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 .

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

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

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 .

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

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

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 .

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

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 .

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

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 .

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

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

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

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

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

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 .

B-CELL_LINE O 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 O B-EXP_ASSAY B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O 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 ) 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 .

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

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 .

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

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 .

B-CELL_LINE O O 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 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 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 .

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

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-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

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

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 B-GENEPROD O O O B-SUBCELLULAR O B-CELL_LINE 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 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 O B-GENEPROD O O B-SUBCELLULAR O O 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 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 B-GENEPROD O O O B-SUBCELLULAR O B-CELL_LINE 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 B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SUBCELLULAR O O 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 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 ) .

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

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

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

(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 .

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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 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 .

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

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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

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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 the 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 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 .

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

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

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and the nuclear 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 ) and the nuclear 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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the hTau - induced suppression of NMDARs protein 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 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 .

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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 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 .

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

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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 and Insl3 of testes from 13 - week old WT males , ARLmon / Y and AR - / Y . Expression levels 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 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 ) .

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

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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 cells co - transfected with the mouse Insl3 promotor together with the 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 levels extracted from RNA - seq data for Fkbp5 ( D ) in kidneys used for RNA - seq . Expression levels 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 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 .

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

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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 ) 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 .

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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 staining ( green ) in kidneys of castrated WT males and 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 assay in COS cells transfected with WT LBD , W752R LBD or empty vector ( negative control ) in the presence of the 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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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.


( D ) Membrane association of ubiquitinated 3HA - Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation . Cells of the indicated genotype and expressing myc - tagged ubiquitin were grown as in ( C ) . Lysates were subjected to centrifugation on an optiprep density gradient . Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti - HA antibodies . Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively . Input fraction corresponds to 10 % of the total amount used for IP . " Top " , " mid " , " bottom " indicate the fractions from the gradient . Asterisk marks a faster migrating unspecific band in the input bottom fraction .

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(D) Membrane association of ubiquitinated 3HA-Pgc1 was analyzed by density gradient centrifugation followed by immunoprecipitation. Cells of the indicated genotype and expressing myc-tagged ubiquitin were grown as in (C). Lysates were subjected to centrifugation on an optiprep density gradient. Collected fractions were analyzed by western blotting before and after immunoprecipitation with anti-HA antibodies. Dpm1 and Pgk1 were used as markers for membrane bound and soluble fractions respectively. Input fraction corresponds to 10% of the total amount used for IP. "Top", "mid", "bottom" indicate the fractions from the gradient. Asterisk marks a faster migrating unspecific band in the input bottom fraction.


( A ) Localization of chromosomally expressed GFP - Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells . Arrowheads indicate GFP - Pgc1 labeling at the ER , which is stained by Sec63 - Cherry . LDs were visualized upon staining with the neutral lipid dye MDH . On the bottom , box plot with quantitation of GFP - Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ . GFP - Pgc1 measurements were performed as described in the Materials and Methods section . Scale bar : 5 µm .

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(A) Localization of chromosomally expressed GFP-Pgc1 from the constitutive ADH1 promoter in wt and doa10Δ cells. Arrowheads indicate GFP-Pgc1 labeling at the ER, which is stained by Sec63-Cherry. LDs were visualized upon staining with the neutral lipid dye MDH. On the bottom, box plot with quantitation of GFP-Pgc1 fluorescence intensity at the nuclear envelope in wt and doa10Δ. GFP-Pgc1 measurements were performed as described in the Materials and Methods section. Scale bar: 5 µm.


( C ) Crude membranes from wt and doa10Δ cells expressing 3HA - Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet ( P ) and supernatant ( S ) . ( D ) Crude membranes from wt and doa10Δ cells expressing 3HA - GFP - Pgc1275 - 321 were analyzed as in ( C ) .

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(C) Crude membranes from wt and doa10Δ cells expressing 3HA-Pgc1 were subjected to the indicated treatments and subsequently fractionated into membrane pellet (P) and supernatant (S).(D) Crude membranes from wt and doa10Δ cells expressing 3HA-GFP-Pgc1275-321 were analyzed as in (C).


( A ) Localization of GFP - Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence ( no LDs ) or absence of DOA10 ( no LDs doa10Δ ) . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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(A) Localization of GFP-Pgc1 in are1Δ are2Δ lro1Δ dga1Δ cells in presence (no LDs) or absence of DOA10 (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( B ) GFP - Pgc1 was expressed under the ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δ . Fluorescence microscopy was used to follow GFP - Pgc1 localization over time . LDs were visualized upon staining with the neutral lipid dye MDH . Scale bar : 5 µm .

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(B) GFP-Pgc1 was expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δ. Fluorescence microscopy was used to follow GFP-Pgc1 localization over time. LDs were visualized upon staining with the neutral lipid dye MDH. Scale bar: 5 µm.


( C ) Photo - conversion of tdEOS - Pgc1 expressed under the ADH1 promoter ; LD formation was stimulated by galactose - induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells . The red square marks the photo - converted region . The time ( in minutes ) after photoconversion is indicated . Arrowheads point LDs containing photoconverted tdEOS - Pgc1 . Scale bar 5µm .

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(C) Photo-conversion of tdEOS-Pgc1 expressed under the ADH1 promoter; LD formation was stimulated by galactose-induced expression of DGA1 in are1Δare2Δlro1Δdoa10Δ cells. The red square marks the photo-converted region. The time (in minutes) after photoconversion is indicated. Arrowheads point LDs containing photoconverted tdEOS-Pgc1. Scale bar 5µm.


