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

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A 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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


B 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 .

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


D , E 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 .

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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 .

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 .

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


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

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H 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 .

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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 .

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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 .

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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 .

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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 .

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


A , B ATP 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 .

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


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

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


D Invasion of SW480 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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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


One - 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 ) .

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


One - 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 ) .

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

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


One - 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 ) .

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

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


One - 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 ) .

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

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


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

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


One - 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 ) .

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

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


One - 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 ) .

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

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

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


One - 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 ) .

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

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


One - 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 ) .

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

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

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


One - 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 ) .

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

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


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

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


[ a ] Sagittal sections obtained from mice ( p15 ) of 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 .

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

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

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


[ a ] Kaplan - Meier curves for survival of mice of 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 ) .

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

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


[ b ] Video monitoring 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O

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


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

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


[ e ] Summary histograms ( mean ± 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 .

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[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.


[ 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 ) .

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[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).


Summary 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 ) .

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Summary 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).


A . 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 ) .

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A. 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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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).


A , 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 ) .

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A, 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 .

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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 .

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 ) .

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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 ) .

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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 .

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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.


A , 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 ,

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A, 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, 100µm


C . 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 ) .

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C. 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 ,

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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, 50µm


F . 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 ) .

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F. 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).


A , 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 .

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A, 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 ) .

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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 .

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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 .

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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.


A . 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 .

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A. 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 .

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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 ) .

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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 .

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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 .

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

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 .

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

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 .

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

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.


A , B . 293T cells were transfected with the indicated expression plasmids . At 1 day post - transfection , whole cell lysates were subjected to dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with an anti - FLAG antibody .

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

A, B. 293T cells were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were subjected to dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with an anti-FLAG antibody.


C . A luciferase assay was performed using 293T cells transfected with the IFN - βluciferase promoter and TK - Renilla together with 2 ng of RIG - I wild - type , RIG - I 858Q , RIG - I 858R , RIG - I K909Q , or RIG - I K909R mutant , followed by transfection with poly ( I : C ) ( 1 μg / ml ) for another 12 h .

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

C. A luciferase assay was performed using 293T cells transfected with the IFN-βluciferase promoter and TK-Renilla together with 2 ng of RIG-I wild-type, RIG-I 858Q, RIG-I 858R, RIG-I K909Q, or RIG-I K909R mutant, followed by transfection with poly(I:C) (1 μg/ml) for another 12 h.


D . Fluorescence microscopy , green fluorescence absorbance , and virus replication at 24 hpi in RIG - I - / - MEFs . Cells were transiently transfected with empty vector , RIG - I wild - type , RIG - I K909Q , or RIG - I K909R mutant for 24 h , followed infection with VSV - GFP ( MOI = 1 ) . bar ,

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

D. Fluorescence microscopy, green fluorescence absorbance, and virus replication at 24 hpi in RIG-I-/- MEFs. Cells were transiently transfected with empty vector, RIG-I wild-type, RIG-I K909Q, or RIG-I K909R mutant for 24 h, followed infection with VSV-GFP (MOI = 1). bar, 100µm


E . HDAC6 - / - MEFs were transfected with the indicated expression plasmids . At 1 day post - transfection , whole cell lysates were prepared and used in dsRNA pull - down assays . Pull - down samples were analyzed by western blotting with anti - RIG - I and anti - β - actin antibodies .

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

E. HDAC6-/- MEFs were transfected with the indicated expression plasmids. At 1 day post-transfection, whole cell lysates were prepared and used in dsRNA pull-down assays. Pull-down samples were analyzed by western blotting with anti-RIG-I and anti-β-actin antibodies.


F . 293T cells were transfected with 2 μg of FLAG - tagged RIG - I and 1 , 5 , or 10 μg of V5 - tagged HDAC6 . At 36 h post - transfection , whole cell lysates were prepared for co - immunoprecipitation analysis of V5 - tagged HDAC6 and acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody .

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

F. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I and 1, 5, or 10 μg of V5-tagged HDAC6. At 36 h post-transfection, whole cell lysates were prepared for co-immunoprecipitation analysis of V5-tagged HDAC6 and acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody.


