B : PK vs . PIPLC treatment to determine prion loads in infected cells in 384 - well plates . Both treatments discriminate between RML prion infected CAD5 and non - infectious brain homogenate ( NBH ) treated CAD5 cells . * * * * = p - value < 0 . 0001 ( Student ' s t - test ) . Shown are mean ± SD , n ≤ 30 individually cultured wells .

O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O B-EXP_ASSAY B-TISSUE O O O O O B-CELL_LINE O O O O O O O O O O O O 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: PK vs. PIPLC treatment to determine prion loads in infected cells in 384-well plates. Both treatments discriminate between RML prion infected CAD5 and non-infectious brain homogenate (NBH) treated CAD5 cells. **** = p-value < 0.0001 (Student's t-test). Shown are mean ± SD, n ≤ 30 individually cultured wells.


C : The infectivity of PIPLC and PK - treated cell lysates was determined by infecting CAD5 cells with lysates as indicated . The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate . PIPLC - treated cells and PK - treated lysates showed similar infectivity titers . Prion - infected and NBH , as well as RML on CAD5ΔPrnp were used for control .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_LINE O O O O O

C: The infectivity of PIPLC and PK-treated cell lysates was determined by infecting CAD5 cells with lysates as indicated. The signal intensity of the highest dilution was measured and compared to untreated RML CAD5 cell lysate. PIPLC-treated cells and PK-treated lysates showed similar infectivity titers. Prion-infected and NBH, as well as RML on CAD5ΔPrnp were used for control.


D : RML - infected CAD5 and GT - 1 / 7 cells were transfected with non - targeting ( NT ) or Prnp targeting siRNAs in a 384 - well plate and subjected to PK or PIPLC treatment . Z ' - factors were calculated for each condition . Shown are mean ± SD , n = 22 individually cultured wells .

O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

D: RML-infected CAD5 and GT-1/7 cells were transfected with non-targeting (NT) or Prnp targeting siRNAs in a 384-well plate and subjected to PK or PIPLC treatment. Z'-factors were calculated for each condition. Shown are mean ± SD, n = 22 individually cultured wells.


A : Western blot showing Hnrnpk siRNA transfection ( 96 hrs . ) decreases Hnrnpk protein levels while increases PrPSc in RML prion infected GT - 1 / 7 . Prnp siRNAs suppressed both PrPC and PrPSc as expected . ⍺ : anti Quantifications are reported as normalized to Actin and in comparison , to NT . PK - western blot is quantified relative to NT .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

A: Western blot showing Hnrnpk siRNA transfection (96 hrs.) decreases Hnrnpk protein levels while increases PrPSc in RML prion infected GT-1/7. Prnp siRNAs suppressed both PrPC and PrPSc as expected. ⍺: anti Quantifications are reported as normalized to Actin and in comparison, to NT. PK-western blot is quantified relative to NT.


B : Western Blot of PSA - treated uninfected and infected GT - 1 / 7 cells . Increasing concentration of PSA leads to a more prominent reduction of PrPSc in mouse cells . PK - western blot is quantified relative to DMSO .

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B: Western Blot of PSA-treated uninfected and infected GT-1/7 cells. Increasing concentration of PSA leads to a more prominent reduction of PrPSc in mouse cells. PK-western blot is quantified relative to DMSO.


C : mRNA levels of Hnrnpk and Prnp following siRNA and PSA treatment . FPKM : Fragments per kilobase of transcript per million mapped reads . Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA levels as well as an increase in Prnp mRNA levels . No difference is seen between DMSO treated and PSA treated cells for either Hnrnpk levels of Prnp levels . n = 2 per treatment group .

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C: mRNA levels of Hnrnpk and Prnp following siRNA and PSA treatment. FPKM: Fragments per kilobase of transcript per million mapped reads. Hnrnpk siRNAs lead to a decrease in Hnrnpk mRNA levels as well as an increase in Prnp mRNA levels. No difference is seen between DMSO treated and PSA treated cells for either Hnrnpk levels of Prnp levels. n = 2 per treatment group.


D : Quantification of PrPSc levels in RML GT - 1 / 7 cells following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

D: Quantification of PrPSc levels in RML GT-1/7 cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


E : Brightfield microscopy images of the effect of siRNA mediated HNRNPK downregulation on prion - induced vacuolation in PG127 hovS cells . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA . ovPRNP siRNA transfected , as well as uninfected cells were used as controls Downregulation of ovPRNP in the infected hovS eliminates the vacuoles ; Right panel : Quantification of vacuoles of NT , HNRNPK and ovPRNP siRNA treated PG127 hovS . Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells . Values represent mean ± SD . * p = 0 . 0113 , * * * * p = < 0 . 0001 ( Dunnett ' s multiple comparisons test ) . Scale bar = 100 µm . n = 3 technical replicates .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O 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-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Brightfield microscopy images of the effect of siRNA mediated HNRNPK downregulation on prion-induced vacuolation in PG127 hovS cells. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT siRNA. ovPRNP siRNA transfected, as well as uninfected cells were used as controls Downregulation of ovPRNP in the infected hovS eliminates the vacuoles; Right panel: Quantification of vacuoles of NT, HNRNPK and ovPRNP siRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. * p = 0.0113, **** p = <0.0001 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


F : Quantification of PrPSc levels in PG127 hovS cells following treatment with 1 µM PSA in comparison to DMSO . Each dot represents an experiment . Shown are mean ± SD .

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F: Quantification of PrPSc levels in PG127 hovS cells following treatment with 1 µM PSA in comparison to DMSO. Each dot represents an experiment. Shown are mean ± SD.


G : Same as in E , using shRNAs instead of siRNA . Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA . Uninfected cells were used as controls Right panel : Quantification of vacuoles of NT and HNRNPK shRNA treated PG127 hovS . Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells . Values represent mean ± SD . * * * p = < 0 . 0009 ( Dunnett ' s multiple comparisons test ) . Scale bar = 100 µm . n = 3 technical replicates .

O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G: Same as in E, using shRNAs instead of siRNA. Downregulation of HNRNPK leads to an enhanced cytopathological vacuolation phenotype when compared to NT shRNA. Uninfected cells were used as controls Right panel: Quantification of vacuoles of NT and HNRNPK shRNA treated PG127 hovS. Cells from pictures at three different positions in the well were manually counted and the amount of vacuolated cells was normalized to the total amount of cells. Values represent mean ± SD. *** p = <0.0009 (Dunnett's multiple comparisons test). Scale bar = 100 µm. n = 3 technical replicates.


H : Quantification of PrPSc levels in PG127 hovS cells following treatment with shRNA against HNRNPK and 1 µM PSA in comparison to DMSO and NT . Each dot represents an experiment . * = p - value < 0 . 027 ( Student ' s t - test ) . Shown are mean ± SD .

O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H: Quantification of PrPSc levels in PG127 hovS cells following treatment with shRNA against HNRNPK and 1 µM PSA in comparison to DMSO and NT. Each dot represents an experiment. *= p-value < 0.027 (Student's t-test). Shown are mean ± SD.


