Immunofluorescence showing the Ago3 signals ( green ) in Siwi - depleted BmN4 cells ( Siwi KD ) and Siwi KD cells where Flag - Siwi ( red ) was expressed . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . The panels on the right - hand side show high magnification images of the part indicated by the white dashed line box in the images on the left - hand side . This arrangement applies also for panel C . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) . A significant difference was found plus / minus Flag - Siwi expression by the Fisher ' s exact test ( P < 0 . 05 ) .

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

Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. The panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This arrangement applies also for panel C. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).


Western blots show that the degree of Ago3 phosphorylation was reduced upon Siwi KA mutant as well as Siwi WT and DDH mutant expression .

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

Western blots show that the degree of Ago3 phosphorylation was reduced upon Siwi KA mutant as well as Siwi WT and DDH mutant expression.


Immunofluorescence showing the Ago3 signals ( green ) in Siwi - depleted BmN4 cells ( Siwi KD ) and Siwi KD cells where Flag - Siwi KA mutant ( red ) was expressed . DAPI shows the nuclei ( blue ) . Scale bar : 10 μm . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) . A significant difference was found plus / minus Flag - Siwi expression by the Fisher ' s exact test ( P < 0 . 05 ) .

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

Immunofluorescence showing the Ago3 signals (green) in Siwi-depleted BmN4 cells (Siwi KD) and Siwi KD cells where Flag-Siwi KA mutant (red) was expressed. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105). A significant difference was found plus/minus Flag-Siwi expression by the Fisher's exact test (P < 0.05).


( C , D ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( green ) . Right panels show top view images across the centriolar length confirming the distal localization of SFI1 at centrioles . The white arrowhead indicates SFI1 distal dot at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of SFI1 alongside the centriole is shown in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and SFI1 (green). Right panels show top view images across the centriolar length confirming the distal localization of SFI1 at centrioles. The white arrowhead indicates SFI1 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of SFI1 alongside the centriole is shown in (D).


( E , F ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) and Centrin 2 / 3 ( Cetn2 / 3 , grey ) . Right panels show top view images across the centriolar length confirming the distal localization of Centrin2 / 3 at centrioles . The white arrowhead indicates Centrin2 / 3 distal dot at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of Centrin2 / 3 alongside the centriole is shown in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and Centrin 2/3 (Cetn2/3, grey). Right panels show top view images across the centriolar length confirming the distal localization of Centrin2/3 at centrioles. The white arrowhead indicates Centrin2/3 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of Centrin2/3 alongside the centriole is shown in (F).


( G , H ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) and Centrin 3 ( Cetn3 , cyan ) . Right panels show top view images across the centriolar length confirming the distal localization of Centrin 3 at centrioles . The white arrowhead indicates Centrin 3 distal dot at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of Centrin 3 alongside the centriole is shown in ( H ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta) and Centrin 3 (Cetn3, cyan). Right panels show top view images across the centriolar length confirming the distal localization of Centrin 3 at centrioles. The white arrowhead indicates Centrin 3 distal dot at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of Centrin 3 alongside the centriole is shown in (H).


( I , J ) Representative confocal images of expanded centrioles from RPE - 1 cells stained for α / β - tubulin ( αβTub , magenta ) , SFI1 ( green ) and Centrin 2 / 3 ( Cetn3 , grey ) . Right panels show top view images confirming the distal localization of SFI1 and Centrin 2 / 3 at centrioles . Scale bars : 200 nm and 100 nm ( right panels ) . Average position of SFI1 and Centrin2 / 3 alongside the centriole is shown in ( J ) .

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

(I, J) Representative confocal images of expanded centrioles from RPE-1 cells stained for α/β-tubulin (αβTub, magenta), SFI1 (green) and Centrin 2/3 (Cetn3, grey). Right panels show top view images confirming the distal localization of SFI1 and Centrin 2/3 at centrioles. Scale bars: 200 nm and 100 nm (right panels). Average position of SFI1 and Centrin2/3 alongside the centriole is shown in (J).


( A - F ) Representative confocal images of expanded U2OS centrioles treated with siCT ( A , D ) , siSFI1 ( B , E ) and siPOC5 ( C , F ) stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( A - C , green ) or Centrin2 / 3 ( D - F , grey ) . Insets show top views of expanded centrioles at different positions along the centriole ( P = proximal , C = central and D = distal ) . Note that in the absence of SFI1 and Centrin staining at the distal tip , orientation of the centriole was decided based on the larger diameter of the proximal region compared to the distal one , as previously observed in cryo - tomography ( Greenan et al , 2020 ) . White arrowheads points to the distal dot of SFI1 and Centrin that disappear in SFI1 - depleted ( yellow arrowheads ) but not in POC5 - depleted centrioles . Scale bars : 200 nm and 100 nm ( inset ) . Longitudinal and radial localization of SFI1 and Centrin2 / 3 in siCT ( A , D ) , siSFI1 ( B , E ) and siPOC5 ( C , F ) are presented below the corresponding image . N = 25 , 40 and 60 centrioles from 3 independent experiments . N = 29 , 34 , 34 centrioles from 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative confocal images of expanded U2OS centrioles treated with siCT (A, D), siSFI1 (B, E) and siPOC5 (C, F) stained for α/β-tubulin (αβTub, magenta) and SFI1 (A-C, green) or Centrin2/3 (D-F, grey). Insets show top views of expanded centrioles at different positions along the centriole (P= proximal, C= central and D= distal). Note that in the absence of SFI1 and Centrin staining at the distal tip, orientation of the centriole was decided based on the larger diameter of the proximal region compared to the distal one, as previously observed in cryo-tomography (Greenan et al, 2020). White arrowheads points to the distal dot of SFI1 and Centrin that disappear in SFI1-depleted (yellow arrowheads) but not in POC5-depleted centrioles. Scale bars: 200 nm and 100 nm (inset). Longitudinal and radial localization of SFI1 and Centrin2/3 in siCT (A, D), siSFI1 (B, E) and siPOC5 (C, F) are presented below the corresponding image. N=25, 40 and 60 centrioles from 3 independent experiments. N=29, 34, 34 centrioles from 3 independent experiments.


( K - N ) Representative images of expanded U2OS cells expressing GFP alone or GFP + SFI1 - RR and treated with siCT or siSFI1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( K , green ) or Centrin2 / 3 ( M , grey ) . Yellow asterisk indicates the centriole lacking SFI1 and Centrin distal dots . Scale bar : 250 nm . Expression of GFP + SFI1 - RR in siSFI1 treated cell rescues the presence of SFI1 and Centrin at the distal tip of the centriole .

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

(K-N) Representative images of expanded U2OS cells expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and SFI1 (K, green) or Centrin2/3 (M, grey). Yellow asterisk indicates the centriole lacking SFI1 and Centrin distal dots. Scale bar: 250 nm. Expression of GFP+SFI1-RR in siSFI1 treated cell rescues the presence of SFI1 and Centrin at the distal tip of the centriole.


( O , P ) Representative confocal images of expanded Cetn2 WT , Cetn KO or Cetn2 rescue RPE - 1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and Centrin2 ( O , grey ) or SFI1 ( P , green ) . Arrowheads indicate the presence ( white ) or the absence ( yellow ) of Centrin2 and SFI1 . Scale bar : 200nm . Quantification shows the total absence of Centrin2 in the KO cells correlating with the absence of SFI1 . Both Centrin2 and SFI1 localization are rescued when Centrin2 is re - expressed in KO cells .

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

(O, P) Representative confocal images of expanded Cetn2 WT, Cetn KO or Cetn2 rescue RPE-1. Cells were stained for α/β-tubulin (αβTub, magenta) and Centrin2 (O, grey) or SFI1 (P, green). Arrowheads indicate the presence (white) or the absence (yellow) of Centrin2 and SFI1. Scale bar: 200nm. Quantification shows the total absence of Centrin2 in the KO cells correlating with the absence of SFI1. Both Centrin2 and SFI1 localization are rescued when Centrin2 is re-expressed in KO cells.


