKO compared to LM ( E , * p = 0 . 0356 ) . F : mEPSC mIPSC sample traces of recordings from superficial CA1 pyramidal . G

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A: mEPSC and mIPSC sample traces of recordings from deep CA1 pyramidal cells. B - E: Bar graphs of mEPSC and mIPSC frequencies and amplitudes in deep CA1 pyramidal cells. mEPSC frequency was not different in deep CA1 pyramidal neurons of adult (4 months) NexCre cTKO versus littermate controls (B, p = 0.6415). mEPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM control cells (C, *p = 0.0363). mIPSC frequency was not different in deep CA1 pyramidal neurons of adult (4 months) NexCre cTKO versus littermate controls (D, p = 0.0564) and mIPSC amplitude was increased in deep CA1 pyramidal cells of NexCre cTKO compared to LM (E, *p = 0.0356). F: mEPSC and mIPSC sample traces of recordings from superficial CA1 pyramidal cells. G


A : Diurnal behavior in home cage ( HC ) . NexCre cTKOs show overshooting activity in phase . graphs illustrate average locomotion per hour . [ Sidak ' s multiple comparisons , geno F ( 1 , 32 ) = 9 . 503 , p = 0 . 0042 ; timepoint F ( 6 . 102 , 195 . 3 ) = 10 . 51 , p < 0 . 0001 ; timepoint × geno F ( 47 , 1504 ) = 5 . 874 , p < 0 . 0001 ] .

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A: Diurnal behavior in the home cage (HC). NexCre cTKOs show overshooting activity in the dark phase. Line graphs illustrate the average locomotion per hour. [Sidak's multiple comparisons test, geno F(1,32) = 9.503, p = 0.0042; timepoint F(6.102,195.3) = 10.51, p < 0.0001; timepoint × geno F(47,1504) = 5.874, p < 0.0001].


I : Sociability in three chambers ( 3CT ) . LM controls spend more time with mouse stranger 1 ( S1 ) whereas NexCre cTKOs spend comparable time exploring novel object ( NO ) or S1 . Bottom : schematic representation of . [ Paired Student ' s t - comparison between chambers LM * * * p = 0 . 0002 ; Wilcoxon between chambers NexCre cTKO ns p = 0 . 5412 ] .

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I: Sociability in the three chambers test (3CT). LM controls spend more time with mouse stranger 1 (S1) whereas NexCre cTKOs spend comparable time exploring the novel object (NO) or S1. Bottom: schematic representation of test. [Paired Student's t-test comparison between chambers LM ***p = 0.0002; Wilcoxon test between chambers NexCre cTKO ns p = 0.5412].


C : T - Maze alternation shows that spontaneous alternation is absent in NexCre cTKOs . [ geno F ( 1 , 16 ) = 18 . 29 , p = 0 . 0006 ] . Chance level is indicated by a dotted .

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C: T-Maze alternation test shows that spontaneous alternation is absent in NexCre cTKOs. [geno F(1,16) = 18.29, p = 0.0006]. Chance level is indicated by a dotted line.


N : Morris Water Maze ( MWM ) , escape latency . During place navigation training , NexCre cTKO showed no evidence of learning . [ geno F ( 1 , 18 ) = 30 . 53 , p < 0 . 0001 ; day F ( 4 , 72 ) = 13 . 35 , p < 0 . 0001 ; day × geno F ( 4 , 72 ) = 11 . 77 p < 0 . 0001 ] .

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N: Morris Water Maze (MWM), escape latency. During place navigation training, NexCre cTKO animals showed no evidence of learning. [geno F(1,18) = 30.53, p < 0.0001; day F(4,72) = 13.35, p < 0.0001 ; day × geno F(4,72) = 11.77 p < 0.0001].


A expression of LINC00839 in CRC tissues normal mucosa tissues in UALCAN database . B , C , D expression of LINC00839 in unpaired CRC normal samples in LnCAR database ( GSE37364 , GSE71187 , GSE33113 ) . Data information : In A , B , C , D middle lines represent median , upper lower lines represent upper lower quartiles . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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A The expression of LINC00839 in CRC tissues and normal mucosa tissues in the UALCAN database. B, C, D The expression of LINC00839 in unpaired CRC and normal samples in the LnCAR database (GSE37364, GSE71187, and GSE33113). Data information: In A, B, C, D middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


E , F expression of LINC00839 in tumour tissues NATs from CRC cohort ( n = 45 ) . nmCRC denotes CRC without metastases , mCRC denotes CRC with metastases . Data information : In E , F , data are presented as mean ± SD were analysed by Student ' s t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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E, F The expression levels of LINC00839 in tumour tissues and NATs from the CRC cohort (n = 45). nmCRC denotes CRC patients without metastases, and mCRC denotes CRC patients with metastases. Data information: In E, F, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


G Representative ISH of LINC00839 in paraffin - embedded samples of CRC tumour tissues NATs . ( a ) Expression of LINC00839 in tumour tissues NATs . Scale bars , 100 μm . ( b ) Magnification of normal mucosal ( c ) magnification of tumour . Scale bars , 50 μm . ( d ) Weakly positive of LINC00839 in normal mucosa . Scale bars , 50 μm . ( e ) Strong positive of LINC00839 in tumours ( f ) magnification of local . Scale bars , 100 μm .

