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

O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-SUBCELLULAR B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 of expanded centrioles during ciliogenesis from siCT siSFI1 RPE - 1 treated . were stained for α / β - tubulin ( magenta ) CP110 ( yellow ) . ( J ) of ciliated observed in U - ExM under indicated conditions . ( K ) of CP110 capped / uncapped centrioles under indicated conditions .

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

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

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

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

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

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

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 expressing ectopic OAS1 - 3 ( OAS2hi , OAS3hi , OAS1hi ) without IFNβ treatment or control HME ( vec ) treated with indicated concentrations of IFNβ for 2 days ( to compare of induced ectopically expressed OASs ) . β - actin 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.


of percentages of dead PC3 DKO or PC3 DKO with high expression of remaining OAS , treated with 75 µM H2O2 , 40 μM p - Benzoquinone ( pBQ ) or 2 mM methyl methane sulfonate ( MMS ) . number of dead ( 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 .

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

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.


of percentages of dead HME DKO HME DKO with high expression of remaining OAS , treated with 500 μM H2O2 , KO1 - 3 OAS1 - 3hi treated with 750 μM H2O2 . Data were obtained 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 .

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

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

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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 activator ( pIC ) 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 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 3 ' , 5 ' - pA3 were used as competitors . Bottom : Dot - blot of synthetic PAR 2 - 5A - PAR . In vitro synthesized PAR PAR - 2 - 5 - A were applied in 1 : 2 dilutions ( left to right ) to nylon 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 , probed with mouse anti - PAR 10H ( left panel ) or rabbit anti - 2 - 5A after stripping ( right panel ) . XC : Xylene Cyanol ( apparent 150 nucleotides ) , BPB : Bromophenol Blue ( apparent 35 nucleotides ) . Lane 1 , purified PARP1 alone ; lane 2 , PAR reaction : PARP1 , activated , NAD + , histone ; lane 3 , 2 - 5A reaction : OAS1 , pIC , ATP ; lane 4 , 2 - 5A PAR reactions : OAS1 , pIC , ATP PARP1 , activated , NAD , histone . After incubation at 37oC for 60 min , 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 of PARP1 auto - modified with PAR in vitro in absence or presence of OAS1 . Reaction mixtures were resolved by SDS - PAGE , transferred to a charged nylon , 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 NAD ; lane 3 , 2 - 5A reaction : OAS1 , pIC , ATP ; lane 4 , 2 - 5A PAR reactions : OAS1 , pIC , ATP with PARP1 , activated , NAD histone ; lane 5 , 2 - 5A PAR reactions without ATP . large smeary signals are due to PAR chains attached to PARP1 smaller sharp bands are free PARP1 . same blot was probed with anti - PAR , anti - 2 - 5A 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 of 2 - 5A precipitated with anti - PAR antibodies from lysates of H2O2 - treated HME . were treated with 500 M H2O2 for 12 min , 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 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 right . Experiments were repeated two times with similar results .

B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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 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 of 2 - 5A precipitated with anti - PAR antibodies from lysates of HME control , KO1 TKO , control pre - incubated with olaparib , treated with H2O2 for 12 min . Samples were probed with anti - PAR or anti - 2 - 5A antibodies . Ponceau of is shown as loading control . Experiments were repeated four times with similar results .

