Figure 5 ( 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 + cells in crypt epithelium from DT treated Atoh1CreERT2 ; Lgr5DTR - GFP ; ROSA26tdTomato mice . ( 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 + 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 + 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 .

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

Figure 5(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+ cells in crypt epithelium from DT treated Atoh1CreERT2;Lgr5DTR-GFP;ROSA26tdTomato mice.(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+ 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+ 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.


Figure 6 ( b ) Atoh1 + lineage labelling post - DSS and DSS + DT . Data information : Scale bars ( b ) = 200μm ( 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 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 + 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 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 .

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

Figure 6(b) Atoh1+ lineage labelling post-DSS and DSS+DT. Data information: Scale bars (b)=200μm (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 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+ 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 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.


Figure 1B : 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 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 ) .

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

Figure 1B: 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 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).


Figure 2C : Representative 3D reconstructions of CA1 pyramidal neurons from deep ( left , dark blue ) and superficial ( right , light blue ) radial layers . 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 :

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

Figure 2C: Representative 3D reconstructions of CA1 pyramidal neurons from deep (left, dark blue) and superficial (right, light blue) radial layers.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:


Figure 3A , 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

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

Figure 3A, 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


Figure 4A : 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

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

Figure 4A: 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


Figure 5A : 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 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 ] .

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

Figure 5A: 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 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].


Figure 6C : 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 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 ] .

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

Figure 6C: 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 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].


Figure 1A 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 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 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 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 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 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 analysis of overall survival rate of the ISH CRC cohort . 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 ) .

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

Figure 1A 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 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 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 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 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 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 analysis of overall survival rate of the ISH CRC cohort.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).


Figure 2C 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 . 000D 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 . 000E 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 . 000F 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 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 .

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

Figure 2C 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 < 0.000D 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 < 0.000E 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 < 0.000F 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 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


Figure 3A An RNA pull - down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells . 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 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 line ) and its antisense sequence ( dotted line ) were used for the RNA pull - down assay and the subsequent WB . 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 , 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 The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB . H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co - IP . I The interaction between Ruvb1 and Tip60 in cells overexpressing wild - type LINC00839 and LINC - Δ5 was confirmed by Co - IP . 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 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 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 .

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

Figure 3A An RNA pull-down assay was performed to identify proteins that bind to LINC00839 in HCT116 cells.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 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 line) and its antisense sequence (dotted line) were used for the RNA pull-down assay and the subsequent WB.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, 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 The effect of LINC00839 on the expression of Tip60 and Ruvb1 was determined by WB.H Interaction of endogenous Ruvb1 and Tip60 in CRC cells was confirmed by Co-IP.I The interaction between Ruvb1 and Tip60 in cells overexpressing wild-type LINC00839 and LINC-Δ5 was confirmed by Co-IP.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 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 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.


Figure 4B 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 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 , and TFAM in LINC00839 - overexpressing and LINC00839 - KD cells as determined by WB . 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 . 05H Correlation analysis of LINC00839 and NRF1 mRNA levels in the TCGA and GTEx databases ( Pearson ) . 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 and LINC00839 - KD cells was measured by WB . 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 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 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 .

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

Figure 4B 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 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, and TFAM in LINC00839-overexpressing and LINC00839-KD cells as determined by WB.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 < 0.05H Correlation analysis of LINC00839 and NRF1 mRNA levels in the TCGA and GTEx databases (Pearson).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 and LINC00839-KD cells was measured by WB.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 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 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.


Figure 5A , 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 and EMT in SW480 and LoVo cells as determined by WB . 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 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 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 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 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 the overall survival rate in patients with CRC ( n = 120 ) . NRF1 expression was stratified as high and low according to the median expression level .

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

Figure 5A, 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 and EMT in SW480 and LoVo cells as determined by WB.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 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 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 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 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 the overall survival rate in patients with CRC (n = 120). NRF1 expression was stratified as high and low according to the median expression level.


Figure 1One - 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 - 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 ) . 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 ) .

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

Figure 1One-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-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).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).


Figure 2One - 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 - 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 - 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 .

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

Figure 2One-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-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-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.


Figure 3One - 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 - 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 - 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 ) .

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

Figure 3One-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-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-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).


Figure 4One - 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 - 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 - 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 ) .

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

Figure 4One-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-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-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).


Figure 5One - 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 - 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 ) .

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

Figure 5One-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-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).


Figure 1 [ 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 ] 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 .

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

Figure 1 [a] 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] 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.


Figure 2 [ 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 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 ) .

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

Figure 2 [a] 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 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).