( A ) Representative confocal images of expanded duplicating centrioles from siCT - and siSFI1 - treated U2OS cells . Cells were stained for SFI1 ( green ) and α / β - tubulin ( αβTub , magenta ) . Inset shows a distal position of the mother centriole where SFI1 signal is visible . White arrowhead indicates the position of SFI1 distal dot in the procentriole of control cell , which is lost in SFI1 - depleted cells ( yellow arrowhead ) . Scale bars : 200 nm . ( B ) Quantification of the percentage of SFI1 - negative procentrioles . ( C ) Quantification of the percentage of duplicating centrioles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of expanded duplicating centrioles from siCT- and siSFI1- treated U2OS cells. Cells were stained for SFI1 (green) and α/β-tubulin (αβTub, magenta). Inset shows a distal position of the mother centriole where SFI1 signal is visible. White arrowhead indicates the position of SFI1 distal dot in the procentriole of control cell, which is lost in SFI1-depleted cells (yellow arrowhead). Scale bars: 200 nm. (B) Quantification of the percentage of SFI1-negative procentrioles. (C) Quantification of the percentage of duplicating centrioles.


( D ) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells . Cells were stained for HsSAS - 6 ( yellow ) and α / β - tubulin ( αβTub , magenta ) . Quantification shows no difference in the percentage of HsSAS - 6 - positive centrioles in siSFI1 - depleted cells compared to control cells . ( E ) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells . Cells were stained for STIL ( yellow ) and α / β - tubulin ( αβTub , magenta ) . Quantification shows no difference in the percentage of STIL - positive centrioles in siSFI1 compared to control cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells. Cells were stained for HsSAS-6 (yellow) and α/β-tubulin (αβTub, magenta). Quantification shows no difference in the percentage of HsSAS-6-positive centrioles in siSFI1-depleted cells compared to control cells. (E) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells. Cells were stained for STIL (yellow) and α/β-tubulin (αβTub, magenta). Quantification shows no difference in the percentage of STIL-positive centrioles in siSFI1 compared to control cells.


( F ) Top views of expanded U2OS centrioles treated with siCT or siSFI1 stained for α / β - tubulin ( αβTub , magenta ) . Scale bars : 200 nm . ( G ) Roundness index of centrioles from siCT and siSFI1 - treated cells . ( H ) Length ( circle ) and diameter ( square ) of expanded centrioles in siCT or siSFI1 - treated cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top views of expanded U2OS centrioles treated with siCT or siSFI1 stained for α/β-tubulin (αβTub, magenta). Scale bars: 200 nm. (G) Roundness index of centrioles from siCT and siSFI1-treated cells. (H) Length (circle) and diameter (square) of expanded centrioles in siCT or siSFI1-treated cells.


( I ) Representative confocal images of expanded U2OS centrioles from siCT and siSFI1 - treated cells stained for α / β - tubulin ( αβTub , magenta ) . White stars point to broken microtubule wall . Scale bars : 200 nm . ( J ) Percentage of abnormal centrioles in the indicated conditions .

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

(I) Representative confocal images of expanded U2OS centrioles from siCT and siSFI1-treated cells stained for α/β-tubulin (αβTub, magenta). White stars point to broken microtubule wall. Scale bars: 200 nm. (J) Percentage of abnormal centrioles in the indicated conditions.


( K ) Top views of expanded procentrioles from U2OS cells treated with siCT or siSFI1 and stained for α / β - tubulin ( αβTub , magenta ) . ( L ) Roundness index of procentrioles from siCT and siSFI1 - treated cells .

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

(K) Top views of expanded procentrioles from U2OS cells treated with siCT or siSFI1 and stained for α/β-tubulin (αβTub, magenta). (L) Roundness index of procentrioles from siCT and siSFI1-treated cells.


( M ) Representative confocal images of expanded U2OS centrioles from siCT - and siSFI1 - treated cells stained for α / β - tubulin ( αβTub , magenta ) and Centrin ( Cetn2 / 3 , grey ) . White dashed lines delimitate the proximal , central and distal regions . White star points to the broken microtubule wall . ( N ) Centrin coverage ( % of the total tubulin length ) along the centriole in the indicated conditions .

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

(M) Representative confocal images of expanded U2OS centrioles from siCT- and siSFI1-treated cells stained for α/β-tubulin (αβTub, magenta) and Centrin (Cetn2/3, grey). White dashed lines delimitate the proximal, central and distal regions. White star points to the broken microtubule wall. (N) Centrin coverage (% of the total tubulin length) along the centriole in the indicated conditions.


( O ) Representative images of expanded U2OS expressing GFP alone or GFP + SFI1 - RR and treated with siCT or siSFI1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and SFI1 ( green ) or Centrin2 / 3 ( grey ) . Red arrowhead indicates abnormal centriole . ( P ) Percentage of abnormal centrioles in the indicated conditions .

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

(O) Representative images of expanded U2OS expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and SFI1 (green) or Centrin2/3 (grey). Red arrowhead indicates abnormal centriole. (P) Percentage of abnormal centrioles in the indicated conditions.


( F ) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and SFI1 ( green ) . Yellow asterisks indicate the absence of SFI1 at the distal tip of the centrioles . Red arrowhead indicates abnormal centriole . Scale bar : 250 nm . ( G ) Percentage of RPE - 1 cells with centrioles SFI1 - positive ( intact SFI1 ) , partially depleted ( partial SFI1 ) or totally missing SFI1 ( no SFI1 ) at the distal dot in siSFI - treated cells . ( H ) Percentage of abnormal centrioles in RPE - 1 cells treated with siSFI1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and SFI1 (green). Yellow asterisks indicate the absence of SFI1 at the distal tip of the centrioles. Red arrowhead indicates abnormal centriole. Scale bar: 250 nm. (G) Percentage of RPE-1 cells with centrioles SFI1-positive (intact SFI1), partially depleted (partial SFI1) or totally missing SFI1 (no SFI1) at the distal dot in siSFI-treated cells. (H) Percentage of abnormal centrioles in RPE-1 cells treated with siSFI1.


( I ) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and CP110 ( yellow ) . ( J ) Percentage of ciliated cells observed in U - ExM under the indicated conditions . ( K ) Percentage of CP110 capped / uncapped centrioles under the indicated conditions .

(I) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CP110 (yellow). (J) Percentage of ciliated cells observed in U-ExM under the indicated conditions. (K) Percentage of CP110 capped/uncapped centrioles under the indicated conditions.


( L ) Representative widefield images of expanded RPE - 1 during ciliogenesis , expressing GFP alone or GFP + SFI1 - RR and treated with siCT or siSFI1 . Cells were stained for α / β - tubulin ( αβTub , magenta ) and CP110 ( yellow ) . Arrowheads indicate a cilium . ( M ) Percentage of ciliated cells observed in U - ExM in the indicated conditions . ( N ) Percentage of CP110 capped / uncapped centrioles in the indicated conditions .

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

(L) Representative widefield images of expanded RPE-1 during ciliogenesis, expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and CP110 (yellow). Arrowheads indicate a cilium. (M) Percentage of ciliated cells observed in U-ExM in the indicated conditions. (N) Percentage of CP110 capped/uncapped centrioles in the indicated conditions.