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

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 were treated with H2O2 for indicated times stained with anti - PAR anti - 2 - 5A . Nuclei were visualized with DAPI . densities of 2 - 5A PAR signals in nuclei are plotted . Signals in 40 + 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 - . 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 ) TKO were treated with H2O2 for 12 min , incubated on ice for 7 min with PBS or detergent - containing Buffer 1 , to extract unbound , fixed with methanol / acetone , stained with anti - 2 - 5A 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 , TKO were treated with H2O2 for 12 min . In untreated , no PAR was detected . densities of signals in nuclei relative to untreated vector control are plotted . Signals in 40 or more 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 - . 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 ) KO1 were treated with H2O2 for indicated times stained with anti - 2 - 5A 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 PAR in HME 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 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 ) OAS single knockout ( KO1 - 3 ) , pre - treated with IFNβ for 2 days to induce OAS expression , fixed , stained with anti - OAS1 , 2 , or 3 . 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 were treated with IFNβ for 2 days to induce OAS expression , fixed 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 expressing Flag - tagged OAS1 , 2 , or 3 were fixed 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 ) were stained with anti - OAS1 - 3 nuclei were visualized by with DAPI . intensities of OAS1 - 3 signals in nuclei are plotted . masks were made on DAPI phalloidin , respectively . Experiments were repeated at least two times ; at least 40 per condition were counted . Data are presented as distribution plot with mean value + / - SD ; statistical significance was determined by unpaired Kolmogorov - Smirnov t - . Scale bar , 10μm .

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

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 ) KO1 were pre - treated with IFNβ for 2 days , followed by a 10 - min treatment with H2O2 . , - bound soluble fractions of lysates were analyzed by Western method ; with anti - AIF anti - HDAC2 was used to characterize fractions , respectively . Anti - PAR detects PAR - modified , which appear as smears .

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

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 OAS1hi ( O1hi ) were pre - treated with IFNβ for 48 h . , - bound soluble fractions of lysates were analyzed by Western method . Anti - AIF anti - HDAC2 were used to characterize fractions , respectively ; * indicates lanes where specific signals are detected .

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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 of Western blots for PAR , performed with lysates of HME control ( vec ) OAS single KO ( KO1 - 3 ) , treated with H2O2 for 15 or 40 min . β - actin is shown as a loading control . Signal 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 - .

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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 ) 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 - .

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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 , 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 position of PARylated PARP1 ( middle ) , OAS2 induction by IFNβ , β - actin as a loading control are shown . Experiments were repeated twice .

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

B-CELL_LINE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 were treated with 750 μM of H2O2 , with without pre - treatment with olaparib . accumulation of dead was measured with an IncuCyte ZOOM instrument . Means of triplicates ± SD . * * * , p < 0 . 01 in two - way ANOVA . data represent results of three experiments .

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

B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD 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 OAS1hi were incubated with olaparib for 30 min , then treated with 500 or 1000 μM of H2O2 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 ) in Student ' s t - .

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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 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 indicated times analyzed by Western method . Loading controls : β - actin overall were visualized by stain - free technology . experiment was repeated three times ; a representative image is shown .

O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O 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.


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

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

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

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 of AIF accumulation in nuclei of HME control ( vec ) OAS single - knockout ( KO ) , HME control ( vec ) OAS double - triple - knockout ( DKOs TKO ) , 24 h after treatment with 1000 μM H2O2 . Data information : Nuclei were visualized by DAPI . Arrowheads : AIF translocated to nuclei . Untreated control are shown on 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 ) ;

O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 of AIF accumulation in nuclei of PC3 DKO2 / 3 PC3 DKO2 / 3 + 1 , 24 h after treatment with 100 , 200 or 400 μM H2O2 . Data information : Nuclei were visualized by DAPI . Arrowheads : AIF translocated to nuclei . Untreated control are shown on 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 of each genotype as assessed by Student ' s t - . Scale bars , 10μm .

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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 - 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 S2 ) were monitored . empty vector was transfected as a control ( Ctrl ) .

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(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 phosphorylated STAT1 at Tyr701 ( pY - STAT1 ) in whole extracts ( C , D ) fraction ( E , F ) measured by Western blotting ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney ) . * , p < 0 . 05 vs Ctrl .

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

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(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 dimer formation in fraction ( Nu ) measured by Western blotting .

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(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 detected by luciferase ( n = 4 ) . Data information : Data were presented as mean ± SD ( Mann - Whitney ) . * , p < 0 . 05 , * * * , p < 0 . 001 vs Ctrl .

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(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 - binding activity in HEK293 measured by electrophoretic mobility shift ( EMSA ) . * , indicates STAT1 / .