I : Gene set overrepresentation analysis of differentially expressed genes ( log2FC - 0 . 25 ≥ or 0 . 25 ≤ and FDR ≤ 0 . 05 ) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT - 1 / 7 cells analyzed by RNAseq . Differentially regulated genes ( up in siRNA treatment and down in PSA or vice versa ) were overlapped and used for pathway analysis . No significantly enriched pathways are detected for upregulated genes in Hnrnpk and downregulated in PSA treatment . For the opposing direction , an enrichment of genes involved in glucose metabolism was detected .

O O O O O O O O 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-GENEPROD O O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

I: Gene set overrepresentation analysis of differentially expressed genes (log2FC -0.25 ≥ or 0.25 ≤ and FDR ≤ 0.05) for siRNA mediated Hnrnpk downregulation or PSA treatment in RML GT-1/7 cells analyzed by RNAseq. Differentially regulated genes (up in siRNA treatment and down in PSA or vice versa) were overlapped and used for pathway analysis. No significantly enriched pathways are detected for upregulated genes in Hnrnpk and downregulated in PSA treatment. For the opposing direction, an enrichment of genes involved in glucose metabolism was detected.


A : Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO . PSA treatment was started two weeks after infection and continued until lysis . PSA reduced the amount of PrPSc . Right panel : Quantification of the Western Blot ; n = 5 biological replicates . Values : mean ± SD . * p = 0 . 0211 ( Student ' s t - test ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O 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: Western Blot of RML prion infected COCS treated with 1 µM PSA or DMSO. PSA treatment was started two weeks after infection and continued until lysis. PSA reduced the amount of PrPSc. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values: mean ± SD. * p = 0.0211 (Student's t-test).


B : Western Blot analysis of NBH - treated COCS . PSA treatment was identical to the samples of A . PSA did not affect PrPC expression . Right panel : Quantification of the Western Blot ; n = 5 biological replicates . Values represent mean ± SD . n . s . = not significant ( Student ' s t - test ) . ⍺ :

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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: Western Blot analysis of NBH-treated COCS. PSA treatment was identical to the samples of A. PSA did not affect PrPC expression. Right panel: Quantification of the Western Blot; n = 5 biological replicates. Values represent mean ± SD. n.s. = not significant (Student's t-test). ⍺: anti


C : Negative geotaxis climbing assay in prion infected Drosophila . Flies were treated with DMSO , 0 . 5 mM PSA , 0 . 75 mM PSA , or 1 mM PSA at the larval stage and during adulthood for the duration of the assay . Climbing ability was assessed on groups of flies ( n = 3 x 15 ) three times a week and expressed as a performance index . Statistical analysis on the difference between PG127 prion infected versus control prion - free treatment group data in each graph was performed by an unpaired t test : DMSO : p = 0 . 0002 ; PSA : 0 . 5 mM : p = 0 . 0186 ; 0 . 75 mM : p = n . s . ; 1 mM : p = n . s . n . s . = not significant . Shown are mean ±

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C: Negative geotaxis climbing assay in prion infected Drosophila. Flies were treated with DMSO, 0.5 mM PSA, 0.75 mM PSA, or 1 mM PSA at the larval stage and during adulthood for the duration of the assay. Climbing ability was assessed on groups of flies (n=3 x 15) three times a week and expressed as a performance index. Statistical analysis on the difference between PG127 prion infected versus control prion-free treatment group data in each graph was performed by an unpaired t test: DMSO: p = 0.0002; PSA: 0.5 mM: p = 0.0186; 0.75 mM: p = n.s.; 1 mM: p = n.s. n.s. = not significant. Shown are mean ± SD


D : RT - QuIC analysis of whole - head homogenates of prion infected Drosophila . For each sample , 10 male and 10 female heads from the same treatment group were homogenized , 1 : 20 diluted and applied to the RT - QuIC . Shown are the RT - QuIC reactions of three independent homogenates per treatment group , each assessed in quadruplicates . For quantification , the lag - time of each reaction was calculated and plotted in a graph . Shown are mean ± SD . The assays were performed for 100 hours , samples not yielding a positive reaction are considered negative . * * * = p ≤ 0 . 0006 , n . s . = not significant , flies fed with NBH and treated with DMSO were used as a negative control , a standard prion - free and a prion - containing sample were used as assay controls . To control for potential interference of DMSO or PSA with the RT - QuIC reaction , prion positive sample was spiked with 1 µM PSA .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O

D: RT-QuIC analysis of whole-head homogenates of prion infected Drosophila. For each sample, 10 male and 10 female heads from the same treatment group were homogenized, 1:20 diluted and applied to the RT-QuIC. Shown are the RT-QuIC reactions of three independent homogenates per treatment group, each assessed in quadruplicates. For quantification, the lag-time of each reaction was calculated and plotted in a graph. Shown are mean ± SD. The assays were performed for 100 hours, samples not yielding a positive reaction are considered negative. *** = p ≤ 0.0006, n.s. = not significant, flies fed with NBH and treated with DMSO were used as a negative control, a standard prion-free and a prion-containing sample were used as assay controls. To control for potential interference of DMSO or PSA with the RT-QuIC reaction, prion positive sample was spiked with 1 µM PSA.


B Expression levels of GlcNAc in wild - type ( WT ) GAS , ΔgacG , H , I , J , K , and L were measured as the sWGA fluorescence intensity ( normalized to WT GAS ) .

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B Expression levels of GlcNAc in wild-type (WT) GAS, ΔgacG, H, I, J, K, and L were measured as the sWGA fluorescence intensity (normalized to WT GAS).


B , C Accumulation of LC3 - II . ( B ) Representative western blot of LC3 - II during non - infection ( NI ) , WT GAS , ΔgacI , and ΔgacI : : gacI . ( C ) LC3 - II intensity normalized to NI .

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B, C Accumulation of LC3-II. (B) Representative western blot of LC3-II during non-infection (NI), WT GAS, ΔgacI, and ΔgacI::gacI. (C) LC3-II intensity normalized to NI.


D , E Recruitment of ubiquitin in GAS - infected cells . HeLa cells were infected with WT GAS , ΔgacI , and ΔgacI : : gacI for 4 h , fixed , and immunostained for ubiquitin ( FK2 : magenta ) . Cellular and bacterial DNA were stained with DAPI ( cyan ) . ( D ) Representative confocal images and ( E ) percentage of cells with ubiquitin - positive GAS . Scale bar , 10 μm .

O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O B-CELL_LINE O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O

D, E Recruitment of ubiquitin in GAS-infected cells. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (D) Representative confocal images and (E) percentage of cells with ubiquitin-positive GAS. Scale bar, 10 μm.


G , H Localization of ubiquitin in sWGA - treated GAS . HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h , fixed , and immunostained for ubiquitin ( magenta ) . ( G ) Representative confocal images and ( H ) percentages of cell with ubiquitin - positive GAS . Scale bar , 10 μm .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O B-ORGANISM O B-CELL_LINE O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O

G, H Localization of ubiquitin in sWGA-treated GAS. HeLa WT were infected with the indicated GAS strains masked with sWGA for 4 h, fixed, and immunostained for ubiquitin (magenta). (G) Representative confocal images and (H) percentages of cell with ubiquitin-positive GAS. Scale bar, 10 μm.