( A - D ) Representative widefield images of expanded centrioles during ciliogenesis from siCT ( A , B ) and siSFI1 ( C , D ) RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and CEP164 ( red hot ) . Note the abnormal organization of the distal appendage protein CEP164 in siSFI1 - treated cells . ( E - H ) Representative widefield images of expanded centrioles during ciliogenesis from siCT ( E , F ) and siSFI1 ( G , H ) RPE - 1 treated cells . Cells were stained for α / β - tubulin ( magenta ) and CEP90 ( cyan ) . ( I ) Frequency distribution of the number of dots per centriole formed by CEP164 in indicated conditions . ( J ) Frequency distribution of the number of dots per centriole formed by CEP90 under the indicated conditions . ( K ) Percentage of cells presenting abnormal CEP164 and CEP90 under the indicated conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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- D) Representative widefield images of expanded centrioles during ciliogenesis from siCT (A, B) and siSFI1 (C, D) RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CEP164 (red hot). Note the abnormal organization of the distal appendage protein CEP164 in siSFI1-treated cells. (E-H) Representative widefield images of expanded centrioles during ciliogenesis from siCT (E, F) and siSFI1 (G, H) RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CEP90 (cyan). (I) Frequency distribution of the number of dots per centriole formed by CEP164 in indicated conditions. (J) Frequency distribution of the number of dots per centriole formed by CEP90 under the indicated conditions. (K) Percentage of cells presenting abnormal CEP164 and CEP90 under the indicated conditions.


Characterization of OAS1 - 3 expression in parental and OAS knockout PC3 cells , obtained using CRISPR - Cas9 technology . Left : Western blot ( WB ) analyses of the expression of OAS1 , 2 , and 3 in clones of OAS single - , double - or triple - knockout PC3 cells ( KO , DKO , TKO ) , treated with IFNβ ( 500 IU / ml ) for 24 h , to increase OAS expression . sgOAS1 - 3 - single guide RNA for OAS1 - 3 . β - actin is shown as a loading control . Right : analysis of the expression of OAS1 , 2 , and 3 in PC3 DKO cells with increased expression of the remaining OAS , without IFNβ treatment . OAS1 expression in DKO2 / 3 cells was measured by qRT - PCR due to insufficient sensitivity of anti - OAS1 ( see Fig EV1A ) , OAS2 and 3 expression was detected by WB . The gene expression data are presented as mean + SD of triplicates . * * , p < 0 . 001 in Student ' s t - test .

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

Characterization of OAS1-3 expression in parental and OAS knockout PC3 cells, obtained using CRISPR-Cas9 technology. Left: Western blot (WB) analyses of the expression of OAS1, 2, and 3 in clones of OAS single-, double- or triple-knockout PC3 cells (KO, DKO, TKO), treated with IFNβ (500 IU/ml) for 24 h, to increase OAS expression. sgOAS1-3 - single guide RNA for OAS1-3. β-actin is shown as a loading control. Right: analysis of the expression of OAS1, 2, and 3 in PC3 DKO cells with increased expression of the remaining OAS, without IFNβ treatment. OAS1 expression in DKO2/3 cells was measured by qRT-PCR due to insufficient sensitivity of anti-OAS1 (see Fig EV1A), OAS2 and 3 expression was detected by WB. The gene expression data are presented as mean + SD of triplicates.**, p<0.001 in Student's t-test.


Characterization of OAS1 - 3 expression in parental and OAS knockout HME cells , obtained using CRISPR - Cas9 technology . Left : WB analyses of the expression of OAS1 , 2 , and 3 in selected clones of HME cells in which OAS1 - 3 were knocked out , to produce OAS single - , double - or triple - knockout cells ( KO , DKO , TKO ) , treated with 100 IU / ml IFNβ for 24 h to increase the levels of OAS expression . sgOAS1 - 3 - single guide RNA for OAS1 - 3 . β - actin is shown as a loading control . Right : The expression of OAS1 , 2 , and 3 in HME DKO cells with increased expression of the remaining OAS , without IFNβ treatment . The expression of OAS2 or 3 was determined by WB analysis ( asterisk , non - specific band ) . OAS1 expression in DKO2 / 3 cells was measured by qRT - PCR due to insufficient sensitivity of anti - OAS1 . The gene expression data are presented as mean + SD of triplicates . * * , p < 0 . 001 in Student ' s t - test .

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

Characterization of OAS1-3 expression in parental and OAS knockout HME cells, obtained using CRISPR-Cas9 technology. Left: WB analyses of the expression of OAS1, 2, and 3 in selected clones of HME cells in which OAS1-3 were knocked out, to produce OAS single-, double- or triple-knockout cells (KO, DKO, TKO), treated with 100 IU/ml IFNβ for 24 h to increase the levels of OAS expression. sgOAS1-3 - single guide RNA for OAS1-3. β-actin is shown as a loading control. Right: The expression of OAS1, 2, and 3 in HME DKO cells with increased expression of the remaining OAS, without IFNβ treatment. The expression of OAS2 or 3 was determined by WB analysis (asterisk, non-specific band). OAS1 expression in DKO2/3 cells was measured by qRT-PCR due to insufficient sensitivity of anti-OAS1. The gene expression data are presented as mean + SD of triplicates. **, p<0.001 in Student's t-test.


OAS expression was assessed by WB with anti OAS1 - 3 in HME cells expressing ectopic OAS1 - 3 ( OAS2hi , OAS3hi , OAS1hi ) cells without IFNβ treatment or control HME cells ( vec ) treated with the indicated concentrations of IFNβ for 2 days ( to compare the levels of induced and ectopically expressed OASs ) . β - actin and Ponceau stain are shown as loading controls ( # - transfer artifact ) ; ns , non - specific band .

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OAS expression was assessed by WB with anti OAS1-3 in HME cells expressing ectopic OAS1-3 (OAS2hi, OAS3hi, OAS1hi) cells without IFNβ treatment or control HME cells (vec) treated with the indicated concentrations of IFNβ for 2 days (to compare the levels of induced and ectopically expressed OASs). β-actin and Ponceau stain are shown as loading controls (# - transfer artifact); ns, non-specific band.


Analysis of the percentages of dead PC3 DKO cells or PC3 DKO cells with high expression of the remaining OAS , treated with 75 µM H2O2 , 40 μM p - Benzoquinone ( pBQ ) or 2 mM methyl methane sulfonate ( MMS ) . The number of dead cells ( Cytotox Green - permeable ) was monitored every 3 h using an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * , p < 0 . 001 ; * , p < 0 . 05 ; ns , no significance in two - way ANOVA . Representative data , which were reproduced more than 3 times , are shown .

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Analysis of the percentages of dead PC3 DKO cells or PC3 DKO cells with high expression of the remaining OAS, treated with 75 µM H2O2, 40 μM p-Benzoquinone (pBQ) or 2 mM methyl methane sulfonate (MMS). The number of dead cells (Cytotox Green-permeable) was monitored every 3 h using an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.001; *, p<0.05; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.


Analysis of the percentages of dead HME DKO cells and HME DKO cells with high expression of the remaining OAS , treated with 500 μM H2O2 , and KO1 - 3 and OAS1 - 3hi cells treated with 750 μM H2O2 . Data were obtained and presented as in ( A ) . Means of triplicates ± SD . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; ns , no significance in two - way ANOVA . Representative data , which were reproduced more than 3 times , are shown .

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Analysis of the percentages of dead HME DKO cells and HME DKO cells with high expression of the remaining OAS, treated with 500 μM H2O2, and KO1-3 and OAS1-3hi cells treated with 750 μM H2O2. Data were obtained and presented as in (A). Means of triplicates ± SD. ***, p<0.001; **, p<0.01; ns, no significance in two-way ANOVA. Representative data, which were reproduced more than 3 times, are shown.


Survival of HME cells , measured by the MTT cell survival assay . Left : control ( vec ) and KO1 - 3 cells were treated with 250 , 500 or 750 μM of H2O2 and analyzed after 24 h ; Right : control , OAS1hi and OAS KO were treated with 500 μM of H2O2 and analyzed after 72 h . The data are presented as mean + SD of triplicates . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 in Student ' s t - test .

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Survival of HME cells, measured by the MTT cell survival assay. Left: control (vec) and KO1-3 cells were treated with 250, 500 or 750 μM of H2O2 and analyzed after 24 h; Right: control, OAS1hi and OAS KO were treated with 500 μM of H2O2 and analyzed after 72 h . The data are presented as mean + SD of triplicates. ***, p<0.001; **, p<0.01; *, p<0.05 in Student's t-test.