Figure 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 ) and Parp1 - / - / Xrcc1Nes - Cre ( n = 10 slices from 3 mice ) . Each horizontal bar corresponds to one slice with the colour code indicating cumulative SLEs .

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

Figure 3 [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.


Figure 4 [ 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 .

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

Figure 4 [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.


Figure 5 [ a ] Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes - Cre mouse pups , immunostained for ADP - ribose ( red ) , NeuN to identify neurons ( green ) , and counterstained with DAPI ( blue ) . Cells were pretreated with PARP inhibitor ( 10 µM ) or vehicle for 2 h prior to fixation , with PARG inhibitor ( 10 μM ) additionally present for the final hour . Scale bar 10 µm . [ b ] Histogram of mean ( ± SEM ) relative pan - ADP - ribose fluorescence in NeuN - positive hippocampal neurons pretreated with PARP inhibitor ( 5 µM ) or vehicle for 5 h prior to fixation , with PARG inhibitor ( 10 μM ) additionally present for the final hour . Neurons were cultured from WT ( n = 6 mice , > 180 cells per condition ) , Xrcc1Nes - Cre ( n = 6 , > 180 ) , Parp1 + / - / Xrcc1Nes - Cre ( n = 3 , > 90 ) , and Parp1 - / - / Xrcc1Nes - Cre ( n = 3 , > 90 ) . * indicates significant differences from WT ( Kruskal - Wallis ANOVA , p = 0 . 0013 and Dunn ' s post - hoc tests ) .

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

Figure 5 [a] Representative images of indirect immunofluorescence of DIV6 hippocampal neurons cultured from P1 WT and Xrcc1Nes-Cre mouse pups, immunostained for ADP-ribose (red), NeuN to identify neurons (green), and counterstained with DAPI (blue). Cells were pretreated with PARP inhibitor (10 µM) or vehicle for 2 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Scale bar 10 µm. [b] Histogram of mean (± SEM) relative pan-ADP-ribose fluorescence in NeuN-positive hippocampal neurons pretreated with PARP inhibitor (5 µM) or vehicle for 5 h prior to fixation, with PARG inhibitor (10 μM) additionally present for the final hour. Neurons were cultured from WT (n = 6 mice, > 180 cells per condition), Xrcc1Nes-Cre (n = 6, > 180), Parp1+/-/Xrcc1Nes-Cre (n = 3, > 90), and Parp1-/-/Xrcc1Nes-Cre (n = 3, > 90). * indicates significant differences from WT (Kruskal-Wallis ANOVA, p = 0.0013 and Dunn's post-hoc tests).


Figure 6Summary histogram of the mean ( ± SEM ) of the integrated fluorescence responses for all individual synapses for each condition . Synapse number is the same as in ( D - F ) . Responses are significantly higher in Xrcc1Nes - Cre synapses versus both WT and Parp1 - / - / Xrcc1Nes - Cre ( one - way ANOVA , * p < 0 . 0033 , and pairwise student ' s t - tests ) .

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

Figure 6Summary histogram of the mean (± SEM) of the integrated fluorescence responses for all individual synapses for each condition. Synapse number is the same as in (D-F). Responses are significantly higher in Xrcc1Nes-Cre synapses versus both WT and Parp1-/-/Xrcc1Nes-Cre (one-way ANOVA, *p < 0.0033, and pairwise student's t-tests).


Figure 1A . The survival of age - and sex - matched HDAC6 + / + mice and HDAC6 - / - mice was monitored for 9 days after intravenous VSV - Indiana infection ( 2 × 108 pfu / mouse ; n = 12 per group ; log - rank test ) . B . Determination of the viral load in organs by standard plaque assay . HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - Indiana , and the brain and spleen were collected at 5 dpi ( 2 × 108 pfu / mouse ; n = 6 per group ; Mann - Whitney test ) . C . Determination of viral loads in organs by Q - PCR of VSV viral transcripts . The brain and spleen were collected at 5 dpi after HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - Indiana ( 2 × 108 pfu / mouse ; n = 9 per group ; Mann - Whitney test ) . D . The viral load in the serum was assessed by standard plaque assay . HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - GFP . Serum was collected at the indicated time points and analyzed ( 4 × 108 pfu / mouse ; n = 3 per group ; Student ' s t - test ) . E . ELISA of IFN - β ( left ) and IL - 6 ( right ) in the serum of mice described in ( d ) ( Student ' s t - test ) . F . ELISA of serum IFN - β ( left ) and IL - 6 ( right ) after poly ( I : C ) injection . HDAC6 + / + and HDAC6 - / - mice were intravenously injected with poly ( I : C ) . Serum was collected at the indicated time points and analyzed ( 200 μg / mouse ; n = 3 per group ; Student ' s t - test ) .