G . 293T cells were transfected with 2 μg of FLAG - tagged RIG - I . At 24 h post - transfection , cells were infected with VSV - GFP ( MOI = 0 . 1 ) . At 2 and 4 h post - infection , whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . Intensity of ac - 909K normalized to Flag - RIG - I was quantified .

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

G. 293T cells were transfected with 2 μg of FLAG-tagged RIG-I. At 24 h post-transfection, cells were infected with VSV-GFP (MOI = 0.1). At 2 and 4 h post-infection, whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to Flag-RIG-I was quantified.


H . whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 ( on RIG - I wild - type ) . Samples were analyzed by western blotting with a K909 acetylation - specific antibody . Intensity of ac - 909K normalized to RIG - I was quantified .

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

H. whole cell lysates were prepared for immunoprecipitation analysis of acetylated K909 (on RIG-I wild-type). Samples were analyzed by western blotting with a K909 acetylation-specific antibody. Intensity of ac-909K normalized to RIG-I was quantified.


A . The structural modeling result of RIG - I / dsRNA complex and positions of the lysine residue were presented . RIG - I helicase A and B are shown as green and pale blue ribbons . The dsRNA strands were presented as purple and yellow rods , respectively . Two lysine residues , K907 and K909 , on RIG - I that interact with dsRNA are indicated as violet sticks . K907 forms hydrogen bonding with neither 5 ´ - ppp dsRNA nor K907 . B , C . Acetylated K909 and K907 on RIG - I are indicated as green sticks . There were hydrogen bonding between NH2 of K907 and oxygen of acetyl - K909 .

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

A. The structural modeling result of RIG-I/dsRNA complex and positions of the lysine residue were presented. RIG-I helicase A and B are shown as green and pale blue ribbons. The dsRNA strands were presented as purple and yellow rods, respectively. Two lysine residues, K907 and K909, on RIG-I that interact with dsRNA are indicated as violet sticks. K907 forms hydrogen bonding with neither 5´-ppp dsRNA nor K907.B, C. Acetylated K909 and K907 on RIG-I are indicated as green sticks. There were hydrogen bonding between NH2 of K907 and oxygen of acetyl-K909.


( A ) Intracellular endogenous Aβ42 ( green ) , Ankyrin - G ( AnkG ; magenta ) and GFP ( blue ) in siBin1 - , siCD2AP - and siControl - treated primary cortical neurons ( neurons ) expressing GFP immunolabelled at 9 DIV with anti - Aβ42 ( clone 12F4 ) and anti - AnkG , analysed by spinning - disk confocal microscopy . The white rectangles indicate the dendrites ( Dd ) and axons ( Ax ) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG ( magenta ) and GFP ( blue ) , respectively . Scale bars , 10 µm . ( B ) Aβ42 line profiles in dendrites ( Dd ) of siControl ( grey line ) , siBin1 ( blue line ) and siCD2AP ( red line ) neurons shown in ( c ) . ( C ) Aβ42 line profiles in axons ( Ax ) of siControl ( grey line ) , siBin1 ( blue line ) and siCD2AP ( red line ) neurons shown in ( c ) .

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(A) Intracellular endogenous Aβ42 (green), Ankyrin-G (AnkG; magenta) and GFP (blue) in siBin1- , siCD2AP- and siControl-treated primary cortical neurons (neurons) expressing GFP immunolabelled at 9 DIV with anti-Aβ42 (clone 12F4) and anti-AnkG, analysed by spinning-disk confocal microscopy. The white rectangles indicate the dendrites (Dd) and axons (Ax) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG (magenta) and GFP (blue), respectively. Scale bars, 10 µm.(B) Aβ42 line profiles in dendrites (Dd) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).(C) Aβ42 line profiles in axons (Ax) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).