An autoradiograph showing radiolabeled PAR2 - 5A bands that were formed in reactions of OAS1 with the activator ( pIC ) and the acceptor PAR ( lanes 11 - 14 ) . Lanes 1 - 2 OAS1 only , lanes 3 - 6 OAS1 with activator pIC ; lanes 7 - 10 OAS1 with substrate PAR ; lane 15 negative control without OAS1 . 32P - α - ATP was added to all reactions .

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An autoradiograph showing radiolabeled PAR2-5A bands that were formed in reactions of OAS1 with the activator (pIC) and the acceptor PAR (lanes 11-14). Lanes 1-2 OAS1 only, lanes 3-6 OAS1 with activator pIC; lanes 7-10 OAS1 with substrate PAR; lane 15 negative control without OAS1. 32P-α-ATP was added to all reactions.


Top : specificity of anti - 2 ' , 5 ' A , as shown by ELISA . Synthetic 2 ' , 5 ' - p3A3 , PAR2 - 5A # 1 , PAR2 - 5A # 2 ( 2 different preparations of PAR2 - 5A ) . PAR and 3 ' , 5 ' - pA3 were used as competitors . Bottom : Dot - blot of synthetic PAR and 2 - 5A - PAR . In vitro synthesized PAR and PAR - 2 - 5 - A were applied in 1 : 2 dilutions ( left to right ) to nylon membrane and probed with anti - 2 - 5A or anti - PAR .

Top: specificity of anti-2',5'A, as shown by ELISA. Synthetic 2',5'-p3A3, PAR2-5A #1, PAR2-5A #2 (2 different preparations of PAR2-5A). PAR and 3',5'-pA3 were used as competitors. Bottom: Dot-blot of synthetic PAR and 2-5A-PAR. In vitro synthesized PAR and PAR-2-5-A were applied in 1:2 dilutions (left to right) to nylon membrane and probed with anti-2-5A or anti-PAR.


Immunostaining of PAR - 2 - 5A synthesized in vitro . Reaction mixtures were resolved by electrophoresis in a polyacrylamide - 8 M urea denaturing gel , transferred to a charged nylon membrane , and probed with mouse anti - PAR 10H ( left panel ) or rabbit anti - 2 - 5A after stripping ( right panel ) . XC : Xylene Cyanol ( apparent size 150 nucleotides ) , BPB : Bromophenol Blue ( apparent size 35 nucleotides ) . Lane 1 , purified PARP1 alone ; lane 2 , PAR reaction : PARP1 , activated DNA , NAD + , and histone ; lane 3 , 2 - 5A reaction : OAS1 , pIC , ATP ; lane 4 , 2 - 5A and PAR reactions : OAS1 , pIC , ATP and PARP1 , activated DNA , NAD , histone . After incubation at 37oC for 60 min , the proteins were removed by digestion with proteinase K .

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Immunostaining of PAR-2-5A synthesized in vitro. Reaction mixtures were resolved by electrophoresis in a polyacrylamide-8 M urea denaturing gel, transferred to a charged nylon membrane, and probed with mouse anti-PAR 10H (left panel) or rabbit anti-2-5A after stripping (right panel). XC: Xylene Cyanol (apparent size 150 nucleotides), BPB: Bromophenol Blue (apparent size 35 nucleotides). Lane 1, purified PARP1 alone; lane 2, PAR reaction: PARP1, activated DNA, NAD+, and histone; lane 3, 2-5A reaction: OAS1, pIC, ATP; lane 4, 2-5A and PAR reactions: OAS1, pIC, ATP and PARP1, activated DNA, NAD, histone. After incubation at 37oC for 60 min, the proteins were removed by digestion with proteinase K.


Western analysis of PARP1 auto - modified with PAR in vitro in the absence or presence of OAS1 . Reaction mixtures were resolved by SDS - PAGE , transferred to a charged nylon membrane , and probed with mouse anti - PARP1 . Reactions were set up as in ( C ) , but without proteinase K digestion . Lane 1 , PARP1 alone ; lane 2 , PAR reaction : PARP1 , activated DNA and NAD ; lane 3 , 2 - 5A reaction : OAS1 , pIC , and ATP ; lane 4 , 2 - 5A and PAR reactions : OAS1 , pIC , and ATP with PARP1 , activated DNA , NAD and histone ; lane 5 , 2 - 5A and PAR reactions without ATP . The large smeary signals are due to PAR chains attached to PARP1 and the smaller sharp bands are free PARP1 . The same blot was probed with anti - PAR , anti - 2 - 5A and anti - OAS1 .

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Western analysis of PARP1 auto-modified with PAR in vitro in the absence or presence of OAS1. Reaction mixtures were resolved by SDS-PAGE, transferred to a charged nylon membrane, and probed with mouse anti-PARP1. Reactions were set up as in (C), but without proteinase K digestion. Lane 1, PARP1 alone; lane 2, PAR reaction: PARP1, activated DNA and NAD; lane 3, 2-5A reaction: OAS1, pIC, and ATP; lane 4, 2-5A and PAR reactions: OAS1, pIC, and ATP with PARP1, activated DNA, NAD and histone; lane 5, 2-5A and PAR reactions without ATP. The large smeary signals are due to PAR chains attached to PARP1 and the smaller sharp bands are free PARP1. The same blot was probed with anti-PAR, anti-2-5A and anti-OAS1.


Western analysis of 2 - 5A precipitated with anti - PAR antibodies from lysates of H2O2 - treated HME cells . Cells were treated with 500 M H2O2 for 12 min , and lysates were incubated with control ( mouse IgG3 ) or mouse anti - PAR . Samples were probed with anti - PAR , anti - 2 - 5A or anti - PARP1 antibodies . Flow - thr . : flow - through , WCL : whole cell lysate , IP : immuno - precipitate . Right panels ( IP ) show exposures for 10 x longer than left panels ( WCL , flow - thr ) . Apparent MW ( kDa ) is indicated on the right . Experiments were repeated two times with similar results .

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Western analysis of 2-5A precipitated with anti-PAR antibodies from lysates of H2O2-treated HME cells. Cells were treated with 500 M H2O2 for 12 min, and lysates were incubated with control (mouse IgG3) or mouse anti-PAR. Samples were probed with anti-PAR, anti-2-5A or anti-PARP1 antibodies. Flow-thr.: flow-through, WCL: whole cell lysate, IP: immuno-precipitate. Right panels (IP) show exposures for 10 x longer than left panels (WCL, flow-thr). Apparent MW (kDa) is indicated on the right. Experiments were repeated two times with similar results.


Western analysis of 2 - 5A precipitated with anti - PAR antibodies from lysates of HME control , KO1 and TKO cells , and control cells pre - incubated with olaparib , treated with H2O2 for 12 min . Samples were probed with anti - PAR or anti - 2 - 5A antibodies . Ponceau staining of the membrane is shown as loading control . Experiments were repeated four times with similar results .

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Western analysis of 2-5A precipitated with anti-PAR antibodies from lysates of HME control, KO1 and TKO cells, and control cells pre-incubated with olaparib, treated with H2O2 for 12 min. Samples were probed with anti-PAR or anti-2-5A antibodies. Ponceau staining of the membrane is shown as loading control. Experiments were repeated four times with similar results.


HME cells were treated with H2O2 for the indicated times and stained with anti - PAR and anti - 2 - 5A . Nuclei were visualized with DAPI . The densities of the 2 - 5A and PAR fluorescence signals in the nuclei are plotted . Signals in 40 + cells per sample were measured . Data are presented as distribution plot with mean value + / - SD ; * * * , p < 0 . 001 ; * , p < 0 . 5 ; ns , non - significant ; as determined by unpaired Kolmogorov - Smirnov t - test . Scale bar , 10μm .