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

Figure 1A. The survival of age- and sex-matched HDAC6+/+ mice and HDAC6-/- mice was monitored for 9 days after intravenous VSV-Indiana infection (2 × 108 pfu/mouse; n = 12 per group; log-rank test).B. Determination of the viral load in organs by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-Indiana, and the brain and spleen were collected at 5 dpi (2 × 108 pfu/mouse; n = 6 per group; Mann-Whitney test).C. Determination of viral loads in organs by Q-PCR of VSV viral transcripts. The brain and spleen were collected at 5 dpi after HDAC6+/+and HDAC6-/- mice were intravenously infected with VSV-Indiana (2 × 108 pfu/mouse; n = 9 per group; Mann-Whitney test).D. The viral load in the serum was assessed by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-GFP. Serum was collected at the indicated time points and analyzed (4 × 108 pfu/mouse; n = 3 per group; Student's t-test).E. ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (d) (Student's t-test).F. ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6-/- mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student's t-test).


Figure 2A , B . virus replication at 12 and 24 hpi , in HDAC6 + / + and HDAC6 - / - BMDMs in response to VSV - GFP ( MOI = 10 ) infection ( A ) and PR8 - GFP ( MOI = 5 ) infection ( B ) . C . Virus replication in HDAC6 + / + and HDAC6 - / - BMDMs in response to HSV - GFP ( MOI = 2 ) infection . D . Virus replication in HDAC6 + / + and HDAC6 - / - PBMCs in response to VSV - GFP ( MOI = 10 ) infection . E . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( A ) and ( B ) , and in HDAC6 + / + and HDAC6 - / - BMDMs treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) . F . Immunoblot analysis of the phosphorylated and inactive forms of IRF3 , IKBα , TBK1 , RIG - I , MAVS , HDAC6 , and β - actin at the indicated times ( 0 , 2 , 4 , 8 , and 16 h ) in HDAC6 + / + and HDAC6 - / - BMDMs . BMDMs were stimulated with PR8 - GFP ( MOI = 3 ) . G . Induction of mRNA for type I IFN , IL - 6 , and other IFN - related antiviral genes in HDAC6 + / + and HDAC6 - / - BMDMs in response to a RIG - I agonist stimulation at 6 h . HDAC6 + / + and HDAC6 - / - BMDMs were stimulated with 5 ' ppp - dsRNA ( 0 . 5 µg / ml ) for 6 h .

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

Figure 2A, B. virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- BMDMs in response to VSV-GFP (MOI = 10) infection (A) and PR8-GFP (MOI = 5) infection (B).C. Virus replication in HDAC6+/+ and HDAC6-/- BMDMs in response to HSV-GFP (MOI = 2) infection.D. Virus replication in HDAC6+/+ and HDAC6-/- PBMCs in response to VSV-GFP (MOI = 10) infection.E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6-/- BMDMs treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6-/- BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3).G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6-/- BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6-/- BMDMs were stimulated with 5'ppp-dsRNA (0.5 µg/ml) for 6 h.


Figure 3A , B . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in control and HDAC6 knockdown RAW264 . 7 cells in response to VSV - GFP ( MOI = 1 ) infection ( A ) and PR8 - GFP ( MOI = 1 ) infection ( B ) . bar , 100µmC . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( A ) and ( B ) , and in control and HDAC6 knockdown RAW264 . 7 cells treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) . D , E . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in HDAC6 + / + and HDAC6 - / - MEFs in response to VSV - GFP ( MOI = 1 ) infection ( D ) and PR8 - GFP ( MOI = 1 ) infection ( E ) . bar , 50µmF . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( D ) and ( E ) , and in HDAC6 + / + and HDAC6 - / - MEFs transfected with poly ( I : C ) ( 1 µg / ml ) .

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

Figure 3A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in control and HDAC6 knockdown RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µmC. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).D, E. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- MEFs in response to VSV-GFP (MOI = 1) infection (D) and PR8-GFP (MOI = 1) infection (E). bar, 50µmF. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (D) and (E), and in HDAC6+/+ and HDAC6-/- MEFs transfected with poly(I:C) (1 µg/ml).


Figure 4A , B . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 cells in response to VSV - GFP ( MOI = 1 ) infection ( A ) and PR8 - GFP ( MOI = 1 ) infection ( B ) . bar , 100µm . C . ELISA of IFN - b ( upper ) and IL - 6 ( lower ) levels in the supernatant of ( A ) , ( B ) , and in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 cells treated with poly ( I : C ) ( 20 μg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) . D , E . Luciferase assay in 293T cells transfected with an IFN - β luciferase promoter and TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) , followed by PR8 - GFP ( MOI = 2 ) infection ( D ) or by poly ( I : C ) ( E ) ( 1 μg / ml ) transfection for another 12 h . F , G . Luciferase assay in 293T cells transfected with RIG - I ( F ) , MDA - 5 ( G ) , an IFN - β luciferase promoter , and TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) . 24 h later , IFN - β activity was measured by luciferase reporter assay .