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G Representative ISH staining of LINC00839 in paraffin-embedded samples of CRC tumour tissues and NATs. (a) Expression of LINC00839 in tumour tissues and NATs. Scale bars, 100 μm. (b) Magnification of normal mucosal area and (c) magnification of the tumour area. Scale bars, 50 μm. (d) Weakly positive staining of LINC00839 in normal mucosa. Scale bars, 50 μm. (e) Strong positive staining of LINC00839 in tumours and (f) magnification of the local area. Scale bars, 100 μm.


H expression score of tumours NATs in ISH cohort ( n = 222 ) . Data information : In H data are shown as mean ± SD were analysed by Student ' s t . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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H The expression score of the tumours and NATs in the ISH cohort (n = 222). Data information: In H the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


I Differential expression of LINC00839 in nonmetastatic CRC ( nmCRC ) metastatic CRC ( mCRC ) . Data information : In I , data are shown as mean ± SD were analysed by Student ' s t . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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I Differential expression of LINC00839 in nonmetastatic CRC patients (nmCRC) and metastatic CRC patients (mCRC). Data information: In I, the data are shown as the mean ± SD and were analysed by Student's t test. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


J Expression of LINC00839 in CRC with early - stage ( stage I II ) advanced - stage ( stage III IV ) disease . Data information : In J , middle lines represent median , upper lower lines represent upper lower quartiles . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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J Expression of LINC00839 in CRC patients with early-stage (stage I and II) and advanced-stage (stage III and IV) disease. Data information: In J, middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


Kaplan - Meier of overall survival rate of ISH CRC cohort .

Kaplan-Meier analysis of overall survival rate of the ISH CRC cohort.


L Kaplan - Meier of overall disease - free survival rate of ISH CRC cohort . M Kaplan - Meier of disease - free survival rate of CRC in LnCAR database ( GSE33113 ) .

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L Kaplan-Meier analysis of overall disease-free survival rate of the ISH CRC cohort. M Kaplan-Meier analysis of the disease-free survival rate of CRC patients in the LnCAR database (GSE33113).


C Colony formation was performed with LINC00839 - overexpressing vector control . , data are presented as mean ± SD were analysed by Student ' s t , n = 3 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

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C Colony formation assay was performed with LINC00839-overexpressing cells and vector control cells. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


D Wound - healing was performed with LINC00839 - overexpressing vector control . Scale bars , 100 μm . , data are presented as mean ± SD were analysed by Student ' s t , n = 3 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

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D Wound-healing assay was performed with LINC00839-overexpressing cells and vector control cells. Scale bars, 100 μm. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


E Matrigel invasion was performed with LINC00839 - overexpressing . Scale bars , 100 μm . , data are presented as mean ± SD were analysed by Student ' s t , n = 3 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

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E Matrigel invasion assay was performed with LINC00839-overexpressing cells. Scale bars, 100 μm. , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


F CDX model showing effect of LINC00839 on tumour growth . Representative image of tumours harvested from mouse model ( upper panel ) , quantitative of tumour tumour volume ( medium panel ) , representative of of Ki - 67 in tumours ( bottom panel ) ( n = 6 for each cohort , results are presented as mean ± SD , Student ' s t ( medium left low right panel ) ANOVA ( medium right ) . Scale bars , 100 μm ( n = 6 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The CDX model showing the effect of LINC00839 on tumour growth. Representative image of tumours harvested from the mouse model (upper panel), the quantitative analysis of tumour weight and tumour volume (medium panel), and representative images and quantification of IHC staining of Ki-67 in tumours (bottom panel) (n = 6 for each cohort, and the results are presented as the mean ± SD, Student's t test (medium left and low right panel) and ANOVA (medium right). Scale bars, 100 μm (n = 6 biological replicates . Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


. G CRC orthotopic mouse model revealed effect of LINC00839 on tumour development . Representative of tumours ( yellow arrowheads ) liver metastasis ( blue arrowheads ) in model ( upper panel ) , of tumour volume number of mice with liver metastasis ( medium panel ) , H & E of tumours metastases ( n = 6 for each cohort ) . Scale bars , 100 μm ( n = 6 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 .