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(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 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 , increased of STAT1 pY - STAT1 in hippocampal total extracts fraction were detected in hTau group by Western blotting ( n = 6 , Mann - Whitney ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

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

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(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 ) representative of STAT1 pY - STAT1 in brain of AD probed by co - immunohistochemical quantitative ( hematoxylin stains nuclei , purple ; DAB stains target , brown ; n = 5 - 6 slices ) . Arrowheads indicated typical 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 .

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

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(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 ) . Data information : Data were presented as mean ± SD . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP , wt or Ctrl .

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(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 hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by immunohistochemical .

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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 hippocampal CA3 of 3 - month - old STAT1flox / flox mice . One month later , downregulation of STAT1 was confirmed by Western

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

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

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(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 ( 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 .

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(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 infection , paired - ( PPR ) was recorded in hippocampal CA3 of hTau or STAT1 knockdown mice ( I ) . IO curve of fEPSP recorded on acute hippocampal slices ( J ) . slope of fEPSP after HFS recorded on hippocampal slices of hTau or STAT1 knockdown mice ( K ) . LTP magnitude was calculated as average ( normalized to baseline ) of 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 of variance ( ANOVA ) followed by Bonferroni ' s post hoc for I - K , one - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc for others ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP ; # , p < 0 . 05 , # # # , p < 0 . 001 vs hTau .

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

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

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(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 mRNA of GluN1 , GluN2A GluN2B detected by qRT - PCR in hippocampal CA3 . Data information : Data were presented as mean ± SD ( n = 4 ; Mann - Whitney ) . * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau .

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

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

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

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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 predicted promoter element ( GASs ) for STAT1 in promoter ( - 2000 - + 299bp ) of GluN2B ( E ) GASs or mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 for 24 h , then luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , 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 predicted promoter element ( GASs ) for STAT1 in promoter ( - 2000 - + 299bp ) of GluN2A ( G , I ) . GASs or mutant ( MUT ) plasmids were co - transfected respectively with WT - STAT1 or its empty vector ( Ctrl ) into HEK293 for 24 h , then luciferase activity was measured ( right panels ) . N = 4 for each group . Data information : Data were presented as mean ± SD ( two - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs Ctrl ; # # # , p < 0 . 001 vs wildtype reporters .

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

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(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 hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts ( C fraction ( D measured by Western blotting ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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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 hTau - induced STAT1 phosphorylation at pY - STAT1 ( Tyr701 ) in total extracts E ) fraction F ) measured by Western blotting ( n = 3 ) . alteration of pS - STAT1 ( Ser727 ) confirms efficacy of JNK1 inhibitors . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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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 fraction ( H ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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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 ) fraction J ) ( n = 3 ) . Data information : Data were presented as mean ± SD , * , p < 0 . 05 vs Ctrl , eGFP or WT .

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

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(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 memory were detected by MWM . ( A ) representative image confirms expression of AAV - hTau AAV - Y701F - STAT1 . Scale bar , 200 μm , or 100 μm for enlarged .

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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 memory were detected by MWM . ( B ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial learning deficits shown by 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 of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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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 memory were detected by MWM . ( C - E ) Overexpression of Y701F - STAT1 mitigated hTau - induced spatial memory impairment shown by decreased latency to reach platform ( C ) , increased crossing time in platform site ( D ) time spent in target quadrant ( E ) measured at day 6 by removed platform ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m , two - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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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 memory were detected by MWM . ( F ) Expression of Y701F - STAT1 did not change swimming speed of mice in water maze task ( n = 7 - 10 each group ) . Data information : Data were presented as mean ± s . e . m two - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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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 average ( normalized to baseline ) of 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 of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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(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.


Simultaneous expression of Y701F - STAT1 rescued hTau - induced suppression of NMDARs expression measured by Western blotting in hippocampal CA3 of C57 mice ( n = 4 ) .

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Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4).


Simultaneous expression of Y701F - STAT1 rescued hTau - induced suppression of NMDARs ( K ) expression measured by Western blotting in hippocampal CA3 of C57 mice ( n = 4 ) . Data information : Data were presented as mean ± SD two - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein (K) expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4). 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.