I , J Recruitment of p62 to gacI mutants . HeLa cells were infected with WT GAS , ΔgacI , and ΔgacI : : gacI for 4 h , fixed , and immunostained for p62 ( magenta ) . Cellular and bacterial DNA were stained with DAPI ( cyan ) . ( I ) Representative confocal images and ( J ) percentage of cells with p62 - positive GAS . Scale bar , 10 μm .

O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O

I, J Recruitment of p62 to gacI mutants. HeLa cells were infected with WT GAS, ΔgacI, and ΔgacI::gacI for 4 h, fixed, and immunostained for p62 (magenta). Cellular and bacterial DNA were stained with DAPI (cyan). (I) Representative confocal images and (J) percentage of cells with p62-positive GAS. Scale bar, 10 μm.


A , B Co - localization of GFP - FBXO2 and mCherry - LC3 in GAS - infected cells . HeLa cells transfected with the indicated EmGFP - FBXO ( green ) and mCherry - LC3 ( magenta ) were infected with WT GAS for 2 h ; scale bar , 10 μm . Representative confocal images ( A ) and ( B ) percentages of FBXO - positive cells .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O

A, B Co-localization of GFP-FBXO2 and mCherry-LC3 in GAS-infected cells. HeLa cells transfected with the indicated EmGFP-FBXO (green) and mCherry-LC3 (magenta) were infected with WT GAS for 2 h; scale bar, 10 μm. Representative confocal images (A) and (B) percentages of FBXO-positive cells.


E , F Localization of EmGFP - FBXO2 in GAS - infected cells . HeLa cells transfected with GFP - FBXO2 ( green ) were infected with the indicated GAS strains for 2 or 4 h at an MOI of 100 ; scale bar , 10 μm . Representative confocal images at 4 h of WT GAS , ΔgacI , and ΔgacI : : gacI ( E ) and ( F ) percentages of FBXO2 ‐ positive cells .

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E, F Localization of EmGFP-FBXO2 in GAS-infected cells. HeLa cells transfected with GFP-FBXO2 (green) were infected with the indicated GAS strains for 2 or 4 h at an MOI of 100; scale bar, 10 μm. Representative confocal images at 4 h of WT GAS, ΔgacI, and ΔgacI::gacI (E) and (F) percentages of FBXO2‐positive cells.


B , HeLa cells transfected the indicated GFP - FBXO2 mutants ( green ) were infected with WT GAS for 4 h ; scale bar , 10 μm . Representative confocal images ( B )

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B, HeLa cells transfected the indicated GFP-FBXO2 mutants (green) were infected with WT GAS for 4 h; scale bar, 10 μm. Representative confocal images (B)


E , F Recruitment of FBXO2 to intracellular GAS in SKP1 / CUL1 / ROC1 knockdown cells . HeLa cells transfected with GFP - FBXO2 ( green ) and the indicated siRNAs were infected with GAS , fixed at 4 h ; scale bar , 10 μm . Representative confocal images ( E ) and percentages of FBXO2 localized to GAS ( F ) .

O O O O O B-GENEPROD O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY O B-ORGANISM O O O O

E, F Recruitment of FBXO2 to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with GFP-FBXO2 (green) and the indicated siRNAs were infected with GAS, fixed at 4 h; scale bar, 10 μm. Representative confocal images (E) and percentages of FBXO2 localized to GAS (F).


D HeLa WT or FBXO2 - KO cells transfected with mCherry - Gal3 ( magenta ) were infected with WT GAS for 2 or 4 h , and percentages of Gal3 - positive GAS - containing cells were quantified .

O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O

D HeLa WT or FBXO2-KO cells transfected with mCherry-Gal3 (magenta) were infected with WT GAS for 2 or 4 h, and percentages of Gal3-positive GAS-containing cells were quantified.


E - J Localization of ubiquitin , LC3 and p62 in FBXO2 KO cells . HeLa WT or FBXO2 - KO cells were infected with WT GAS for 4 h , fixed , and immunostained for ubiquitin ( FK2 : magenta ) or LC3 ( magenta ) or p62 ( magenta ) . Representative confocal images ( E , G , and I ) and percentages of cells with ubiquitin , LC3 , or p62 - positive GAS ( F , H , and J ) .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O

E - J Localization of ubiquitin, LC3 and p62 in FBXO2 KO cells. HeLa WT or FBXO2-KO cells were infected with WT GAS for 4 h, fixed, and immunostained for ubiquitin (FK2: magenta) or LC3 (magenta) or p62 (magenta). Representative confocal images (E, G, and I) and percentages of cells with ubiquitin, LC3, or p62 -positive GAS (F, H, and J).


M , N GlcNAc recognition of FBXO2 required for GAS xenophagy . Non - transfected HeLa WT cells and HeLa FBXO2 - KO cells transfected GFP , GFP - FBXO2 , GFP - FBXO2 YW / AA were infected with GAS WT for 4 h , fixed , and immunostained for ubiquitin . Percentages of cells with LC3 ( M ) or ubiquitin ( N ) - positive GAS were quantified .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-ORGANISM O O O O O B-CELL_LINE O O O B-CELL_LINE B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O

M, N GlcNAc recognition of FBXO2 required for GAS xenophagy. Non-transfected HeLa WT cells and HeLa FBXO2-KO cells transfected GFP, GFP-FBXO2, GFP-FBXO2 YW/AA were infected with GAS WT for 4 h, fixed, and immunostained for ubiquitin. Percentages of cells with LC3 (M) or ubiquitin (N) -positive GAS were quantified.


A - C Recruitment of LC3 and ubiquitin to intracellular GAS in SKP1 / CUL1 / ROC1 knockdown cells . HeLa cells transfected with mCherry - LC3 ( magenta ) and the indicated siRNAs were infected with GAS for 4 h . Cells were immunostained for ubiquitin ( FK2 ) ; scale bar , 10 μm . Representative confocal images ( A ) and percentages of cells with ubiquitin ( B ) and LC3 ( C ) - positive GAS .

O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR B-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O B-ORGANISM O

A-C Recruitment of LC3 and ubiquitin to intracellular GAS in SKP1/CUL1/ROC1 knockdown cells. HeLa cells transfected with mCherry-LC3 (magenta) and the indicated siRNAs were infected with GAS for 4 h. Cells were immunostained for ubiquitin (FK2); scale bar, 10 μm. Representative confocal images (A) and percentages of cells with ubiquitin (B) and LC3 (C) -positive GAS.


D , E Neddylated cullin inhibitor : MLN4924 - treated HeLa cells were infected with WT GAS , and fixed at 4 h . Cells were immunostained for ubiquitin ( FK2 : magenta ) or LC3 ( magenta ) ; scale bar , 10 μm . Representative images and quantification of cells with ubiquitin - positive GAS ( D ) and LC3 - positive GAS ( E ) .

O O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O B-GENEPROD O O B-ORGANISM O O O O

D, E Neddylated cullin inhibitor: MLN4924-treated HeLa cells were infected with WT GAS, and fixed at 4 h. Cells were immunostained for ubiquitin (FK2: magenta) or LC3 (magenta); scale bar, 10 μm. Representative images and quantification of cells with ubiquitin-positive GAS (D) and LC3-positive GAS (E).


F Lysates from HeLa cells infected with GAS were immunoprecipitated with an anti - lipoteichoic acid antibody and Protein G - Magnetic Beads . Immunoprecipitates were analyzed by immunoblotting .