( D ) Quantification of Aβ42 ( 12F4 ) intensity in cell body ( CB ) , dendrite ( Dd ) and axon ( Ax ) ( n = 5 , NCB = 44 - 53 , NDd = 90 - 120 , NAx = 60 - 74 ; * * * * PCB < 0 . 0001 siBin1 vs . siControl , * * * PCB < 0 . 001 siCD2AP vs . siControl , * * * * PDd < 0 . 0001 siBin1 vs . siControl , * * * * PDd < 0 . 0001 siCD2AP vs . siControl , * * * * PAx < 0 . 0001 siBin1 vs . siControl , t - test , mean ± SEM ) .

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(D) Quantification of Aβ42 (12F4) intensity in cell body (CB), dendrite (Dd) and axon (Ax) (n=5, NCB=44-53, NDd=90-120, NAx=60-74; ****PCB<0.0001 siBin1 vs. siControl, ***PCB<0.001 siCD2AP vs. siControl, ****PDd<0.0001 siBin1 vs. siControl, ****PDd<0.0001 siCD2AP vs. siControl, ****PAx<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).


( E ) Quantification of extracellular endogenous Aβ40 , Aβ42 and of Aβ42 / Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1 , siCD2AP or siControl neurons ( n = 6 ; * PAβ40 = 0 . 0270 siBin1 vs . siControl , * PAβ42 / 40 = 0 . 0378 siBin1 vs . siControl , * PAβ42 / 40 = 0 . 0463 siCD2AP vs . siControl , t - test , mean ± SEM ) .

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

(E) Quantification of extracellular endogenous Aβ40, Aβ42 and of Aβ42/Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1, siCD2AP or siControl neurons (n=6; *PAβ40=0.0270 siBin1 vs. siControl, *PAβ42/40=0.0378 siBin1 vs. siControl, *PAβ42/40=0.0463 siCD2AP vs. siControl, t-test, mean ± SEM).


( F ) Endogenous APP and APP - CTFs levels by western blot with anti - APP antibody ( Y188 ) of siBin1 - , siCD2AP - or siControl - treated neurons at 9 DIV . ( G ) Quantification of APP and APP - CTFs levels normalized to APP ( n = 4 ; * PAPP = 0 . 0355 siBin1 vs . siControl , * * * PAPPCTFs / APP < 0 . 001 siBin1 vs . siControl , * * * * PAPPCTFs / APP < 0 . 0001 siCD2AP vs . siControl , t - test , mean ± SEM ) .

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(F) Endogenous APP and APP-CTFs levels by western blot with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons at 9 DIV.(G) Quantification of APP and APP-CTFs levels normalized to APP (n=4; *PAPP=0.0355 siBin1 vs. siControl, ***PAPPCTFs/APP<0.001 siBin1 vs. siControl, ****PAPPCTFs/APP<0.0001 siCD2AP vs. siControl, t-test, mean ± SEM).


N2a cells treated with siBin1 , siCD2AP or siControl . ( B ) Endocytosed APP detected after a 10 min pulse with 22C11 and APP - RFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( C ) The amount of endocytosed APP fluorescence at 10 min was quantified and normalized to APP - RFP fluorescence ( n = 3 , NsiControl = 56 , NsiBin1 = 70 , NsiCD2AP = 42 ; mean ± SEM ) . ( D ) Non - degraded APP detected after 10 min pulse and 60 min chase with 22C11 and APP - RFP ( insets ) , analysed by epifluorescence microscopy . Cells are outlined in white . Scale bars , 10 µm . ( E ) APP degradation was assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 ( 10 min pulse ) in siControl cells normalized to APP - RFP fluorescence ( n60min = 3 , NsiControl = 68 , NsiBin1 = 43 , NsiCD2AP = 64 ; * * * * PAPP60min < 0 . 0001 , t - test , mean ± SEM ) .

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N2a cells treated with siBin1, siCD2AP or siControl.(B) Endocytosed APP detected after a 10 min pulse with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(C) The amount of endocytosed APP fluorescence at 10 min was quantified and normalized to APP-RFP fluorescence (n=3, NsiControl=56, NsiBin1=70, NsiCD2AP =42; mean ± SEM).(D) Non-degraded APP detected after 10 min pulse and 60 min chase with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Cells are outlined in white. Scale bars, 10 µm.(E) APP degradation was assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 (10 min pulse) in siControl cells normalized to APP-RFP fluorescence (n60min=3, NsiControl=68, NsiBin1=43, NsiCD2AP=64; ****PAPP60min<0.0001, t-test, mean ± SEM).