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HME cells were treated with H2O2 for the indicated times and stained with anti-PAR and anti-2-5A. Nuclei were visualized with DAPI. The densities of the 2-5A and PAR fluorescence signals in the nuclei are plotted. Signals in 40+ cells per sample were measured. Data are presented as distribution plot with mean value +/- SD; ***, p<0.001; *, p<0.5; ns, non-significant; as determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.


HME control ( vector ) and TKO cells were treated with H2O2 for 12 min , incubated on ice for 7 min with PBS or detergent - containing Buffer 1 , to extract unbound proteins , fixed with methanol / acetone , and stained with anti - 2 - 5A and anti - PAR . Scale bar , 10μm .

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HME control (vector) and TKO cells were treated with H2O2 for 12 min, incubated on ice for 7 min with PBS or detergent-containing Buffer 1, to extract unbound proteins, fixed with methanol/acetone, and stained with anti-2-5A and anti-PAR. Scale bar, 10μm.


HME vector control ( vec ) , DKO , and TKO cells were treated with H2O2 for 12 min . In untreated cells , no PAR staining was detected . The densities of fluorescence signals in the nuclei relative to the untreated vector control are plotted . Signals in 40 or more cells per sample were measured . Data are presented as distribution plot with mean value + / - SD ; * * * , p < 0 . 001 ; * , p < 0 . 5 ; ns , non - significant as determined by unpaired Kolmogorov - Smirnov t - test . Scale bar , 10μm .

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HME vector control (vec), DKO, and TKO cells were treated with H2O2 for 12 min. In untreated cells, no PAR staining was detected. The densities of fluorescence signals in the nuclei relative to the untreated vector control are plotted. Signals in 40 or more cells per sample were measured. Data are presented as distribution plot with mean value +/- SD; ***, p<0.001; *, p<0.5; ns, non-significant as determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.


HME control ( vector ) and KO1 cells were treated with H2O2 for the indicated times and stained with anti - 2 - 5A and anti - PAR . Scale bar , 10μm .

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HME control (vector) and KO1 cells were treated with H2O2 for the indicated times and stained with anti-2-5A and anti-PAR. Scale bar, 10μm.


Accumulation of 2 - 5A and PAR in HME cells pre - incubated with 10 or 100 nM olaparib for 30 min before treatment with H2O2 for 12 min , as visualized by immunostaining with anti - 2 - 5A and anti - PAR . Scale bar , 10μm .

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Accumulation of 2-5A and PAR in HME cells pre-incubated with 10 or 100 nM olaparib for 30 min before treatment with H2O2 for 12 min, as visualized by immunostaining with anti-2-5A and anti-PAR. Scale bar, 10μm.


HME control ( vec ) and OAS single knockout cells ( KO1 - 3 ) , pre - treated with IFNβ for 2 days to induce OAS expression , fixed , and stained with anti - OAS1 , 2 , or 3 . Staining with secondary antibodies is shown as a negative control . Scale bar , 10μm .

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HME control (vec) and OAS single knockout cells (KO1-3), pre-treated with IFNβ for 2 days to induce OAS expression, fixed, and stained with anti-OAS1, 2, or 3. Staining with secondary antibodies is shown as a negative control. Scale bar, 10μm.


HME control cells were treated with IFNβ for 2 days to induce OAS expression , fixed and stained with anti - OAS1 , 2 , or 3 . Scale bar , 10μm .

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HME control cells were treated with IFNβ for 2 days to induce OAS expression, fixed and stained with anti-OAS1, 2, or 3. Scale bar, 10μm.


HME cells expressing Flag - tagged OAS1 , 2 , or 3 were fixed and stained with anti - Flag . Scale bar , 10μm .

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HME cells expressing Flag-tagged OAS1, 2, or 3 were fixed and stained with anti-Flag. Scale bar, 10μm.


Untreated HME ( left ) or PC3 ( right ) cells were stained with anti - OAS1 - 3 and the nuclei were visualized by staining with DAPI . The intensities of OAS1 - 3 fluorescence signals in the nuclei are plotted . Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining , respectively . Experiments were repeated at least two times ; at least 40 cells per condition were counted . Data are presented as distribution plot with mean value + / - SD ; statistical significance was determined by unpaired Kolmogorov - Smirnov t - test . Scale bar , 10μm .

Untreated HME (left) or PC3 (right) cells were stained with anti-OAS1-3 and the nuclei were visualized by staining with DAPI. The intensities of OAS1-3 fluorescence signals in the nuclei are plotted. Nuclear and cytoplasmic masks were made based on DAPI and phalloidin staining, respectively. Experiments were repeated at least two times; at least 40 cells per condition were counted. Data are presented as distribution plot with mean value +/- SD; statistical significance was determined by unpaired Kolmogorov-Smirnov t-test. Scale bar, 10μm.


HME control ( vec ) and KO1 cells were pre - treated with IFNβ for 2 days , followed by a 10 - min treatment with H2O2 . Cytoplasmic , membrane - bound and soluble nuclear fractions of cell lysates were analyzed by the Western method ; staining with anti - AIF and anti - HDAC2 staining was used to characterize membrane and nuclear fractions , respectively . Anti - PAR detects all PAR - modified proteins , which appear as smears .

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HME control (vec) and KO1 cells were pre-treated with IFNβ for 2 days, followed by a 10-min treatment with H2O2. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method; staining with anti-AIF and anti-HDAC2 staining was used to characterize membrane and nuclear fractions, respectively. Anti-PAR detects all PAR-modified proteins, which appear as smears.


HME control ( v ) , KO1 and OAS1hi ( O1hi ) cells were pre - treated with IFNβ for 48 h . Cytoplasmic , membrane - bound and soluble nuclear fractions of cell lysates were analyzed by the Western method . Anti - AIF and anti - HDAC2 were used to characterize membrane and nuclear fractions , respectively ; * indicates lanes where specific signals are 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HME control (v), KO1 and OAS1hi (O1hi) cells were pre-treated with IFNβ for 48 h. Cytoplasmic, membrane-bound and soluble nuclear fractions of cell lysates were analyzed by the Western method. Anti-AIF and anti-HDAC2 were used to characterize membrane and nuclear fractions, respectively; * indicates lanes where specific signals are detected.


Representative images and analysis of Western blots for PAR , performed with lysates of HME control ( vec ) OAS single KO cells ( KO1 - 3 ) , treated with H2O2 for 15 or 40 min . β - actin is shown as a loading control . Signal intensity was measured using ImageJ software . Average lane densities , normalized to vector from three independent experiments , are plotted below as mean + SD , * p < 0 . 05 as assessed by Student ' s t - test .

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

Representative images and analysis of Western blots for PAR, performed with lysates of HME control (vec) OAS single KO cells (KO1-3), treated with H2O2 for 15 or 40 min. β-actin is shown as a loading control. Signal intensity was measured using ImageJ software. Average lane densities, normalized to vector from three independent experiments, are plotted below as mean + SD, * p < 0.05 as assessed by Student's t-test.


Same as ( A ) , with lysates of HME control ( vec ) cells and cells expressing ectopic OAS1 , OAS2 or OAS3 ( OAS1 - 3hi ) . Experiments were repeated at least three times . Average lane densities normalized to vector are plotted below below as mean + SD , * p < 0 . 05 as assessed by Student ' s t - test .

Same as (A), with lysates of HME control (vec) cells and cells expressing ectopic OAS1, OAS2 or OAS3 (OAS1-3hi). Experiments were repeated at least three times. Average lane densities normalized to vector are plotted below below as mean + SD, * p < 0.05 as assessed by Student's t-test.


HME cells , pre - incubated with IFNβ for 2 days to increase OAS abundance , were treated with H2O2 for 0 - 60 min at 37oC . Western blots of PAR accumulation ( top ) , shift in the position of PARylated PARP1 ( middle ) , OAS2 induction by IFNβ , and β - actin as a loading control are shown . Experiments were repeated twice .