O O O B-CELL_LINE O O O B-ORGANISM O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O

F Lysates from HeLa cells infected with GAS were immunoprecipitated with an anti-lipoteichoic acid antibody and Protein G-Magnetic Beads. Immunoprecipitates were analyzed by immunoblotting.


( A ) BMDMs were treated with E . coli ( MOI = 100 ) for 30 minutes and further cultured for various length of time as indicated . The dynamic of ENT3 expression was determined by measuring Slc29a3 transcripts via RT - qPCR . ( B ) Phagocytosis inhibitor , Cytochalasin D ( CytoD ) , pretreated BMDMs were incubated with E . coli ( MOI = 100 ) and subjected for Slc29a3 expression analysis via RT - qPCR 6 hours post - infection . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDMs were treated with E. coli (MOI=100) for 30 minutes and further cultured for various length of time as indicated. The dynamic of ENT3 expression was determined by measuring Slc29a3 transcripts via RT-qPCR. (B) Phagocytosis inhibitor, Cytochalasin D (CytoD), pretreated BMDMs were incubated with E. coli (MOI=100) and subjected for Slc29a3 expression analysis via RT-qPCR 6 hours post-infection. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( D ) Different bacterial components were applied as stimulants to BMDMs . Slc29a3 expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation . ( E ) BMDMs were treated with different doses of LPS for 30 minutes , harvested 6 hours post - stimulation , and subjected for RT - qPCR analysis . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Different bacterial components were applied as stimulants to BMDMs. Slc29a3 expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation. (E) BMDMs were treated with different doses of LPS for 30 minutes, harvested 6 hours post-stimulation, and subjected for RT-qPCR analysis. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( C ) WT or ( D ) MyD88 - / - BMDMs were stimulated with 10 ng / ml LPS or live E . coli ( MOI = 100 ) in the presence of 10 µM TBK inhibitor , MRT 67307 , and analyzed for Slc29a3 expression . Data information : The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) WT or (D) MyD88-/- BMDMs were stimulated with 10 ng/ml LPS or live E. coli (MOI=100) in the presence of 10 µM TBK inhibitor, MRT 67307, and analyzed for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( E ) The 10 ng / ml LPS - treated BMDMs were examined for Ifnb1 expression after 30 minutes of stimulation and a total of 6 hours incubation . Data information : The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The 10 ng/ml LPS-treated BMDMs were examined for Ifnb1 expression after 30 minutes of stimulation and a total of 6 hours incubation. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( F ) BMDMs were treated with 100 U / ml of IFN - β for various length of time and subjected for Slc29a3 expression . Data information : The results shown are combined from three biological replicates ( n = 3 ) , unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) BMDMs were treated with 100 U/ml of IFN-β for various length of time and subjected for Slc29a3 expression. Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( B - E ) BMDMs from WT , IFNAR1 - / - ( B ) , or STAT1 - / - ( C ) mice were treated with 10 ng / ml LPS , live E . coli ( MOI = 100 ) , or 100 U / ml IFN - β , harvested and examined for Slc29a3 induction . BMDMs from WT , IFNAR1 - / - ( D ) or STAT1 - / - ( E ) mice were treated with 100 U / ml IFN - β , 50 µg / ml poly ( I : C ) , or EMCV ( MOI = 10 ) , harvested and examined for Slc29a3 induction . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown in B to E are from three biological replicates ( n = 3 ) Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) BMDMs from WT, IFNAR1-/- (B), or STAT1-/- (C) mice were treated with 10 ng/ml LPS, live E. coli (MOI=100), or 100 U/ml IFN-β, harvested and examined for Slc29a3 induction. BMDMs from WT, IFNAR1-/- (D) or STAT1-/- (E) mice were treated with 100 U/ml IFN-β, 50 µg/ml poly(I:C), or EMCV (MOI=10), harvested and examined for Slc29a3 induction. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in B to E are from three biological replicates (n=3) Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( F - H ) The expression dynamic of Slc29a3 was profiled in BMDMs treated with two different doses of IFN - α ( F ) , IFN - β ( G ) , IFN - γ ( H ) for various length of time as indicated . Data information : The expression level was calculated relative to Rpl19 and normalized to untreated group as 1 . The results shown in F to H from two biological replicates ( n = 2 ) . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-H) The expression dynamic of Slc29a3 was profiled in BMDMs treated with two different doses of IFN-α (F), IFN-β (G), IFN-γ (H) for various length of time as indicated. Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in F to H from two biological replicates (n=2). Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( A ) The induction fold change ( treated / untreated ) of Slc29a3 and selected ISG transcripts were curated from INTERFEROME database . Shown is data extracted from 250 IU / ml IFN - β treated BMDMs . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O 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) The induction fold change (treated/untreated) of Slc29a3 and selected ISG transcripts were curated from INTERFEROME database. Shown is data extracted from 250 IU/ml IFN-β treated BMDMs. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( D - F ) Primers targeting 3 ' flanking region 2 ( D ) , 3 ' flanking region 1 ( E ) , and promoter region ( F ) were used to quantify the STAT1 binding in BMDMs upon 100 U / ml IFN - β after 4 hour stimulation via qChIP assay . Data information : The results in D - F were generated from two biological replicates ( n = 2 ) with four technical repeats each ( n = 4 ) . One representative example is shown . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-F) Primers targeting 3' flanking region 2 (D), 3' flanking region 1 (E), and promoter region (F) were used to quantify the STAT1 binding in BMDMs upon 100 U/ml IFN-β after 4 hour stimulation via qChIP assay. Data information: The results in D-F were generated from two biological replicates (n=2) with four technical repeats each (n=4). One representative example is shown. Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( A - C ) ( A ) BMDMs were infected with EMCV ( MOI = 10 ) and harvested at indicated time points . ( B ) THP - 1 derived macrophages were challenged with EV71 ( MOI = 5 ) and collected at different time points . ( C ) BMDMs were co - cultured with 50 µg / ml poly ( I : C ) with various length of time and harvested . The mRNA expression of Slc29a3 or SLC29A3 was measured by RT - qPCR . The expression level was calculated relative to Rpl19 or RPLP0 and normalized to untreated group at time 0 as 1 . Results are combined from three biological replicates ( n = 3 ) Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) (A) BMDMs were infected with EMCV (MOI=10) and harvested at indicated time points. (B) THP-1 derived macrophages were challenged with EV71 (MOI=5) and collected at different time points. (C) BMDMs were co-cultured with 50 µg/ml poly (I:C) with various length of time and harvested. The mRNA expression of Slc29a3 or SLC29A3 was measured by RT-qPCR. The expression level was calculated relative to Rpl19 or RPLP0 and normalized to untreated group at time 0 as 1. Results are combined from three biological replicates (n=3) Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( D , E ) ENT3fl / fl and Cx3cr1cre / + ENT3fl / fl mice ( n = 12 ) were challenged with EMCV ( 104 PFU , i . p . ) and monitored for the survival ( D ) and disease severity ( E ) for two weeks . Survival results were analyzed by log - rank ( Mantel - Cox ) test , * p < 0 . 05 . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice (n=12) were challenged with EMCV (104 PFU, i.p.) and monitored for the survival (D) and disease severity (E) for two weeks. Survival results were analyzed by log-rank (Mantel-Cox) test, *p < 0.05. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( F , G ) ENT3fl / fl and Cx3cr1cre / + ENT3fl / fl mice were challenged with 107 PFU EMCV and measured the viral titer in the brain at post - infection day 3 by plaque assay ( n = 6 ) . Shown are representative plaque assay images ( F ) and the quantification ( G ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice were challenged with 107 PFU EMCV and measured the viral titer in the brain at post-infection day 3 by plaque assay (n=6). Shown are representative plaque assay images (F) and the quantification (G). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( A ) WT and ENT3 - / - BMDMs were infected with EMCV ( MOI = 10 ) , and the culture supernatants were harvested 24 hours post - infection to measure the production of IFN - α , IFN - β , and IFN - γ by ELISA . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10), and the culture supernatants were harvested 24 hours post-infection to measure the production of IFN-α, IFN-β, and IFN-γ by ELISA. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.