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

. G The CRC orthotopic mouse model revealed the effect of LINC00839 on tumour development. Representative images of primary tumours (yellow arrowheads) and liver metastasis (blue arrowheads) in the model (upper panel), analysis of tumour volume and the number of mice with liver metastasis (medium panel), and H&E staining of tumours and metastases (n = 6 for each cohort). Scale bars, 100 μm (n = 6 biological replicates . Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000


A An pull - down was performed to identify that bind to LINC00839 in HCT116 .

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

A An RNA pull-down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells.


B pull - down WB showed that biotinylated LINC00839 can bind to Ruvb1 in HCT116 . Dot blot of - interactions indicating equal amounts of used in .

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

B RNA pull-down assay and WB showed that biotinylated LINC00839 can bind to Ruvb1 in HCT116 cells. Dot blot of RNA-protein interactions indicating equal amounts of RNA used in the assay.


C RIP experiments were performed to confirm binding of LINC00839 to Ruvb1 . IgG was used as a negative control antibody . Primers specific for U6 GAPDH were used as negative control primers . Data information : In C , data are presented as mean ± SD were analysed by unpaired t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RIP experiments were performed to confirm the binding of LINC00839 to Ruvb1. IgG was used as a negative control antibody. Primers specific for U6 and GAPDH were used as negative control primers. Data information: In C, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


D Different fragments of LINC00839 ( solid ) its antisense ( dotted ) were used for pull - down subsequent WB .

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

D Different fragments of LINC00839 (solid line) and its antisense sequence (dotted line) were used for the RNA pull-down assay and the subsequent WB.


E Full LINC00839 , antisense of LINC00839 , LINC00839 lacking nucleotides 1033 - 1290 were used for pull - down subsequent WB .

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

E Full length LINC00839, antisense sequence of LINC00839, and LINC00839 sequence lacking nucleotides 1033-1290 were used for the RNA pull-down assay and the subsequent WB.


F RIP experiments were performed by incubating Tip60 , INO80 , SRCAP , PIH1D1 antibodies with HCT116 lysates . Data information : In F , data are presented as mean ± SD were analysed by unpaired t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

F RIP experiments were performed by incubating Tip60, INO80, SRCAP, and PIH1D1 antibodies with HCT116 cell lysates. Data information: In F, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


G effect of LINC00839 on expression of Tip60 Ruvb1 was determined by WB .

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

G The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB.


H Interaction of endogenous Ruvb1 Tip60 in CRC was confirmed by Co - IP .

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

H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co-IP.


I interaction between Ruvb1 Tip60 in overexpressing wild - type LINC00839 LINC - Δ5 was confirmed by Co - IP .

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

I The interaction between Ruvb1 and Tip60 in cells overexpressing wild-type LINC00839 and LINC-Δ5 was confirmed by Co-IP.


J interaction between Ruvb1 Tip60 in LINC00839 - KD expressing LINC - Δ5 was confirmed by Co - IP .

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

J The interaction between Ruvb1 and Tip60 in LINC00839-KD cells and cells expressing LINC-Δ5 was confirmed by Co-IP.


K Acetylase activity assays were performed with LINC00839 - overexpressing LINC00839 - knockdown . Data information : In data are presented as mean ± SD were analysed by unpaired t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

K Acetylase activity assays were performed with LINC00839-overexpressing and LINC00839-knockdown cells. Data information: In the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


L Acetylase activity was measured in SW480 LoVo subjected to different treatments . Data information : In L , data are presented as mean ± SD were analysed by unpaired t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

L Acetylase activity was measured in SW480 and LoVo cells subjected to different treatments. Data information: In L, the data are presented as the mean ± SD and were analysed by unpaired t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


B of DEGs related to OXPHOS mitochondrial biogenesis . Expression are shown in log2 scale of normalized values ( see colour gradient ) ranging from low ( blue ) to high ( red ) expression .

O O O O O O O O O O O O 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 Heatmap of DEGs related to OXPHOS and mitochondrial biogenesis. Expression levels are shown in log2 scale of normalized intensity values (see colour gradient) ranging from low (blue) to high (red) expression.


D , E ATP NAD / NADH were measured in LINC00839 - overexpressing LINC00839 - KD ( mean ± SD , Student ' s t , n = 3 biological replicates ) . Data information : Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

D, E The ATP levels and the NAD/NADH ratio were measured in LINC00839-overexpressing and LINC00839-KD cells (mean ± SD, Student's t test, n = 3 biological replicates). Data information: Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


F Expression of MT - ND5 , MT - CYB , MT - CO1 , TFAM in LINC00839 - overexpressing LINC00839 - KD as determined by WB .

F Expression of MT-ND5, MT-CYB, MT-CO1, and TFAM in LINC00839-overexpressing and LINC00839-KD cells as determined by WB.


G of NRF1 expression in CRC tissues ( T ) normal tissues ( N ) GEPIA database . Data information : In G , middle lines represent median , upper lower lines represent upper lower quartiles ; data were analysed by ANOVA . Across experiments , P values are as follows : ns = not significant , * P < 0 .