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

HME cells, pre-incubated with IFNβ for 2 days to increase OAS abundance, were treated with H2O2 for 0-60 min at 37oC. Western blots of PAR accumulation (top), shift in the position of PARylated PARP1 (middle), OAS2 induction by IFNβ, and β-actin as a loading control are shown. Experiments were repeated twice.


HME control ( vec ) and KO1 cells were pretreated with 0 - 500 IU / ml IFNβ for 2 days , and then treated with 500 μM H2O2 for 10 or 30 min . Western blots of PAR accumulation ( top , high and medium exposure ; middle , low exposure ) , OAS1 - 3 induction by IFNβ , and β - actin as a loading control are shown . The experiment was repeated four times ; ns , non - specific band .

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

HME control (vec) and KO1 cells were pretreated with 0-500 IU/ml IFNβ for 2 days, and then treated with 500 μM H2O2 for 10 or 30 min. Western blots of PAR accumulation (top, high and medium exposure; middle, low exposure), OAS1-3 induction by IFNβ, and β-actin as a loading control are shown. The experiment was repeated four times; ns, non-specific band.


HME cells were treated with 750 μM of H2O2 , with and without pre - treatment with olaparib . The accumulation of dead cells was measured with an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * , p < 0 . 01 in two - way ANOVA . The data represent the results of three experiments .

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

HME cells were treated with 750 μM of H2O2, with and without pre-treatment with olaparib. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ***, p<0.01 in two-way ANOVA. The data represent the results of three experiments.


HME control ( vec ) and KO1 - 3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min . PAR accumulation was assessed by the Western method , with anti - PAR . β - actin is shown as a loading control .

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

HME control (vec) and KO1-3 cells were incubated with olaparib for 30 min followed by treatment with 500 μM H2O2 for 15 min. PAR accumulation was assessed by the Western method, with anti-PAR. β-actin is shown as a loading control.


HME vec , KO1 and OAS1hi cells were incubated with olaparib for 30 min , then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h . Bars represent means of triplicates ± SD from three experiments . * p < 0 . 05 ; * * p < 0 . 01 ( relative to H2O2 - treated vec cells ) in Student ' s t - test .

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

HME vec, KO1 and OAS1hi cells were incubated with olaparib for 30 min, then treated with 500 or 1000 μM of H2O2 and allowed to grow for 72 h. Bars represent means of triplicates ± SD from three experiments. * p<0.05; ** p<0.01 (relative to H2O2-treated vec cells) in Student's t-test.


KO1 cells transfected with vector ( KO1 - vec ) , wild - type OAS1 ( KO1 - OAS1 wt ) , or mutant OAS1 ( D75A / D77A , KO1 - OAS1 mut ) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method . Loading controls : β - actin and overall protein levels were visualized by stain - free technology . The experiment was repeated three times ; a representative image is shown .

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

KO1 cells transfected with vector (KO1-vec), wild-type OAS1 (KO1-OAS1 wt), or mutant OAS1 (D75A/D77A, KO1-OAS1 mut) were treated with 500 μM of H2O2 for the indicated times and analyzed by the Western method. Loading controls: β-actin and overall protein levels were visualized by stain-free technology. The experiment was repeated three times; a representative image is shown.


Quantification of the relative abundance of PAR in ( D ) . Images obtained in three independent experiments were analyzed ; the data are plotted as mean + SD * , p < 0 . 05 by Student ' s t - test .

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

Quantification of the relative abundance of PAR in (D). Images obtained in three independent experiments were analyzed; the data are plotted as mean + SD *, p<0.05 by Student's t-test.


KO1 cells transfected with vector , wild - type OAS1 ( OAS1 wt ) , or mutant OAS1 ( D75A / D77A , OAS1 mut ) were treated with 500 μM of H2O2 . The accumulation of dead cells was measured with an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * * , p < 0 . 001 in two - way ANOVA . The data represent the results of three experiments .

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

KO1 cells transfected with vector, wild-type OAS1 (OAS1 wt), or mutant OAS1 (D75A/D77A, OAS1 mut) were treated with 500 μM of H2O2. The accumulation of dead cells was measured with an IncuCyte ZOOM instrument. Means of triplicates ± SD. ****, p<0.001 in two-way ANOVA. The data represent the results of three experiments.


A , B . Representative images and analysis of AIF accumulation in the nuclei of HME control ( vec ) and OAS single - knockout cells ( KO ) , and HME control ( vec ) and OAS double - and triple - knockout cells ( DKOs and TKO ) , 24 h after treatment with 1000 μM H2O2 . Data information : Nuclei were visualized by DAPI staining . Arrowheads : AIF translocated to nuclei . Untreated control cells are shown on the left . Right panels , AIF - positive nuclei were counted from pictures taken from three independent fields in two independent experiments . Means ± SD are plotted . HME : * p < 0 . 005 ; * * p < 0 . 001 compared to H2O2 - treated control ( vec ) cells ;

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

A, B. Representative images and analysis of AIF accumulation in the nuclei of HME control (vec) and OAS single-knockout cells (KO), and HME control (vec) and OAS double- and triple-knockout cells (DKOs and TKO), 24 h after treatment with 1000 μM H2O2. Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. HME: * p<0.005; ** p<0.001 compared to H2O2-treated control (vec) cells;


C . Representative images and analysis of AIF accumulation in the nuclei of PC3 DKO2 / 3 and PC3 DKO2 / 3 + 1 cells , 24 h after treatment with 100 , 200 or 400 μM H2O2 . Data information : Nuclei were visualized by DAPI staining . Arrowheads : AIF translocated to nuclei . Untreated control cells are shown on the left . Right panels , AIF - positive nuclei were counted from pictures taken from three independent fields in two independent experiments . Means ± SD are plotted . PC3 : * * * p < 0 . 0001 compared to untreated cells of each genotype as assessed by Student ' s t - test . Scale bars , 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images and analysis of AIF accumulation in the nuclei of PC3 DKO2/3 and PC3 DKO2/3+1 cells, 24 h after treatment with 100, 200 or 400 μM H2O2. Data information: Nuclei were visualized by DAPI staining. Arrowheads: AIF translocated to nuclei. Untreated control cells are shown on the left. Right panels, AIF-positive nuclei were counted from pictures taken from three independent fields in two independent experiments. Means ±SD are plotted. PC3: *** p<0.0001 compared to untreated cells of each genotype as assessed by Student's t-test. Scale bars, 10μm.


( A , B ) Overexpression of wildtype full - length human tau ( hTau , also termed tau441 or tau40 or tau2N4R ) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II , in which 96 transcription factors ( Appendix Table S1 and S2 ) were monitored . The empty vector was transfected as a control ( Ctrl ) .

(A, B) Overexpression of wildtype full-length human tau (hTau, also termed tau441 or tau40 or tau2N4R) induced significant alterations of 9 transcription factors screened by using Transcription Factors Activation Profiling Plate Array II, in which 96 transcription factors (Appendix Table S1 and S2) were monitored. The empty vector was transfected as a control (Ctrl).


( C - F ) Expression of hTau ( probed by HT7 ) increased total and the phosphorylated STAT1 at Tyr701 ( pY - STAT1 ) in cell whole extracts ( C , D ) and the nuclear fraction ( E , F ) measured by Western blotting ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . * , p < 0 . 05 vs Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Expression of hTau (probed by HT7) increased total and the phosphorylated STAT1 at Tyr701 (pY-STAT1) in cell whole extracts (C, D) and the nuclear fraction (E, F) measured by Western blotting (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 vs Ctrl.


( G ) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control ( eGFP ) ( n = 5 ) . Scale bar , 10 μm . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . , * * , p < 0 . 01 vs Ctrl .

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

(G) The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control (eGFP) (n=5). Scale bar, 10 μm. Data information: Data were presented as mean ± SD (Mann-Whitney test). , **, p<0.01 vs Ctrl.


( H ) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction ( Nu ) measured by Western blotting .