( D , E ) The mitochondrial respiratory capacity ( D ) and glycolysis capacity ( E ) of WT and ENT3 - / - BMDMs treated with 1000 U / ml IFN - β for 6 hours were measured by extracellular flux analysis . Shown are the representative results of OCR and ECAR kinetics from six biological replicates ( n = 6 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM .

O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) The mitochondrial respiratory capacity (D) and glycolysis capacity (E) of WT and ENT3-/- BMDMs treated with 1000 U/ml IFN-β for 6 hours were measured by extracellular flux analysis. Shown are the representative results of OCR and ECAR kinetics from six biological replicates (n=6). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.


( G , H ) The replication of EMCV in WT ( G ) and ENT3 - / - BMDMs ( H ) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides ( A , U , C , G ) 9 hours post - infection ( MOI = 10 ) . The expression of EMCV - 2A2B was determined by RT - qPCR . Shown are results from three to four biological replicates ( n = 3 - 4 ) . Data information : In G , H ) the EMCV - 2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1 . Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * * p < 0 . 001 . n . s . no significance . The combined data are shown as mean ± SEM .

O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G, H) The replication of EMCV in WT (G) and ENT3-/- BMDMs (H) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 9 hours post-infection (MOI=10). The expression of EMCV-2A2B was determined by RT-qPCR. Shown are results from three to four biological replicates (n=3-4). Data information: In G, H) the EMCV-2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1. Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.


( A ) Illustration of strategy to track the viral uncoating upon infection . ( B - D ) ( B ) WT and ENT3 - / - BMDMs were infected with Syto82 - labeled EMCV ( MOI = 1 ) and subjected to live imagining 30 , 60 and 90 minutes post - infection . The arrowheads indicated the Syto82 + virus puncta in the cell . Scale bars represent 10 µm . ( C ) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82 + cells ( % ) and ( D ) the mean fluorescent intensity ( MFI ) per Syto82 + cell ( D ) . Results were generated from images of three biological replicates ( n = 3 ) , each with 4 fields analyzed . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Illustration of strategy to track the viral uncoating upon infection. (B-D) (B) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and subjected to live imagining 30, 60 and 90 minutes post-infection. The arrowheads indicated the Syto82+ virus puncta in the cell. Scale bars represent 10 µm. (C) Quantification of Syto82 signal in BMDMs after 60 minutes was performed by enumerating Syto82+ cells (%) and (D) the mean fluorescent intensity (MFI) per Syto82+ cell (D). Results were generated from images of three biological replicates (n=3), each with 4 fields analyzed. Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( E ) WT and ENT3 - / - BMDMs were infected with Syto82 - labeled EMCV ( MOI = 1 ) and co - stained with 100 nM Lysotracker Green , subjected to live imagining 60 minutes post - infection . Data were the representative images of two biological replicates ( n = 2 ) . Scale bars represent 5 µm . ( F ) WT and ENT3 - / - BMDMs were infected with EMCV ( MOI = 10 ) for 2 hours and then enriched the endosomes and lysosomes . EMCV particles in endosome - or lysosome - enriched fractions were quantified by plaque assay . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O 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-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-ORGANISM O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and ENT3-/- BMDMs were infected with Syto82-labeled EMCV (MOI=1) and co-stained with 100 nM Lysotracker Green, subjected to live imagining 60 minutes post-infection. Data were the representative images of two biological replicates (n=2). Scale bars represent 5 µm. (F) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10) for 2 hours and then enriched the endosomes and lysosomes. EMCV particles in endosome- or lysosome-enriched fractions were quantified by plaque assay. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


( G - K ) ( G ) Calu - 3 cells were infected with SARS - CoV - 2 original strain or delta variant at MOI = 0 . 1 for 48 hours , harvested , and examined for SLC29A3 induction . ( H ) ENT3 knockdown in Calu - 3 cells was performed by lentiviral shRNA system . The knockdown efficiency was confirmed by RT - qPCR . ENT3 knockdown Calu - 3 cells were infected with SARS - CoV - 2 delta variant and subjected for the viral replication analysis 24 hours post - infection . The virus released out of cells was measured by plaque assay ( I ) , and the viral titer in the cells was measured by RT - qPCR ( J ) . ( K ) The replication of SARS - CoV - 2 delta variant in scramble and ENT3 knockdown Calu - 3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides ( A , U , C , G ) 24 hours post - infection ( MOI = 0 . 1 ) . The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1 . The SARS - CoV - 2 E gene expression level was calculated relative to RPLP0 to normalize the cell number . Shown are combined results from three biological replicates ( n = 3 ) . Data information : Unpaired two - tailed Student ' s t - test was used for statistical analysis , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , n . s . no significance . Data are shown as mean ± SEM .

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-K) (G) Calu-3 cells were infected with SARS-CoV-2 original strain or delta variant at MOI=0.1 for 48 hours, harvested, and examined for SLC29A3 induction. (H) ENT3 knockdown in Calu-3 cells was performed by lentiviral shRNA system. The knockdown efficiency was confirmed by RT-qPCR. ENT3 knockdown Calu-3 cells were infected with SARS-CoV-2 delta variant and subjected for the viral replication analysis 24 hours post-infection. The virus released out of cells was measured by plaque assay (I), and the viral titer in the cells was measured by RT-qPCR (J). (K) The replication of SARS-CoV-2 delta variant in scramble and ENT3 knockdown Calu-3 cells were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 24 hours post-infection (MOI=0.1). The SLC29A3 expression level was calculated relative to RPLP0 and normalized to control as 1. The SARS-CoV-2 E gene expression level was calculated relative to RPLP0 to normalize the cell number. Shown are combined results from three biological replicates (n=3). Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.


B ( Top ) Semiquantitative RT - PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK - 293 cells with a NT siRNA or a siRNA that specifically targets the UPF1LL isoform . ( Bottom ) Venn diagram ( to scale ) of overlapping targets identified from RNA - seq following total UPF1 or UPF1LL - specific knockdown . Depicted are genes that increased in abundance at least 1 . 4 - fold ( FDR < 0 . 05 ) and met read count cutoffs in both datasets . P value indicates enrichment of genes that increased in abundance at least 1 . 4 - fold ( FDR < 0 . 05 ) with UPF1LL - specific knockdown among those regulated by total UPF1 , as determined by Fisher ' s exact test .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O

B (Top) Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with a NT siRNA or a siRNA that specifically targets the UPF1LL isoform. (Bottom) Venn diagram (to scale) of overlapping targets identified from RNA-seq following total UPF1 or UPF1LL-specific knockdown. Depicted are genes that increased in abundance at least 1.4-fold (FDR < 0.05) and met read count cutoffs in both datasets. P value indicates enrichment of genes that increased in abundance at least 1.4-fold (FDR < 0.05) with UPF1LL-specific knockdown among those regulated by total UPF1, as determined by Fisher's exact test.