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

G Analysis of NRF1 expression in CRC tissues (T) and normal tissues (N) the GEPIA database. Data information: In G, the middle lines represent the median, and the upper and lower lines represent the upper and lower quartiles; the data were analysed by ANOVA. Across experiments, the P values are as follows: ns = not significant, *P < 0.05


H Correlation of LINC00839 NRF1 mRNA in TCGA GTEx databases ( Pearson ) .

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

H Correlation analysis of LINC00839 and NRF1 mRNA levels in the TCGA and GTEx databases (Pearson).


I , Expression of NRF1 in LINC00839 - overexpressin LINC00839 - KD was measured by qPCR Data information : In I , data are presented as mean ± SD were analysed by Student ' s t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Expression of NRF1 in LINC00839-overexpressin and LINC00839-KD cells was measured by qPCR Data information: In I, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


J Expression of NRF1 in LINC00839 - overexpressin LINC00839 - KD was measured by WB .

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

J Expression of NRF1 in LINC00839-overexpressin and LINC00839-KD cells was measured by WB.


K , L enrichment of NRF1 promoter ( P1 - P10 ) by H4K5ac H4K8ac antibodies in LINC00839 - overexpressing control LoVo as determined by CHIP . Normal IgG was used as a negative control . Data information : In K , L , , data are presented as mean ± SD were analysed by Student ' s t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

K, L The enrichment of the NRF1 promoter (P1-P10) by H4K5ac and H4K8ac antibodies in LINC00839-overexpressing and control LoVo cells as determined by CHIP assay. Normal IgG was used as a negative control. Data information: In K, L, , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


M , N enrichment of P2 P3 fragments by H4K5ac H4K8ac antibodies in LoVo as determined by CHIP . Normal IgG was used as a negative control . Data information : In M , N , data are presented as mean ± SD were analysed by Student ' s t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

M, N The enrichment of the P2 and P3 fragments by H4K5ac and H4K8ac antibodies in LoVo cells as determined by CHIP assay. Normal IgG was used as a negative control. Data information: In M, N , the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


O enrichment of NRF1 with a probe targeting LINC00839 relative to a NC probe in HCT116 , as determined by ChIRP . LacZ served as a negative control ( NC ) . Data information : In O , data are presented as mean ± SD were analysed by Student ' s t , n = 3 technical replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

O The enrichment of NRF1 with a probe targeting LINC00839 relative to a NC probe in HCT116 cells, as determined by ChIRP assay. LacZ served as a negative control (NC). Data information: In O, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 technical replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


A , B ATP NAD / NADH ratios were measured in SW480 LoVo . Data information : In A , B , data are presented as mean ± SD were analysed by Student ' s t , n = 3 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O B-MEASURED_VAR I-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 O

A, B ATP levels and NAD/NADH ratios were measured in SW480 and LoVo cells. Data information: In A, B, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


C Expression of genes related to mitochondrial biogenesis EMT in SW480 LoVo as determined by WB .

C Expression of genes related to mitochondrial biogenesis and EMT in SW480 and LoVo cells as determined by WB.


D Invasion of SW480 LoVo was assessed by Matrigel invasion . Scale bars , 100 μm . Data information : In D , data are presented as mean ± SD were analysed by Student ' s t , n = 3 biological replicates . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Invasion of SW480 and LoVo cells was assessed by Matrigel invasion assay. Scale bars, 100 μm. Data information: In D, the data are presented as the mean ± SD and were analysed by Student's t test, n = 3 biological replicates. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


E Representative of tumours ( yellow arrowhead ) metastases in orthotopic implantation models ( n = 6 for each cohort , left panel ) . number of mice with liver metastasis ( blue arrowhead ) was determined . tumour volume in each group was calculated ( right panel ) . H & E of tumours metastases ( bottom panel ) . Scale bars , 200 μm ( n = 6 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of primary tumours (yellow arrowhead) and metastases in the orthotopic implantation models (n = 6 for each cohort, left panel). The number of mice with liver metastasis (blue arrowhead) was determined. The primary tumour volume in each group was calculated (right panel). H&E staining of tumours and metastases (bottom panel). Scale bars, 200 μm (n = 6 biological replicates).


F ISH of LINC00839 of NRF1 , H4K5ac , H4K8ac in paraffin - embedded samples of CRC tumour tissues NATs . Scale bars , 200 μm .

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

F ISH staining of LINC00839 and IHC staining of NRF1, H4K5ac, and H4K8ac in paraffin-embedded samples of CRC tumour tissues and NATs. Scale bars, 200 μm.