Simultaneous expression of Y701F - STAT1 rescued hTau - induced suppression of NMDARs mRNA expression measured by qRT - PCR in hippocampal CA3 of C57 mice ( n = 4 ) . Data information : Data were presented as mean ± SD , two - way of variance ( ANOVA ) followed by Bonferroni ' s post hoc * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 vs eGFP or hTau ; # , p < 0 . 05 , # # , p < 0 . 01 vs hTau .

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Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs mRNA expression measured by qRT-PCR in hippocampal CA3 of C57 mice (n=4). 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.


( E - F ) Serum of T ( E ) LH ( F ) in WT , ARLmon / Y AR - / Y mice at of 13 .

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(E-F) Serum levels of T (E) and LH (F) in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.


( D ) RT - qPCR analyses of Rhox5 Insl3 of testes from 13 - week old WT , ARLmon / Y AR - / Y . Expression are normalized to WT .

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(D) RT-qPCR analyses of Rhox5 and Insl3 of testes from 13-week old WT males, ARLmon/Y and AR-/Y. Expression levels are normalized to WT.


( A ) Expression of genes involved in steroidogenesis normalized to WT . bar graphs show means ± ( data derived from - seq of testes , biological replicates , n = 5 , one - way ANOVA with Tukey ' s multiple comparisons , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns = not significant ) .

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(A) Expression levels of genes involved in steroidogenesis normalized to WT. The bar graphs show means ± SEM (data derived from RNA-seq analysis of testes, biological replicates, n = 5, one-way ANOVA with Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant).


( B ) Serum of A - dione in WT , ARLmon / Y AR - / Y mice at of 13 .

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(B) Serum levels of A-dione in WT males, ARLmon/Y and AR-/Y mice at the age of 13 weeks.


( D ) Reporter gene assays in Hela co - transfected with mouse Insl3 promotor together with WT or mutant mouse AR .

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(D) Reporter gene assays in Hela cells co-transfected with the mouse Insl3 promotor together with the WT or mutant mouse AR.


mRNA expression extracted from - seq data for Fkbp5 ( D ) in kidneys used for - seq . Expression are normalized to WT ORX .

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mRNA expression levels extracted from RNA-seq data for Fkbp5 (D) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.


mRNA expression extracted from - seq data for , Odc1 ( E ) Kap ( F ) in kidneys used for - seq . Expression are normalized to WT ORX .

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mRNA expression levels extracted from RNA-seq data for , Odc1 (E) and Kap (F) in kidneys used for RNA-seq. Expression levels are normalized to WT ORX.


( B ) Specific ligand binding in COS with overload of cold ligand increasing concentrations of radioactive labeled DHT . Values are normalized to expression .

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(B) Specific ligand binding assay in COS cells with overload of cold ligand and increasing concentrations of radioactive labeled DHT. Values are normalized to protein expression.


( C ) Western blot on , whole extracts derived from Hela transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT . Both panels represent same blot . blot was cut in two parts antibodies against HSP90 Lamin A / C were used to confirm fractionation of . For AR visualization ( upper panel ) , longer exposure time was used . # = residual AR expression ( lower panel ) .

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(C) Western blot on cytoplasmic, nuclear and whole cell extracts derived from Hela cells transfected with human WT or W752R AR followed by stimulation with increasing concentrations of DHT. Both panels represent the same blot. The blot was cut in two parts and antibodies against HSP90 and Lamin A/C were used to confirm cellular fractionation of cytoplasmic and nuclear proteins. For AR visualization (upper panel), longer exposure time was used. # = residual AR expression (lower panel).


( B ) AR occupancy ( blue ) of histone marks H3K27ac ( green ) H3K27me3 ( red ) at ARBS within regulatory regions of Fkbp5 , Kap ( both androgen - regulated in kidney ) Tox3 ( androgen - regulated in prostate but not in kidney hence used as a negative control ) were assessed by ChIP - qPCR in kidneys from 13 - week - old ARLmon / Y mice WT .