O 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) Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction (Nu) measured by Western blotting.


( I ) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney test ) . * , p < 0 . 05 , * * * , p < 0 . 001 vs Ctrl .

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

(I) Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay (n=4). Data information: Data were presented as mean ± SD (Mann-Whitney test). *, p<0.05 , ***, p<0.001 vs Ctrl.


( J ) Overexpression of hTau increased STAT1 - DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay ( EMSA ) . * , indicates STAT1 / DNA complex .

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

(J) Overexpression of hTau increased STAT1-DNA binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). *, indicates STAT1/DNA complex.


( A , B ) AAV - hTau - eGFP ( AAV - hTau ) or the empty vector AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , the increased levels of STAT1 and pY - STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting ( n = 6 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

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

(A, B) AAV-hTau-eGFP (AAV-hTau) or the empty vector AAV-eGFP (1.13×1013 v.g./ml) was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, the increased levels of STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting (n=6, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( C , D ) The increased STAT1 and pY - STAT1 in hippocampal total extracts and the nuclear fraction of 12 - month - old hTau transgenic mice measured by Western blotting ( n = 4 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) The increased STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction of 12-month-old hTau transgenic mice measured by Western blotting (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( E , F ) The representative images of STAT1 and pY - STAT1 in the brain of AD patients probed by co - immunohistochemical staining and quantitative analysis ( hematoxylin stains nuclei , purple ; DAB stains the target proteins , brown ; n = 5 - 6 slices ) . Arrowheads indicated typical nuclear staining of STAT1 / pY - STAT1 . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) The representative images of STAT1 and pY-STAT1 in the brain of AD patients probed by co-immunohistochemical staining and quantitative analysis (hematoxylin stains nuclei, purple; DAB stains the target proteins, brown; n=5-6 slices). Arrowheads indicated typical nuclear staining of STAT1/pY-STAT1. Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( G ) The increased AT8 ( pS202 / pT205 ) , STAT1 and pY - STAT1 in cortex total extracts of AD patients measured by Western blotting ( n = 3 , Student ' s t test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

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

(G) The increased AT8 (pS202/pT205), STAT1 and pY-STAT1 in cortex total extracts of AD patients measured by Western blotting (n=3, Student's t test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


( H ) STAT1 mRNA was analyzed by qPCR in AAV - hTau or AAV - eGFP infected hippocampal tissues ( n = 6 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) STAT1 mRNA was analyzed by qPCR in AAV-hTau or AAV-eGFP infected hippocampal tissues (n=6, Mann-Whitney test). Data information: Data were presented as mean ± SD. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP, wt or Ctrl.


AAV - Cre ( 5 × 1012 v . g . / ml ) mixed with AAV - hTau or AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) were stereotaxically injected into the hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by immunohistochemical staining .

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

AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by immunohistochemical staining.


AAV - Cre ( 5 × 1012 v . g . / ml ) mixed with AAV - hTau or AAV - eGFP ( 1 . 13 × 1013 v . g . / ml ) were stereotaxically injected into the hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by Western

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

AAV-Cre (5×1012 v.g./ml) mixed with AAV-hTau or AAV-eGFP (1.13×1013 v.g./ml) were stereotaxically injected into the hippocampal CA3 of 3-month-old STAT1flox/flox mice. One month later, downregulation of STAT1 was confirmed by Western blotting


( C ) Downregulation of STAT1 ameliorated hTau - induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze ( MWM ) test ( n = 9 - 11 for each group ) . Data information : Data were presented as mean ± s . e . m . ( two - way repeated measures analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for C * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Downregulation of STAT1 ameliorated hTau-induced spatial learning deficit shown by the decreased escape latency during 5 consecutive days training in Morris water maze (MWM) test (n=9-11 for each group). Data information: Data were presented as mean ± s.e.m. (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni's post hoc test for C *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( D - G ) Downregulation of STAT1 ameliorated hTau - induced spatial memory deficit shown by the decreased latency to reach the platform quadrant ( D ) , the increased crossing time in the platform site ( E ) and time spent in the target quadrant ( F ) measured at day 6 by removed the platform in MWM test ; no motor dysfunction was seen ( G ) ( n = 9 - 11 for each group ) . Data information : Data were presented as mean ± s . e . m . , one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Downregulation of STAT1 ameliorated hTau-induced spatial memory deficit shown by the decreased latency to reach the platform quadrant (D), the increased crossing time in the platform site (E) and time spent in the target quadrant (F) measured at day 6 by removed the platform in MWM test; no motor dysfunction was seen (G) (n=9-11 for each group). Data information: Data were presented as mean ± s.e.m. , one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( H ) Downregulation of STAT1 ameliorated hTau - induced contextual memory deficits measured at 24 h during contextual fear conditioning test ( n = 8 each group ) . Data information : Data were presented as mean ± s . e . m . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Downregulation of STAT1 ameliorated hTau-induced contextual memory deficits measured at 24 h during contextual fear conditioning test (n=8 each group). Data information: Data were presented as mean ± s.e.m. *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( I - L ) One month after the virus infection , paired - pulse ratio ( PPR ) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice ( I ) . The IO curve of fEPSP recorded on acute hippocampal slices ( J ) . The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice ( K ) . LTP magnitude was calculated as the average ( normalized to baseline ) of the responses recorded 40 - 60 min after conditioning stimulation . ( L ) . ( n = 5 slices from 4 mice for each group ) . Data information : Data were presented as mean ± SD two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for I - K , one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

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

(I-L) One month after the virus infection, paired-pulse ratio (PPR) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice (I). The IO curve of fEPSP recorded on acute hippocampal slices (J). The slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice (K). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (L). (n=5 slices from 4 mice for each group). Data information: Data were presented as mean ± SD two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test for I-K, one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( M - P ) One month after the virus infection , whole cell patch clamp was used to measure the function of NMDA ( at + 40 mV ) and AMPA ( at - 70 mV ) receptors on acute brain slices ( 400 μm ) . The insets show representative sample traces of EPSCs in virus infected neurons ( M ) . The reduced NMDA and unchanged AMPA currents with a reduced NMDA / AMPA ratio were seen in hTau infected neurons , while knockdown of STAT1 restored the hTau - induced NMDA currents ( N - P ) . ( n = 12 neurons from 4 animals for eGFP group ; n = 11 neurons from 4 animals for hTau group ; n = 13 neurons from 4 animals for hTau + CRE group ) . Data information : Data were presented as mean ± SD one - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

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

(M-P) One month after the virus infection, whole cell patch clamp was used to measure the function of NMDA (at +40 mV) and AMPA (at -70 mV) receptors on acute brain slices (400 μm). The insets show representative sample traces of EPSCs in virus infected neurons (M). The reduced NMDA and unchanged AMPA currents with a reduced NMDA/AMPA ratio were seen in hTau infected neurons, while knockdown of STAT1 restored the hTau-induced NMDA currents (N-P). (n=12 neurons from 4 animals for eGFP group; n=11 neurons from 4 animals for hTau group; n=13 neurons from 4 animals for hTau+CRE group). Data information: Data were presented as mean ± SD one-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test for others). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP; #, p<0.05, ###, p<0.001 vs hTau.


( A , B ) Overexpression of AAV - hTau decreased the protein levels of GluN1 , GluN2A and GluN2B detected by Western blotting in the hippocampal CA3 of C57 mice , compared with the AAV - eGFP vector control . ( C , D ) Simultaneous downregulation of STAT1 by infusing AAV - Cre in hippocampal CA3 of STAT1flox / flox mice abolished the hTau - induced inhibition in expression of NMDARs protein . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney test ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau .

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

(A, B) Overexpression of AAV-hTau decreased the protein levels of GluN1, GluN2A and GluN2B detected by Western blotting in the hippocampal CA3 of C57 mice, compared with the AAV-eGFP vector control. (C, D) Simultaneous downregulation of STAT1 by infusing AAV-Cre in hippocampal CA3 of STAT1flox/flox mice abolished the hTau-induced inhibition in expression of NMDARs protein. Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.