G , H , Correlation of expression of LINC00839 NRF1 ( G ) , H4K5ac ( H ) in CRC ( n = 120 ) . Data information : In G , H show Pearson correlation coefficient results . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

G, H, Correlation analysis of the expression of LINC00839 and NRF1 (G), H4K5ac (H) in CRC patients (n = 120). Data information: In G, H show the Pearson correlation coefficient results. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


I Correlation of expression of LINC00839 H4K8ac ( I ) in CRC ( n = 120 ) . Data information : In I show Pearson correlation coefficient results . Across experiments , P values are as follows : ns = not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

I Correlation analysis of the expression of LINC00839 and H4K8ac (I) in CRC patients (n = 120). Data information: In I show the Pearson correlation coefficient results. Across experiments, the P values are as follows: ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


J Correlation of NRF1 expression with overall survival rate in with CRC ( n = 120 ) . NRF1 expression was stratified as high low according to median expression level .

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

J Correlation of NRF1 expression with the overall survival rate in patients with CRC (n = 120). NRF1 expression was stratified as high and low according to the median expression level.


One - stage were injected animally with mRNA encoding dkk1 ( 35 / 45 pg ) . Total was isolated from ten control at neurula stage 17 ( lane 2 ) ten from each dkk1 injected group ( lanes 3 - 4 ) . Various neural AP markers were examined by sqRT - PCR : cement forebrain ( xag1 , xanf1 , otx2 ) , hindbrain ( krox20 , hoxb3 ) , hindbrain / spinal cord border ( hoxb4 ) , spinal cord ( hoxb9 , cdx1 - 2 - 4 , hoxd10 ) . Ef1α serves as a control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNA encoding dkk1 (35/45 pg) protein. Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each dkk1 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with zygotic - expressing Nxfz8 / pCS2 encoding plasmid ( 100 / 200 pg ) . Total was isolated from ten control at neurula stage 17 ( lane 2 ) ten from each Nxfz8 injected group ( lanes 3 - 4 ) . Various neural AP markers were examined by sqRT - PCR : cement forebrain ( xag1 , xanf1 , otx2 ) , hindbrain ( krox20 , hoxb3 ) , hindbrain / spinal cord border ( hoxb4 ) , spinal cord ( hoxb9 , cdx1 - 2 - 4 , hoxd10 ) . Ef1α serves as a control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with the zygotic-expressing Nxfz8/pCS2 encoding plasmid (100/200 pg). Total RNA was isolated from ten control embryos at neurula stage 17 (lane 2) and ten embryos from each Nxfz8 injected group (lanes 3-4). Various neural AP markers were examined by sqRT-PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


from same experiment as in ( A ) underwent in situ hybridization . Expression of hindbrain marker krox20 ( upper two panels ) was reduced by Dkk1 ( 45pg ) in 83 % of ( n = 18 ) . Expression of spinal cord markers ( hoxb9 , cdx4 , hox10 ) was normal , being unchanged in 89 % of ( n = 18 for each marker ) . Control , ( CE ) .

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 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

Embryos from the same experiment as in (A) underwent in situ hybridization. Expression of the hindbrain marker krox20 (upper two panels) was reduced by Dkk1 (45pg) in 83% of the embryos (n=18). Expression of spinal cord markers (hoxb9, cdx4, hox10) was normal, being unchanged in 89% of the embryos (n=18 for each marker). Control embryos, (CE).


One - stage were injected animally with Zic1 - MO ( 25 / 40 ng ) . Total was isolated from ten control at neurula stage 18 ( lane 2 ) ten from each Zic1 - MO injected group ( lanes 3 - 4 ) . Neural AP markers were examined by sqRT - PCR : forebrain ( xanf1 , otx2 ) , hindbrain ( krox20 , hoxb1 , hoxb3 ) , spinal cord ( hoxb9 , cdx1 - 4 ) panneural ( soxd ) . Ef1α is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with the Zic1-MO (25/40 ng). Total RNA was isolated from ten control embryos at neurula stage 18 (lane 2) and ten embryos from each Zic1-MO injected group (lanes 3-4). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with mRNA encoding Zic5 dominant - negative ( 0 . 5 ng ) . Total was isolated from five control at neurula stage 18 ( lane 1 ) five from injected group ( lanes 2 ) . Neural AP markers were examined by sqRT - PCR : hindbrain ( krox20 ) spinal cord ( cdx1 - 4 ) . Ef1α is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 3 ) .

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

One-cell stage embryos were injected animally with mRNA encoding Zic5 dominant-negative (0.5 ng) protein. Total RNA was isolated from five control embryos at neurula stage 18 (lane 1) and five embryos from the injected group (lanes 2). Neural AP markers were examined by sqRT-PCR: hindbrain (krox20) and spinal cord (cdx1-4). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 3).