C Heat map of changes in relative mRNA abundance for genes encoding NMD factors , as determined from RNA - seq following transfection of HEK - 293 cells with a siRNA that targets both UPF1 isoforms ( UPF1total ) or a siRNA that specifically targets the UPF1LL isoform .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O

C Heat map of changes in relative mRNA abundance for genes encoding NMD factors, as determined from RNA-seq following transfection of HEK-293 cells with a siRNA that targets both UPF1 isoforms (UPF1total) or a siRNA that specifically targets the UPF1LL isoform.


D Density plot of changes in relative mRNA abundance as determined by RNA - seq in SMG7ko / SMG6kd cells , relative to a parental cell line treated with control siRNAs Genes were categorized as up - regulated by siUPF1total only , siUPF1LL only , or both siUPF1total and siUPF1LL . Statistical significance was determined by K - W test , with Dunn ' s correction for multiple comparisons .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Density plot of changes in relative mRNA abundance as determined by RNA-seq in SMG7ko/SMG6kd cells, relative to a parental cell line treated with control siRNAs Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


E RT - qPCR analysis of indicated transcripts following transfection of HEK - 293 cells with the indicated siRNAs . Relative fold changes are in reference to NT siRNA . Asterisk ( * ) indicates P < 0 . 05 , as determined by two - way ANOVA . Black dots represent individual data points and error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed line indicates log2 ( fold change ) of + 0 . 5 . PTC + indicates the use of primers specific to transcript isoforms with validated poison

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with the indicated siRNAs. Relative fold changes are in reference to NT siRNA. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed line indicates log2(fold change) of +0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


F Gene ontology analysis of 1621 genes that increased in expression at least 1 . 4 - fold upon UPF1LL - specific knockdown in HEK - 293 cells under normal cellular conditions . Genes may map to multiple categories .

O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O

F Gene ontology analysis of 1621 genes that increased in expression at least 1.4-fold upon UPF1LL-specific knockdown in HEK-293 cells under normal cellular conditions. Genes may map to multiple categories.


B Scatterplot of CLIP - UPF1LL vs CLIP - UPF1SL RIP - seq efficiency . mRNAs were binned according to 3 ' UTR length ( short , medium , or long ) .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

B Scatterplot of CLIP-UPF1LL vs CLIP-UPF1SL RIP-seq efficiency. mRNAs were binned according to 3'UTR length (short, medium, or long).


C Density plot of recovered mRNAs in CLIP - UPF1LL or CLIP - UPF1SL affinity purifications . mRNAs were binned according to 3 ' UTR length . Statistical significance was determined by K - S test .

O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

C Density plot of recovered mRNAs in CLIP-UPF1LL or CLIP-UPF1SL affinity purifications. mRNAs were binned according to 3'UTR length. Statistical significance was determined by K-S test.


D Density plot of recovered mRNAs in CLIP - UPF1LL affinity purifications relative to that of CLIP - UPF1SL . mRNAs were subdivided by PTBP1 and / or hnRNP L motif density within the 3 ' UTR , as indicated by the gradient triangle . Statistical significance was determined by K - W test , with Dunn ' s correction for multiple comparisons .

O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Density plot of recovered mRNAs in CLIP-UPF1LL affinity purifications relative to that of CLIP-UPF1SL. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the 3'UTR, as indicated by the gradient triangle. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


E RT - qPCR analysis of indicated transcripts from CLIP - UPF1 overexpression RNA - seq experiments . Relative fold changes are in reference to the GFP expressing control line . Significance of CLIP - UPF1SL or CLIP - UPF1LL overexpression was compared to the GFP expressing control line . Asterisk ( * ) indicates P < 0 . 05 , as determined by two - way ANOVA . Black dots represent individual data points and error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . For protected mRNAs , the motif density of PTBP1 / hnRNP L within the 3 ' UTR is indicated . PTC + indicates the use of primers specific to transcript isoforms with validated poison

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

E RT-qPCR analysis of indicated transcripts from CLIP-UPF1 overexpression RNA-seq experiments. Relative fold changes are in reference to the GFP expressing control line. Significance of CLIP-UPF1SL or CLIP-UPF1LL overexpression was compared to the GFP expressing control line. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. For protected mRNAs, the motif density of PTBP1/hnRNP L within the 3'UTR is indicated. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


A Semiquantitative RT - PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK - 293 cells with the indicated siRNAs .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O

A Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with the indicated siRNAs.


B RT - qPCR analysis of indicated transcripts following transfection of a NT siRNA or SRSF1 - specific siRNA under conditions of CLIP - UPF1SL or CLIP - UPF1LL overexpression . Relative fold changes are in reference to the GFP expressing control line treated with a NT siRNA . Asterisk ( * ) indicates P < 0 . 05 , as determined by unpaired Student ' s t - test . Black dots represent individual data points and error bars indicate mean ± SD ( n = 3 biological replicates ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RT-qPCR analysis of indicated transcripts following transfection of a NT siRNA or SRSF1-specific siRNA under conditions of CLIP-UPF1SL or CLIP-UPF1LL overexpression. Relative fold changes are in reference to the GFP expressing control line treated with a NT siRNA. Asterisk (*) indicates P < 0.05, as determined by unpaired Student's t-test. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates).


C Density plot of changes in relative mRNA abundance as determined by RNA - seq following SRSF1 overexpression Genes were categorized as up - regulated by siUPF1total only , siUPF1LL only , or both siUPF1total and siUPF1LL . Statistical significance was determined by K - W test , with Dunn ' s correction for multiple comparisons .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Density plot of changes in relative mRNA abundance as determined by RNA-seq following SRSF1 overexpression Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


D Density plot as in ( C ) , with genes binned according to enrichment in the CLIP - UPF1LL or CLIP - UPF1SL affinity purifications .

O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O

D Density plot as in (C), with genes binned according to enrichment in the CLIP-UPF1LL or CLIP-UPF1SL affinity purifications.


B UPF1SLΔCH translocation along an RNA substrate harboring a high - affinity PTBP1 binding site in the absence and presence of PTBP1 . Time to 50 % unwound and relative total % unwound at end - of - assay ( 1200 sec ) are indicated . Results of four technical replicates are shown for each dataset and represent at least three independent experiments . Shaded region indicates SD . C Results as in ( B ) but with UPF1LLΔCH .

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B UPF1SLΔCH translocation along an RNA substrate harboring a high-affinity PTBP1 binding site in the absence and presence of PTBP1. Time to 50% unwound and relative total % unwound at end-of-assay (1200 sec) are indicated. Results of four technical replicates are shown for each dataset and represent at least three independent experiments. Shaded region indicates SD. C Results as in (B) but with UPF1LLΔCH.