( E , F ) Overexpression of AAV - hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1 , GluN2A and GluN2B detected by qRT - PCR in the hippocampal CA3 . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney test ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F) Overexpression of AAV-hTau or simultaneous downregulation of STAT1 changed the mRNA levels of GluN1, GluN2A and GluN2B detected by qRT-PCR in the hippocampal CA3. Data information: Data were presented as mean ± SD (n=4; Mann-Whitney test). *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau.


( A ) Overexpression of AAV - hTau increased binding of STAT1 to the promoter regions of GluN1 , GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay ( ChIP ) ( n = 3 from three independent experiments ) . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overexpression of AAV-hTau increased binding of STAT1 to the promoter regions of GluN1, GluN2A and GluN2B gene in hippocampal CA3 extracts measured by chromatin immunoprecipitation assay (ChIP) (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


( B ) Overexpression of hTau or wildtype STAT1 ( WT - STAT1 ) inhibits the transcription activity of NMDARs compared with the empty vector control ( Ctrl ) measured by luciferase activity assay in HEK293 cells ( n = 3 from three independent experiments ) . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overexpression of hTau or wildtype STAT1 (WT-STAT1) inhibits the transcription activity of NMDARs compared with the empty vector control (Ctrl) measured by luciferase activity assay in HEK293 cells (n=3 from three independent experiments). Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN1 ( C ) The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

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

Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN1 (C) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN2B ( E ) The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2B (E) The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


Diagrams show the predicted GAS promoter element ( GASs ) for STAT1 in the promoter ( - 2000 - + 299bp ) of GluN2A ( G , I ) . The GASs or the mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 cells for 24 h , and then the luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

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

Diagrams show the predicted GAS promoter element (GASs) for STAT1 in the promoter (-2000-+299bp) of GluN2A (G, I). The GASs or the mutant (MUT) plasmids were co-transfected respectively with WT-STAT1 or its empty vector (Ctrl) into HEK293 cells for 24 h, and then the luciferase activity was measured (right panels). N=4 for each group. Data information: Data were presented as mean ± SD (two-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs Ctrl; ###, p<0.001 vs wildtype reporters.


( A , B ) Overexpression of hTau in HEK293 cells for 48 h increased the activity - dependent phosphorylation of JAK2 , JNK1 and ERK1 compared with the empty vector control ( Ctrl ) measured by Western blotting ( n = 3 , student ' s t test ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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

(A, B) Overexpression of hTau in HEK293 cells for 48 h increased the activity-dependent phosphorylation of JAK2, JNK1 and ERK1 compared with the empty vector control (Ctrl) measured by Western blotting (n=3, student's t test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Pharmacological inhibition of ERK1 ( C , D ) for 24 h did not significantly affect the hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts ( C and the nuclear fraction ( D measured by Western blotting ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

Pharmacological inhibition of ERK1 (C, D) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts (C and the nuclear fraction (D measured by Western blotting (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Pharmacological inhibition of JNK1 ( E , F ) for 24 h did not significantly affect the hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts E ) and the nuclear fraction F ) measured by Western blotting ( n = 3 ) . The alteration of pS - STAT1 ( Ser727 ) confirms the efficacy of JNK1 inhibitors . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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

Pharmacological inhibition of JNK1 (E, F) for 24 h did not significantly affect the hTau-induced STAT1 phosphorylation at pY-STAT1 (Tyr701) in total extracts E) and the nuclear fraction F) measured by Western blotting (n=3). The alteration of pS-STAT1 (Ser727) confirms the efficacy of JNK1 inhibitors. Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


Pharmacological inhibition of JAK2 ( G , H ) abolished hTau - induced STAT1 phosphorylation at Tyr701 in total extracts ( G and the nuclear fraction ( H ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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

Pharmacological inhibition of JAK2 (G, H) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts (G and the nuclear fraction (H (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


knockdown JAK2 by siRNA ( I , J ) abolished hTau - induced STAT1 phosphorylation at Tyr701 in total extracts I ) and the nuclear fraction J ) ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

knockdown JAK2 by siRNA (I, J) abolished hTau-induced STAT1 phosphorylation at Tyr701 in total extracts I) and the nuclear fraction J) (n=3). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


( K , L ) The phosphorylated JAK2 level increased in the hippocampus of 12 - month - old hTau transgenic mice ( K ) , and the hippocampus of C57 mice infected with AAV - hTau ( 1 . 13 × 1013 v . g . / ml ) ( L ) ( n = 4 , Mann - Whitney test ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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

(K, L) The phosphorylated JAK2 level increased in the hippocampus of 12-month-old hTau transgenic mice (K), and the hippocampus of C57 mice infected with AAV-hTau (1.13×1013 v.g./ml) (L) (n=4, Mann-Whitney test). Data information: Data were presented as mean ± SD, *, p<0.05 vs Ctrl, eGFP or WT.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( A ) The representative fluorescence image confirms expression of AAV - hTau and AAV - Y701F - STAT1 . Scale bar , 200 μm , or 100 μm for the enlarged .

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

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (A) The representative fluorescence image confirms expression of AAV-hTau and AAV-Y701F-STAT1. Scale bar, 200 μm, or 100 μm for the enlarged.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( B ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial learning deficits shown by the decreased escape latency during water maze training ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m ( two - way repeated measures analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (B) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial learning deficits shown by the decreased escape latency during water maze training (n=7-10 each group). Data information: Data were presented as mean ± s.e.m (two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( C - E ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial memory impairment shown by the decreased latency to reach the platform ( C ) , the increased crossing time in the platform site ( D ) and time spent in the target quadrant ( E ) measured at day 6 by removed the platform ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (C-E) Overexpression of Y701F-STAT1 mitigated hTau-induced spatial memory impairment shown by the decreased latency to reach the platform (C), the increased crossing time in the platform site (D) and time spent in the target quadrant (E) measured at day 6 by removed the platform (n=7-10 each group). Data information: Data were presented as mean ± s.e.m , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


AAV - eGFP ( eGFP ) or AAV - hTau - eGFP ( hTau ) ( 1 . 13 × 1013 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) or AAV - Y701F - STAT1 ( 5 × 1012 v . g . / ml ) plus hTau was stereotaxically injected into hippocampal CA3 of 3 - month - old C57 mice . After one month , learning and memory were detected by MWM test . ( F ) Expression of Y701F - STAT1 did not change the swimming speed of the mice in water maze task ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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

AAV-eGFP (eGFP) or AAV-hTau-eGFP (hTau) (1.13×1013 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) or AAV-Y701F-STAT1 (5×1012 v.g./ml) plus hTau was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After one month, learning and memory were detected by MWM test. (F) Expression of Y701F-STAT1 did not change the swimming speed of the mice in water maze task (n=7-10 each group). Data information: Data were presented as mean ± s.e.m two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.


( G - I ) Simultaneous expression of Y701F - STAT1 did not induced any further change on basal synaptic transmission ( I / O curve ) compared with expression of hTau alone , recorded in hippocampal CA3 ( G ) . LTP magnitude was calculated as the average ( normalized to baseline ) of the responses recorded 40 - 60 min after conditioning stimulation . ( I ) . ( n = 5 slices from 4 mice for each group ) . Data information : Data were presented as mean ± SD , two - way analysis of variance ( ANOVA ) followed by Bonferroni ' s post hoc test * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Simultaneous expression of Y701F-STAT1 did not induced any further change on basal synaptic transmission (I/O curve) compared with expression of hTau alone, recorded in hippocampal CA3 (G). LTP magnitude was calculated as the average (normalized to baseline) of the responses recorded 40-60 min after conditioning stimulation. (I). (n=5 slices from 4 mice for each group). Data information: Data were presented as mean ± SD , two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.