One - stage were injected animally with Zic1 - MO ( 25 ng ) . underwent in situ hybridization . Expression of panneural sox2 marker was normal , forebrain marker xanf1 was expanded in 80 % of , hindbrain representative marker krox20 was reduced in 80 % of representative spinal cord marker cdx4 was expanded in 90 % of . N = 23 were per marker .

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

One-cell stage embryos were injected animally with the Zic1-MO (25 ng). Embryos underwent in situ hybridization. Expression of the panneural sox2 marker was normal, the forebrain marker xanf1 was expanded in 80% of the embryos, the hindbrain representative marker krox20 was reduced in 80% of the embryos and the representative spinal cord marker cdx4 was expanded in 90% of the embryos. N = 23 embryos were per marker.


One - stage were injected animally with mRNAs encoding noggin ( 5 / 20 pg ) / or meis3 ( 0 . 5 ng ) . AC explants were removed from control injected at blastula stage 9 , explants were cultured to neurula stage 17 . Total was isolated from seven control ( lane 2 ) , from eighteen ACs from each group ( lanes 3 - 8 ) . Neural AP markers were examined by sqRT - PCR : forebrain ( xanf1 , otx2 ) , hindbrain ( krox20 , hoxb1 , hoxb3 ) , spinal cord ( hoxb9 , cdx1 - 4 ) panneural ( soxd ) . Ef1α is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural AP markers were examined by sqRT-PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4) and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with mRNA encoding bmp4 ( 0 . 2 ng ) . Total was isolated from five control at neurula stage 17 ( lane 1 ) five from injected group ( lanes 2 ) . Neural mesodermal markers were examined by sqRT - PCR : forebrain ( otx2 ) , hindbrain ( krox20 , hoxb3 ) , spinal cord ( hoxb9 , hoxc10 , cdx4 ) , panneural ( nrp1 , ncam ) mesodermal ( muscle actin - MA ) . ODC is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein. Total RNA was isolated from five control embryos at neurula stage 17 (lane 1) and five embryos from the injected group (lanes 2). Neural and mesodermal markers were examined by sqRT-PCR: forebrain (otx2), hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), panneural (nrp1, ncam) and mesodermal (muscle actin - MA). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with mRNA encoding bmp4 ( 0 . 2 ng ) underwent in situ hybridization . Expression of hindbrain marker hoxb3 was reduced / eliminated by bmp4 ( 0 . 2 ng ) in 68 % of ( n = 19 ) . Expression of spinal cord marker cdx4 was slightly increased / normal , in 100 % of ( n = 19 ) .

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

One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein Embryos underwent in situ hybridization. Expression of the hindbrain marker hoxb3 was reduced/eliminated by bmp4 (0.2 ng) in 68% of the embryos (n=19). Expression of the spinal cord marker cdx4 was slightly increased/normal, in 100% of the embryos (n=19).


One - stage were injected animally with increasing concentrations of bmp4 encoding mRNA ( 5 - 50 pg ) . AC explants were removed from control injected at blastula stage 9 , explants were cultured to neurula stage 16 . Total was isolated from five control ( lane 2 ) , from eighteen ACs from each group ( lanes 3 - 7 ) . Neural , epidermal mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 ) , spinal cord ( hoxb9 , hoxc10 / d10 , cdx1 - 2 - 4 ) , epidermal cytokeratin ( E13 ) , lateral mesoderm ( osr1 - 2 ) dorsal - lateral mesoderm muscle actin ( MA ) . ODC is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-7). Neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10/d10, cdx1-2-4), epidermal cytokeratin (E13), lateral mesoderm (osr1-2) and dorsal-lateral mesoderm muscle actin (MA). ODC is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with mRNAs encoding bmp4 ( 50 / 150 pg ) / or meis3 ( 0 . 7 ng ) . AC explants were removed from control injected at blastula stage 9 , explants were cultured to neurula stage 16 . Total was isolated from five control ( lane 2 ) , from eighteen ACs from each group ( lanes 3 - 8 ) . Neural mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 ) , spinal cord ( hoxb9 , hoxc10 , cdx2 ) , lateral mesoderm ( osr1 ) . Ef1α is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50/150 pg) and/or meis3 (0.7 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-8). Neural and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10, cdx2), and lateral mesoderm (osr1). Ef1α is the control for quantitating the RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with increasing concentrations of bmp4 encoding mRNA ( 75 - 200 pg ) . AC explants were removed from control injected at blastula stage 9 , explants were cultured to gastrula stage 11 - 11 . 5 . Total was isolated from five control ( lane 2 ) , from eighteen ACs from each group ( lanes 3 - 6 ) . Marker expression was determined by sqRT - PCR . Xenopus brachyury ( xbra ) expression was induced by BMP4 in a dose - dependent manner . Marker expression was determined by sqRT - PCR . Neither dorsal ( chd , goosecoid - gsc ) or ventral - lateral ( osr1 ) mesoderm markers were induced by BMP4 . Sox2 / 3 neural markers are expressed in ACs are unchanged by BMP expression . EF1α is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (75-200 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to gastrula stage 11-11.5. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). Marker expression was determined by sqRT-PCR. Xenopus brachyury (xbra) expression was induced by BMP4 in a dose-dependent manner. Marker expression was determined by sqRT-PCR. Neither dorsal (chd, goosecoid-gsc) or ventral-lateral (osr1) mesoderm markers were induced by BMP4. Sox2/3 neural markers are expressed in the ACs and are unchanged by BMP expression. EF1α is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with increasing concentrations of bmp4 encoding mRNA ( 5 - 50 pg ) . AC explants were removed from control injected at blastula stage 9 , explants were cultured to neurula stage 16 . Total was isolated from five control ( lane 2 ) , from eighteen ACs from each group ( lanes 3 - 6 ) . FGF3 - 4 - 8 expression was determined by sqRT - PCR . ODC is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5-50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). FGF3-4-8 expression was determined by sqRT-PCR. ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