N2a cells treated with siBin1 , siCD2AP or siControl . ( G ) Endocytosed BACE1 detected upon 5 min pulse with M1 and BACE1 - GFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( H ) The amount of endocytosed BACE1 per cell was quantified as percentage of siControl normalized to BACE1 - GFP ( n = 3 , NsiControl = 86 , NsiBin1 = 72 , NsiCD2AP = 113 ; mean ± SEM ) . ( I ) Recycled BACE1 detected at the plasma membrane of non - permeabilized cells with M1 , upon a 10 min pulse , acid stripping and 20 min chase , and BACE1 - GFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( J ) The amount of recycled BACE1 was quantified as in ( h ) ( n = 3 , NsiControl = 94 , NsiBin1 = 58 , NsiCD2AP = 109 ; * * * * P < 0 . 0001 siBin1 vs . siControl , t - test , mean ± SEM ) . ( K ) Non - recycled BACE1 detected in acid - stripped permeabilized cells pulse - chased as in ( i ) with M1 and BACE1 - GFP ( insets ) , analysed by epifluorescence microscopy . Scale bars , 10 µm . ( L ) The amount of non - recycled BACE1 was quantified as in ( h ) ( n = 3 , NsiControl = 61 , NsiBin1 = 51 ; * * * * P < 0 . 0001 siBin1 vs . siControl , t - test ; n = 2 , NsiCD2AP = 37 ; mean ± SEM ) .

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N2a cells treated with siBin1, siCD2AP or siControl.(G) Endocytosed BACE1 detected upon 5 min pulse with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(H) The amount of endocytosed BACE1 per cell was quantified as percentage of siControl normalized to BACE1-GFP (n=3, NsiControl=86, NsiBin1=72, NsiCD2AP=113; mean ± SEM).(I) Recycled BACE1 detected at the plasma membrane of non-permeabilized cells with M1, upon a 10 min pulse, acid stripping and 20 min chase, and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(J) The amount of recycled BACE1 was quantified as in (h) (n=3, NsiControl=94, NsiBin1=58, NsiCD2AP=109; ****P<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).(K) Non-recycled BACE1 detected in acid-stripped permeabilized cells pulse-chased as in (i) with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 µm.(L) The amount of non-recycled BACE1 was quantified as in (h) (n=3, NsiControl=61, NsiBin1=51; ****P<0.0001 siBin1 vs. siControl, t-test; n=2, NsiCD2AP=37; mean ± SEM).


( A - D ) APP endocytic trafficking followed in neurons expressing APP - RFP treated with siCD2AP or siControl using a pulse - chase assay with 22C11 , analysed by epifluorescence microscopy . ( A ) Endocytosed APP detected with 22C11 ( 10 min pulse ; top panels ) and APP - RFP ( bottom panels ) . Arrowheads identify axons ( Ax ) and dendrites ( Dd ) magnified on the right . Scale bars , 10 µm . ( B ) The amount of endocytosed APP ( 10 min ) in cell body , dendrites and axons normalized to APP - RFP expression in the cell body quantified as percentage of siControl ( n = 3 , NsiControl = 19 , NsiCD2AP = 20 ; mean ± SEM ) . ( C ) Endocytosed APP detected with 22C11 ( 10 min pulse , 60 min chase ; top panels ) and APP - RFP ( bottom panels ) . Arrowheads identify axons ( Ax ) and dendrites ( Dd ) magnified on the right . Scale bars , 10 µm . ( D ) The amount of endocytosed APP ( 60 min chase ) was quantified as in ( b ) ( n = 3 , NsiControl = 23 , NsiCD2AP = 19 ; * * PCB = 0 . 001 siCD2AP vs . siControl , * * * PDd = 0 . 0004 siCD2AP vs . siControl , t - test ; # P = 0 . 0347 Dendrite vs . Cell body , # # # P = 0 . 0008 Dendrite vs . Axon , 1 - way ANOVA with Tukey ' s multiple comparisons test ; mean ± SEM ) .