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(B) AR occupancy (blue) and levels of the histone marks H3K27ac (green) and H3K27me3 (red) at ARBS within the regulatory regions of Fkbp5, Kap (both androgen-regulated in kidney) and Tox3 (androgen-regulated in prostate but not in kidney and hence used as a negative control) were assessed by ChIP-qPCR in kidneys from 13-week-old ARLmon/Y mice and WT males.


( C ) Representative pictures of immunofluorescence AR ( green ) in kidneys of castrated WT ARLmon / Y mice supplemented with vehicle or T . Nuclei are shown in blue .

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(C) Representative pictures of immunofluorescence AR staining (green) in kidneys of castrated WT males and ARLmon/Y mice supplemented with vehicle or T. Nuclei are shown in blue.


( A ) Double hybrid in COS transfected with WT LBD , W752R LBD or empty vector ( negative control ) in presence of LxxLL fragment of SRC1 .

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(A) Double hybrid assay in COS cells transfected with WT LBD, W752R LBD or empty vector (negative control) in the presence of the LxxLL fragment of SRC1.


( B ) Confocal microscopy of AR - WT - BirA * AR W752R - BirA * expressing HEK293 treated in presence of 50 µM biotin with 100 nM DHT or vehicle ( ethanol ) with or without 0 . 03 µg / ml TET as indicated . AR - BirA * s were detected with anti - AR ( red ) biotinylated with fluorescently labeled streptavidin ( green ) . Nuclei were visualized using DAPI .

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(B) Confocal fluorescence microscopy images of AR-WT-BirA* and AR W752R-BirA*expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle (ethanol) and with or without 0.03 µg/ml TET as indicated. AR-BirA*s were detected with anti-AR (red) and biotinylated proteins with fluorescently labeled streptavidin (green). Nuclei were visualized using DAPI.


( A ) degradation of 3HA - Pgc1 was analyzed in with indicated genotype upon inhibition of synthesis with cycloheximide ( chx ) . A plasmid - borne 3HA - Pgc1 expressed from endogenous promoter was used . 3HA - Pgc1 was detected with anti - HA antibodies . Kar2 was used as a loading control detected with anti - Kar2 antibodies . graph on right shows average of four independent experiments ; error bars represent standard deviation .

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(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype upon inhibition of protein synthesis with cycloheximide (chx). A plasmid-borne 3HA-Pgc1 expressed from the endogenous promoter was used. 3HA-Pgc1 was detected with anti-HA antibodies. Kar2 was used as a loading control and detected with anti-Kar2 antibodies. The graph on the right shows the average of four independent experiments; error bars represent the standard deviation.


( B ) ubiquitination of 3HA - Pgc1 was analyzed in with indicated genotype expressing myc - tagged ubiquitin . 3HA - Pgc1 was immunoprecipitated using anti - HA polyubiquitin - conjugated 3HA - Pgc1 was detected using anti - myc antibody . Input fraction corresponds to 2 % of total amount used for IP .

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(B) The ubiquitination of 3HA-Pgc1 was analyzed in cells with the indicated genotype and expressing myc-tagged ubiquitin. 3HA-Pgc1 was immunoprecipitated using anti-HA and polyubiquitin-conjugated 3HA-Pgc1 was detected using anti-myc antibody. Input fraction corresponds to 2% of the total amount used for IP.


( C ) degradation of 3HA - Pgc1 was analyzed as in ( A ) in bearing CDC48 - sensitive allele cdc48 - 6 . were grown 2 , 5 hours at 37C prior addition of cycloheximide . Inactivation of Cdc48 mutant was confirmed by stabilization of ERAD substrate Erg1 in same .

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(C) The degradation of 3HA-Pgc1 was analyzed as in (A) in cells bearing the CDC48 temperature-sensitive allele cdc48-6. Cells were grown 2,5 hours at 37C prior addition of cycloheximide. Inactivation of Cdc48 mutant protein was confirmed by stabilization of the ERAD substrate Erg1 in the same cells.