One - stage were injected animally with mRNAs encoding bmp4 ( 50 pg ) / or FGF dominant - negative receptor ( FGF - DNR , 1 . 5 ng ) . AC explants were removed from control injected at blastula stage 9 , explants were cultured to neurula stage 16 . Total was isolated from five control ( lane 2 ) , from eighteen ACs from each group ( lanes 3 - 6 ) . In addition to fgf3 , neural , epidermal mesodermal markers were examined by sqRT - PCR : hindbrain ( krox20 , hoxb3 ) , spinal cord ( hoxb9 , hoxc10 , cdx4 ) , lateral mesoderm ( osr1 ) , dorsal - lateral mesoderm muscle actin ( MA ) , epidermal cytokeratin ( E13 ) . ODC is control for quantitating . - RT - PCR was performed on total isolated from control ( lane 1 ) .

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

One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50 pg) and/or FGF dominant-negative receptor (FGF-DNR, 1.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3-6). In addition to fgf3, neural, epidermal and mesodermal markers were examined by sqRT-PCR: hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), lateral mesoderm (osr1), dorsal-lateral mesoderm muscle actin (MA), and epidermal cytokeratin (E13). ODC is the control for quantitating RNA levels. -RT-PCR was performed on total RNA isolated from control embryos (lane 1).


[ a ] Sagittal sections obtained from mice ( p15 ) of indicated genotypes were immunostained for ADP - ribose using pan - ADP - ribose detection reagent MABE1016 . Representative showing of ADP - ribose in hippocampal regions CA1 , CA3 , dentate gyrus ( DG ) , in cerebral cortex . Red dotted boxes highlight elevated ADP - ribose in Xrcc1Nes - Cre cerebellum ( left dotted box ) hippocampus ( right doted box ) . Scale bars : 5 mm , 50 μm . WT ( n = 4 mice ) , Xrcc1Nes - Cre ( n = 4 ) , Parp1 + / - / Xrcc1Nes - Cre ( n = 4 ) , Parp1 - / - / Xrcc1Nes - Cre ( n = 3 ) Parp1 - / - ( n = 3 ) . Summary histograms show mean ± . Pairwise comparisons between WT versus Xrcc1Nes - Cre mice WT versus Parp1 - / - / Xrcc1Nes - Cre mice were conducted by Kruskal - Wallis ANOVA with Dunn ' s post - hoc statistically significant differences ( * p < 0 . 05 ) are shown .

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

[a] Sagittal sections obtained from mice (p15) of the indicated genotypes were immunostained for ADP-ribose using the pan-ADP-ribose detection reagent MABE1016. Representative images showing levels of ADP-ribose in the hippocampal regions CA1, CA3, and dentate gyrus (DG), and in the cerebral cortex. Red dotted boxes highlight the elevated ADP-ribose staining in Xrcc1Nes-Cre cerebellum (left dotted box) and hippocampus (right doted box). Scale bars: 5 mm, 50 μm. WT (n = 4 mice), Xrcc1Nes-Cre (n = 4), Parp1+/- /Xrcc1Nes-Cre (n = 4), Parp1-/-/Xrcc1Nes-Cre (n = 3) and Parp1-/- (n = 3). Summary histograms show mean ± SEM. Pairwise comparisons between WT versus Xrcc1Nes-Cre mice and WT versus Parp1-/-/Xrcc1Nes-Cre mice were conducted by Kruskal-Wallis ANOVA with Dunn's post-hoc test and statistically significant differences (*p<0.05) are shown.


[ b ] extracts from wild type ( WT ) , Xrcc1Nes - Cre , Parp1 - / - forebrain tissue , containing hippocampus cortex , were incubated with 1 mM NAD + for 45 min in presence or absence of PARP inhibitor as indicated , ADP - ribosylation detected by western blotting using poly ( ADP - ribose ) - specific detection reagent MABE1031 . Representative from two or more independent experiments are shown . A western blot showing level of Parp1 Xrcc1 in forebrain tissue extracts is also shown , Inset .