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(A - D) APP endocytic trafficking followed in neurons expressing APP-RFP treated with siCD2AP or siControl using a pulse-chase assay with 22C11, analysed by epifluorescence microscopy.(A) Endocytosed APP detected with 22C11 (10 min pulse; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(B) The amount of endocytosed APP (10 min) in cell body, dendrites and axons normalized to APP-RFP expression in the cell body quantified as percentage of siControl (n=3, NsiControl=19, NsiCD2AP=20; mean ± SEM).(C) Endocytosed APP detected with 22C11 (10 min pulse, 60 min chase; top panels) and APP-RFP (bottom panels). Arrowheads identify axons (Ax) and dendrites (Dd) magnified on the right. Scale bars, 10 µm.(D) The amount of endocytosed APP (60 min chase) was quantified as in (b) (n=3, NsiControl=23, NsiCD2AP=19; **PCB=0.001 siCD2AP vs. siControl, ***PDd=0.0004 siCD2AP vs. siControl, t-test; #P=0.0347 Dendrite vs. Cell body, ###P=0.0008 Dendrite vs. Axon, 1-way ANOVA with Tukey's multiple comparisons test; mean ± SEM).


( E ) Degradation of surface biotinylated APP ( biotin APP ) ( time 0 ) chased for 20 and 60 min in neurons treated with siCD2AP , siBin1 or siControl . Biotin APP and total APP were detected with anti - APP ( Y188 ) by western blot . ( F ) Quantification of biotinylated APP normalized to levels at time 0 ( n = 3 - 4 , * P60min = 0 . 0198 , t - test ; replicates and mean ) .

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(E) Degradation of surface biotinylated APP (biotin APP) (time 0) chased for 20 and 60 min in neurons treated with siCD2AP, siBin1 or siControl. Biotin APP and total APP were detected with anti-APP (Y188) by western blot.(F) Quantification of biotinylated APP normalized to levels at time 0 (n=3-4, *P60min=0.0198, t-test; replicates and mean).


( G and H ) CD2AP ( green ) and APP ( magenta ) localisation in dendrites ( G ) and axons ( I ) of neurons expressing APP - RFP immunolabelled at 11 DIV with anti - CD2AP , analysed by spinning - disk confocal microscopy . Scale bars , 10 µm . The white squares are magnified on the right . Scale bars , 1 µm . ( I ) Quantification of colocalisation between CD2AP and APP in axons and dendrites ( n = 3 - 4 , NDd = 19 , NAx = 17 ; * * * * P < 0 . 0001 , t - test , mean ± SEM ) .

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(G and H) CD2AP (green) and APP (magenta) localisation in dendrites (G) and axons (I) of neurons expressing APP-RFP immunolabelled at 11 DIV with anti-CD2AP, analysed by spinning-disk confocal microscopy. Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(I) Quantification of colocalisation between CD2AP and APP in axons and dendrites (n=3-4, NDd=19, NAx=17; ****P<0.0001, t-test, mean ± SEM).


( J and K ) CD2AP ( green ) and Rab5 ( magenta ) localisation in dendrites ( J ) and axons ( K ) of neurons expressing Rab5 - GFP analysed as in ( G and H ) . Scale bars , 10 µm . The white squares are magnified on the right . Scale bars , 1 µm . ( L ) Quantification of colocalisation between CD2AP and Rab5 - positive endosomes in dendrites ( Dd ) and axons ( Ax ) ( n = 3 , NDd = 17 , NAx = 23 , * * * * P < 0 . 0001 , t - test , mean ± SEM ) .

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

(J and K) CD2AP (green) and Rab5 (magenta) localisation in dendrites (J) and axons (K) of neurons expressing Rab5-GFP analysed as in (G and H). Scale bars, 10 µm. The white squares are magnified on the right. Scale bars, 1 µm.(L) Quantification of colocalisation between CD2AP and Rab5-positive endosomes in dendrites (Dd) and axons (Ax) (n=3, NDd=17, NAx=23, ****P<0.0001, t-test, mean ± SEM).