B Density plots of changes in relative mRNA abundance as determined by RNA - seq following treatment of HEK - 293 cells with 1 µM thapsigargin for 6hr ( left ) or 9hr ( right ) . Genes were categorized as up - regulated by siUPF1total only or siUPF1LL only under basal conditions . Statistical significance was determined by K - W test , with Dunn ' s correction for multiple comparisons .

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B Density plots of changes in relative mRNA abundance as determined by RNA-seq following treatment of HEK-293 cells with 1 µM thapsigargin for 6hr (left) or 9hr (right). Genes were categorized as up-regulated by siUPF1total only or siUPF1LL only under basal conditions. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.


D RT - qPCR analysis of indicated transcripts following transfection of HEK - 293 cells with indicated siRNAs and treatment with 1 µM thapsigargin for 6hr . Relative fold changes are in reference to vehicle - treated , NT siRNA . Black dots represent individual data points and error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . PTC + indicates the use of primers specific to transcript isoforms with validated poison

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D RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 1 µM thapsigargin for 6hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


C RT - qPCR analysis of indicated transcripts following transfection of HEK - 293 cells with indicated siRNAs and treatment with 50 µg / mL puromycin for 4hr . Relative fold changes are in reference to vehicle - treated , NT siRNA . Black dots represent individual data points and error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . PTC + indicates the use of primers specific to transcript isoforms with validated poison

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C RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


A mRNA decay measurement using Roadblock - qPCR RNA was isolated from HEK - 293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg / mL puromycin . mRNA half - lives were estimated by fitting the data to a single - phase exponential decay model . Puromycin treatment was compared to the vehicle control and siUPF1LL was compared to the siNT in the absence and presence of puromycin treatment using the extra - sum - of - squares F test . Error bars indicate mean ± SD ( n = 4 biological replicates ) .

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A mRNA decay measurement using Roadblock-qPCR RNA was isolated from HEK-293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg/mL puromycin. mRNA half-lives were estimated by fitting the data to a single-phase exponential decay model. Puromycin treatment was compared to the vehicle control and siUPF1LL was compared to the siNT in the absence and presence of puromycin treatment using the extra-sum-of-squares F test. Error bars indicate mean±SD (n = 4 biological replicates).


B RT - qPCR analysis of indicated transcripts following transfection of HEK - 293 cells with indicated siRNAs and treatment with 50 µg / mL puromycin for 4hr . Relative fold changes are in reference to vehicle - treated , NT siRNA . Black dots represent individual data points and error bars indicate mean ± SD ( n = 3 biological replicates ) . Dashed lines indicate log2 ( fold change ) of ± 0 . 5 . PTC + indicates the use of primers specific to transcript isoforms with validated poison

B RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons


C Density plot of changes in relative mRNA abundance as determined from RNA - seq following treatment of HEK - 293 cells with 50 µg / mL of puromycin for 4hr . mRNAs were subdivided by PTBP1 and / or hnRNP L motif density within the first 400 nt of 3 ' UTR . Statistical significance was determined by K - W test , with Dunn ' s correction for multiple comparisons . D Density plot as in ( C ) , following UPF1LL - specific knockdown .

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C Density plot of changes in relative mRNA abundance as determined from RNA-seq following treatment of HEK-293 cells with 50 µg/mL of puromycin for 4hr. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the first 400 nt of 3'UTR. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons. D Density plot as in (C), following UPF1LL-specific knockdown.


A - E Echocardiographic analysis of ( A ) posterior wall thickness , ( B ) septum thickness , ( C ) left ventricle end diastolic diameter , ( D ) fractional area change and ( E ) ejection fraction . n = 8 per genotype .

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A-E Echocardiographic analysis of (A) posterior wall thickness, (B) septum thickness, (C) left ventricle end diastolic diameter, (D) fractional area change and (E) ejection fraction. n= 8 per genotype.


F Comparison of TAZ gene expression , normalized to S12 , in heart , liver , and kidney , analyzed in triplicates by quantitative PCR . n = 5 per genotype . G TAZ gene expression in percent . Wild - type is set to 100 % .

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F Comparison of TAZ gene expression, normalized to S12, in heart, liver, and kidney, analyzed in triplicates by quantitative PCR. n= 5 per genotype.G TAZ gene expression in percent. Wild-type is set to 100%.


H , I Lipid species profiles of cardiolipin ( H ) and monolysocardiolipin ( I ) in isolated cardiac mitochondria analyzed by mass spectrometry . n = 4 per genotype .

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H, I Lipid species profiles of cardiolipin (H) and monolysocardiolipin (I) in isolated cardiac mitochondria analyzed by mass spectrometry. n= 4 per genotype.


J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues .

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J Comparison of ratios of total monolysocardiolipin and cardiolipin content in indicated tissues.


A Periodic measurement of the oxygen consumption rate ( OCR ) of mitochondria , isolated from 5 mice per genotype , before and after the administration of the indicated compounds ( left ) . Quantification of OCR in independent experiments , n = 8 per genotype ( right ) .

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A Periodic measurement of the oxygen consumption rate (OCR) of mitochondria, isolated from 5 mice per genotype, before and after the administration of the indicated compounds (left). Quantification of OCR in independent experiments, n= 8 per genotype (right).


B OCR rates of cardiac mitochondria after administration of pyruvate , malate and ADP , n = 7 per genotype .

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B OCR rates of cardiac mitochondria after administration of pyruvate, malate and ADP, n= 7 per genotype.


A Mitochondrial membranes , isolated from cardiac tissue , were solubilized in 1 % digitonin and analyzed by BN - PAGE and Western - blotting with the indicated antibodies .

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A Mitochondrial membranes, isolated from cardiac tissue, were solubilized in 1% digitonin and analyzed by BN-PAGE and Western- blotting with the indicated antibodies.


B Cardiac mitochondria were prepared as in ( A ) and analyzed on a large pore BN - gel for better resolution of supercomplexes .

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B Cardiac mitochondria were prepared as in (A) and analyzed on a large pore BN-gel for better resolution of supercomplexes.


C Cardiac mitochondria were solubilized in 0 . 4 % DDM and complexes were separated by BN - PAGE and analyzed by Western - blotting with the indicated antibodies .

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C Cardiac mitochondria were solubilized in 0.4% DDM and complexes were separated by BN-PAGE and analyzed by Western- blotting with the indicated antibodies.


D Mitochondria were isolated from kidney and liver and analyzed as in ( A ) .

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D Mitochondria were isolated from kidney and liver and analyzed as in (A).


E Quantification of ROS production over time in cardiac mitochondria using the ROS - specific fluorescence - based sensor H2DCFDA . One representative measurement is shown . Analyses were performed in triplicates and confirmed for three animals per genotype . Significance was calculated using Student ' s T - TEST . sh , shTAZ .

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E Quantification of ROS production over time in cardiac mitochondria using the ROS-specific fluorescence-based sensor H2DCFDA. One representative measurement is shown. Analyses were performed in triplicates and confirmed for three animals per genotype. Significance was calculated using Student's T-TEST. sh, shTAZ.


A Enzymatic activity of ubiquinone - cytochrome c oxidase in mitochondria isolated from cardiac tissue . The ubiquinone analogue decylubiquinone was used in this assay . Data were standardized to malate - dehydrogenase activity and presented as percent of wild - type , n = 4 per genotype .