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

[b] Protein extracts from wild type (WT), Xrcc1Nes-Cre, and Parp1-/- forebrain tissue, containing hippocampus and cortex, were incubated with 1 mM NAD+ for 45 min in the presence or absence of PARP inhibitor as indicated, and ADP-ribosylation detected by western blotting using the poly(ADP-ribose)-specific detection reagent MABE1031. Representative images from two or more independent experiments are shown. A western blot showing the level of Parp1 and Xrcc1 in the forebrain tissue extracts is also shown, Inset.


[ a ] Kaplan - Meier curves for survival of mice of indicated genotypes . table shows number of individuals in each group , median survival ( ) , p - values from pairwise curve comparisons ; Und : undetermined ( Log - rank Mantel - Cox ) .

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

[a] Kaplan-Meier curves for survival of mice of the indicated genotypes. The table shows number of individuals in each group, median survival (weeks), and p-values from pairwise curve comparisons; Und: undetermined (Log-rank Mantel-Cox tests).


[ b ] Video monitoring recording of generalised running / bouncing seizures in mice of indicated genotypes from P15 - P19 . point of of Xrcc1Nes - Cre mice by fatal seizure is indicated ( cross ) . Note no seizures from mice of other genotypes were detected . WT ( n = 9 mice ) , Xrcc1Nes - cre ( n = 3 mice ) , Parp1 + / - / Xrcc1Nes - Cre ( n = 3 mice ) , Parp1 - / - / Xrcc1Nes - Cre ( n = 3 ) .

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

[b] Video monitoring and recording of generalised running/bouncing seizures in mice of the indicated genotypes from P15-P19. The point of death of Xrcc1Nes-Cre mice by fatal seizure is indicated (cross). Note no seizures from mice of other genotypes were detected. WT (n = 9 mice), Xrcc1Nes-cre (n = 3 mice), Parp1+/- /Xrcc1Nes-Cre (n = 3 mice), and Parp1-/-/Xrcc1Nes-Cre (n = 3).


[ b ] Heatplots of cumulative SLEs : WT ( n = 11 slices from 3 mice ) , Xrcc1Nes - Cre ( n = 12 slices from 4 mice ) , Parp1 + / - / Xrcc1Nes - Cre ( n = 12 slices from 3 mice ) Parp1 - / - / Xrcc1Nes - Cre ( n = 10 slices from 3 mice ) . Each horizontal bar corresponds to one slice with colour code indicating cumulative SLEs .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O 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] Heatplots of cumulative SLEs: WT (n=11 slices from 3 mice), Xrcc1Nes-Cre (n=12 slices from 4 mice), Parp1+/-/Xrcc1Nes-Cre (n=12 slices from 3 mice) and Parp1-/-/Xrcc1Nes-Cre (n=10 slices from 3 mice). Each horizontal bar corresponds to one slice with the colour code indicating cumulative SLEs.


[ e ] Summary histograms ( mean ± ) of cumulative seizure - like activity at 5 , 10 , 15 min timepoints in cortex , CA1 , CA3 regions of wild type Xrcc1Nes - Cre brain slices from mice treated or not for 5 - 8 days ad libitum with PARP1 inhibitor ( ABT - 888 ) prior to . WT ( n = 8 slices from 4 mice ) , Xrcc1Nes - Cre ( n = 10 from 5 mice ) , WT + PARPi ( n = 6 from 3 mice ) , Xrcc1Nes - Cre + PARPi ( n = 9 from 3 mice ) . Pairwise comparisons at 15 min time point between WT versus Xrcc1Nes - Cre mice WT versus Xrcc1Nes - Cre mice + PARPi were conducted by Kruskal - Wallis with Dunn ' s post - hoc statistically significant differences ( * p < 0 . 05 ) are shown .

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

[e] Summary histograms (mean ± SEM) of cumulative seizure-like activity at 5, 10, and 15 min timepoints in cortex, CA1, and CA3 regions of wild type and Xrcc1Nes-Cre brain slices from mice treated or not for 5-8 days ad libitum with PARP1 inhibitor (ABT-888) prior to analysis. WT (n = 8 slices from 4 mice), Xrcc1Nes-Cre (n = 10 from 5 mice), WT + PARPi (n = 6 from 3 mice), Xrcc1Nes-Cre + PARPi (n = 9 from 3 mice). Pairwise comparisons at the 15 min time point between WT versus Xrcc1Nes-Cre mice and WT versus Xrcc1Nes-Cre mice + PARPi were conducted by Kruskal-Wallis with Dunn's post-hoc tests and statistically significant differences (*p<0.05) are shown.