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

Figure 4A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm.C. ELISA of IFN-b (upper) and IL-6 (lower) levels in the supernatant of (A), (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h.F, G. Luciferase assay in 293T cells transfected with RIG-I (F), MDA-5 (G), an IFN-β luciferase promoter, and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng). 24 h later, IFN-β activity was measured by luciferase reporter assay.


Figure 5A . 293T cells were transfected with poly ( I : C ) and cell lysates were prepared at the indicated times for co - immunoprecipitation analysis of endogenous HDAC6 and RIG - I . Actin was used as the loading control . B . BMDMs were isolated from HDAC6 + / + and HDAC6 - / - mice and were transfected with 5ˊppp - dsRNA . Whole cell lysates were prepared after 8 h of transfection and co - immunoprecipitation analysis of RIG - I and HDAC6 was performed . C . HeLa cells were transfected with 5ˊppp - dsRNA . Cells were stained with anti - RIG - I ( red ) and anti - HDAC6 H300 ( green ) antibodies after 8 h of transfection . Nuclei were stained with DAPI ( blue ) ( bar , 20µm ) . D . Fluorescence microscopy , green fluorescence absorbance and virus replication at 24hpi in RIG - I + / + and RIG - I - / - MEFs . Cells were transfected with si - HDAC6 and si - control for 36 h , followed infection with VSV - GFP ( MOI = 1 ) . bar , 100µm . E . HDAC6 + / + , HDAC6 - / - MEFs , and HDAC6 - / - MEFs reconstituted with HDAC6 wild - type were subjected to immunoprecipitation using an anti - RIG - I antibody and immunoblotted with anti - acetyl - lysine and anti - RIG - I antibodies . Acetyl - tubulin was measured in total lysates to determine HDAC6 deacetylase activity . F . Whole cell lysates from HDAC6 + / + and HDAC6 - / - MEFs were prepared and used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I , anti - HDAC6 , and anti - β - actin antibodies . Intensity of pull - downed RIG - I was quantified . G . Whole cell lysates from HDAC6 - / - MEFs and HDAC6 - / - MEFs reconstituted with HDAC6 wild - type and HDAC6 - CDM were prepared and used in dsRNA pull - down assays . Samples were analyzed by western blotting with anti - RIG - I and anti - HDAC6 antibodies . Intensity of pull - downed RIG - I was quantified .

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Figure 5A. 293T cells were transfected with poly(I:C) and cell lysates were prepared at the indicated times for co-immunoprecipitation analysis of endogenous HDAC6 and RIG-I. Actin was used as the loading control.B. BMDMs were isolated from HDAC6+/+ and HDAC6-/- mice and were transfected with 5ˊppp-dsRNA. Whole cell lysates were prepared after 8 h of transfection and co-immunoprecipitation analysis of RIG-I and HDAC6 was performed.C. HeLa cells were transfected with 5ˊppp-dsRNA. Cells were stained with anti-RIG-I (red) and anti-HDAC6 H300 (green) antibodies after 8 h of transfection. Nuclei were stained with DAPI (blue) (bar, 20µm).D. Fluorescence microscopy, green fluorescence absorbance and virus replication at 24hpi in RIG-I+/+ and RIG-I-/- MEFs. Cells were transfected with si-HDAC6 and si-control for 36 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm.E. HDAC6+/+, HDAC6-/- MEFs, and HDAC6-/- MEFs reconstituted with HDAC6 wild-type were subjected to immunoprecipitation using an anti-RIG-I antibody and immunoblotted with anti-acetyl-lysine and anti-RIG-I antibodies. Acetyl-tubulin was measured in total lysates to determine HDAC6 deacetylase activity.F. Whole cell lysates from HDAC6+/+ and HDAC6-/- MEFs were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I, anti-HDAC6, and anti-β-actin antibodies. Intensity of pull-downed RIG-I was quantified.G. Whole cell lysates from HDAC6-/- MEFs and HDAC6-/- MEFs reconstituted with HDAC6 wild-type and HDAC6-CDM were prepared and used in dsRNA pull-down assays. Samples were analyzed by western blotting with anti-RIG-I and anti-HDAC6 antibodies. Intensity of pull-downed RIG-I was quantified.