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A Enzymatic activity of ubiquinone-cytochrome c oxidase in mitochondria isolated from cardiac tissue. The ubiquinone analogue decylubiquinone was used in this assay. Data were standardized to malate-dehydrogenase activity and presented as percent of wild-type, n= 4 per genotype.


B Cytochrome c reductase activity , standardized to malate - dehydrogenase activity in cardiac mitochondria , were plotted as % of wild - type , n = 3 per genotype .

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B Cytochrome c reductase activity, standardized to malate-dehydrogenase activity in cardiac mitochondria, were plotted as % of wild-type, n= 3 per genotype.


C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria , separated on SDS gel and visualized by Western - blotting .

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C Protein levels of Cox1 and Rieske compared to VDAC3 in three different dilutions of isolated mitochondria, separated on SDS gel and visualized by Western-blotting.


D Respiratory chain complexes in cardiac mitochondria were separated on BN - PAGE and stained for activity of complex I , II , IV and F1FO - ATPase . sh , shTAZ ; Q , ubiquinone .

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D Respiratory chain complexes in cardiac mitochondria were separated on BN-PAGE and stained for activity of complex I, II, IV and F1FO- ATPase. sh, shTAZ; Q, ubiquinone.


A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype .

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A Enzymatic activity of the succinate dehydrogenase in isolated cardiac mitochondria from 3 animals per genotype.


B Mitochondrial membranes were solubilized in 1 % digitonin and analyzed by BN - PAGE and Western - blotting using antibodies against SDH components SDHA and SDHB ( upper panel ) . For a loading control , samples were separated on SDS gel and analyzed by Western - blotting ( lower panel ) .

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B Mitochondrial membranes were solubilized in 1% digitonin and analyzed by BN-PAGE and Western-blotting using antibodies against SDH components SDHA and SDHB (upper panel). For a loading control, samples were separated on SDS gel and analyzed by Western-blotting (lower panel).


C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western - blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm . Lower panel : quantification of signal ratio SDHA / VDAC3 in shTAZ and control ( set to 100 % ) .

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C Steady state protein levels analyzed by SDS gel separation of indicated amounts of mitochondrial protein and subsequent Western- blotting with antibodies against SDHA and VDAC3 or visualizing of Flavin fluorescence after excitation with 488 nm. Lower panel: quantification of signal ratio SDHA/VDAC3 in shTAZ and control (set to 100%).


D Analysis of gene expression in cardiac tissue analyzed in ( C ) by qPCR using primers against indicated mRNA . Data of one representative measurement , normalized to b - actin with WT set to 100 % are shown . Identical results were obtained in further experiments with two different pairs of animals .

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D Analysis of gene expression in cardiac tissue analyzed in (C) by qPCR using primers against indicated mRNA. Data of one representative measurement, normalized to b-actin with WT set to 100% are shown. Identical results were obtained in further experiments with two different pairs of animals.


E Upper panel : SDH complex of cardiac , liver and kidney were analyzed by BN - PAGE as in ( B ) ; lower panel : the same samples were analyzed by SDS gel and Western - blotting .

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E Upper panel: SDH complex of cardiac, liver and kidney were analyzed by BN-PAGE as in (B); lower panel: the same samples were analyzed by SDS gel and Western-blotting.


F , G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney ( F ) and liver ( G ) . Values of 3 animals per genotype are plotted as percent of wild - type . sh , shTAZ .

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F, G Analysis of enzymatic activity of the succinate dehydrogenase related to the malate dehydrogenase activity in mitochondria isolated from kidney (F) and liver (G). Values of 3 animals per genotype are plotted as percent of wild-type. sh, shTAZ.


A Flow cytometry analysis of cardiac troponinT ( cTNT ) expression was performed at day 60 post cardiac differentiation . 98 % of cTNT + cardiomyocytes ( CM ) were obtained after metabolic selection .

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A Flow cytometry analysis of cardiac troponinT (cTNT) expression was performed at day 60 post cardiac differentiation. 98% of cTNT+ cardiomyocytes (CM) were obtained after metabolic selection.


B RT - PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC - CMs of BTHS patient TAZ10 and a healthy control .

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B RT-PCR showing the expression of indicated genes in undifferentiated iPSCs and differentiated iPSC-CMs of BTHS patient TAZ10 and a healthy control.


C Immunostaining of control ( left ) and BTHS iPSC - CMs at day 60 post differentiation for cardiac troponin T ( cTNT ) and Connexin 43 ( Cx43 ) . Scale bars : 50µm .

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C Immunostaining of control (left) and BTHS iPSC-CMs at day 60 post differentiation for cardiac troponin T (cTNT) and Connexin 43 (Cx43). Scale bars: 50µm.


D Quantitation of sarcomere organization . Left panel : Immunostaining of cTNT , MLC2a ( myosin light chain ) and a - actinin at day 60 post cardiac differentiation . Right panel : The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments . Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10 . Significance was analyzed using Student ' s T - TEST * * P = 0 . 002 and * * * P = 0 . 0006 . Scale bars : 50 µm .

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D Quantitation of sarcomere organization. Left panel: Immunostaining of cTNT, MLC2a (myosin light chain) and a-actinin at day 60 post cardiac differentiation. Right panel: The bar graphs at the right panel represent the sarcomere regularity from 3 independent experiments. Error bars represent standard deviation for the comparison between control and BTHS patient TAZ10. Significance was analyzed using Student's T-TEST **P = 0.002 and ***P = 0.0006. Scale bars: 50 µm.


E Measurement of cell surface areas . Cell surface area was measured in single cardiomyocytes ( n = at least 30 measurements each group from N = 3 independent experiments each group ) at day 60 post cardiac differentiation after immunostaining of sarcomeric a - actinin . Scale bars : 50 µm .

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E Measurement of cell surface areas. Cell surface area was measured in single cardiomyocytes (n = at least 30 measurements each group from N = 3 independent experiments each group) at day 60 post cardiac differentiation after immunostaining of sarcomeric a-actinin. Scale bars: 50 µm.


A Oxygen consumption rate ( OCR ) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds .

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A Oxygen consumption rate (OCR) of cardiomyocytes from patient TAZ10 and control at basal conditions and after the administration of the indicated compounds.


B Mitochondrial membranes analyzed by BN - PAGE and Western - blotting using indicated antibodies . For a loading control samples were separated on SDS gel and analyzed by Western - blotting ( lower panel ) .

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B Mitochondrial membranes analyzed by BN-PAGE and Western-blotting using indicated antibodies. For a loading control samples were separated on SDS gel and analyzed by Western-blotting (lower panel).


C Western blot analysis of SDHA protein levels compared to VDAC3 . Quantification of SDHA relative to VDAC3 in percent with control set to 100 % . Error bars indicate range of values of two independent experiments .

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C Western blot analysis of SDHA protein levels compared to VDAC3. Quantification of SDHA relative to VDAC3 in percent with control set to 100%. Error bars indicate range of values of two independent experiments.


D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes , analyzed in ( C ) .

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D Analysis of SDHA gene expression in triplicates by qPCR in cardiomyocytes, analyzed in (C).


[ 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 B-TISSUE O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O 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.