( D ) FRAP experiment of GFP - Pgc1 in wt and doa10Δcells . Representative examples are shown . The bleached areas are marked by red squares and include a LD adjacent to ER . The time ( in seconds ) after photobleaching is indicated . Each graph shows average fluorescence intensities for 10 cells normalized to pre - bleached plotted over time . Error bars indicate standard deviation .

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(D) FRAP experiment of GFP-Pgc1 in wt and doa10Δcells. Representative examples are shown. The bleached areas are marked by red squares and include a LD adjacent to ER. The time (in seconds) after photobleaching is indicated. Each graph shows average fluorescence intensities for 10 cells normalized to pre-bleached plotted over time. Error bars indicate standard deviation.


( A ) The degradation of 3HA - Pgc1 was analyzed in cells with the indicated genotype as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(A) The degradation of 3HA-Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) The degradation of the Doa10 substrates 3HA - Pgc1 and 3HA - Vma12 - Ndc10C ' was analyzed in cells of the indicated genotypes treated with oleic acid . 3HA - Pgc1 and 3HA - Vma12 - Ndc10C ' were detected with anti - HA antibodies . Dpm1 was used as a loading control and detected with anti - Dpm1 antibodies . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(B) The degradation of the Doa10 substrates 3HA-Pgc1 and 3HA-Vma12-Ndc10C' was analyzed in cells of the indicated genotypes treated with oleic acid. 3HA-Pgc1 and 3HA-Vma12-Ndc10C' were detected with anti-HA antibodies. Dpm1 was used as a loading control and detected with anti-Dpm1 antibodies. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( A ) Localization of GFP - tagged Pgc1 derivatives in which the hydrophobic hairpin ( aa275 - 321 ) was replaced by membrane anchor of Scs2 ( Pgc1Scs2MA ) or Bos1 ( Pgc1Bos1MA ) . ER and LDs were visualized by expression of Sec63 - Cherry and MDH staining , respectively . Scale bar : 5 µm .

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(A) Localization of GFP-tagged Pgc1 derivatives in which the hydrophobic hairpin (aa275-321) was replaced by membrane anchor of Scs2 (Pgc1Scs2MA) or Bos1 (Pgc1Bos1MA). ER and LDs were visualized by expression of Sec63-Cherry and MDH staining, respectively. Scale bar: 5 µm.


( B ) The degradation of 3HA - Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig . 1A . The blot is representative of at least three independent experiments .

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(B) The degradation of 3HA-Pgc1 or the indicated chimeras in wt and doa10Δ cells was analyzed as in Fig. 1A. The blot is representative of at least three independent experiments.


( C ) The degradation of 3HA - GFP - Pgc1275 - 321 in wt and doa10Δ cells was analyzed as in Fig . 1A . The graph shows the average of four independent experiments ; error bars represent the standard deviation .

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(C) The degradation of 3HA-GFP-Pgc1275-321 in wt and doa10Δ cells was analyzed as in Fig. 1A. The graph shows the average of four independent experiments; error bars represent the standard deviation.


( D ) Localization of 3HA - GFP - Pgc1275 - 321 in doa10Δ cells . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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(D) Localization of 3HA-GFP-Pgc1275-321 in doa10Δ cells. The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


( A ) The degradation of 3HA - Pgc1 - GPAT160 - 216 , containing GPAT4 hydrophobic hairpin ( aa160 - 216 ) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(A) The degradation of 3HA-Pgc1-GPAT160-216, containing GPAT4 hydrophobic hairpin (aa160-216) replacing the one of Pgc1 was analyzed in cells with the indicated genotype as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( B ) Localization of GFP - Pgc1 - GPAT4160 - 216 in cells with the indicated genotype . Arrowheads indicate GFP - Pgc1 - GPAT4160 - 216 labeling at the ER , marked with Sec63 - Cherry . LDs were visualized upon staining with MDH . Scale bar : 5 µm .

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(B) Localization of GFP-Pgc1-GPAT4160-216 in cells with the indicated genotype. Arrowheads indicate GFP-Pgc1-GPAT4160-216 labeling at the ER, marked with Sec63-Cherry. LDs were visualized upon staining with MDH. Scale bar: 5 µm.


( C ) The degradation of 3HA - Pgc1 - GPAT160 - 216 in cells with the indicated genotype was analyzed as in Fig . 1A . The graph shows the average of three independent experiments ; error bars represent the standard deviation .

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(C) The degradation of 3HA-Pgc1-GPAT160-216 in cells with the indicated genotype was analyzed as in Fig. 1A. The graph shows the average of three independent experiments; error bars represent the standard deviation.


( D ) Localization of GFP - Pgc1 - GPAT160 - 216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant ( no LDs doa10Δ ) . The ER was visualized by expression of Sec63 - Cherry . Scale bar : 5 µm .

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(D) Localization of GFP-Pgc1-GPAT160-216 in are1Δ are2Δ lro1Δ dga1Δ doa10Δ mutant (no LDs doa10Δ). The ER was visualized by expression of Sec63-Cherry. Scale bar: 5 µm.


B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young ( 2 months ) and old ( 22 months ) mice ( n = 3 mice per group ) . Scale bar 200 µm .

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B Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lungs from young (2 months) and old (22 months) mice (n = 3 mice per group). Scale bar 200 µm.


C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young ( 20 - 35 years old ) and old ( 60 - 80 years old ) humans ( n = 4 - 7 individuals per group ) . Scale bar , 200 µm .

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

C Representative microphotographs and quantitative analyses of ACE2 immunohistochemical staining in lung parenchyma of young (20-35 years old) and old (60-80 years old) humans (n = 4-7 individuals per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs from young and old mice ( n = 3 - 6 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs from young and old mice (n = 3-6 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


E Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1 - positive and CD31 - positive endothelia in lungs from young and old humans ( n = 4 - 7 individuals per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

E Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic TTF1-positive and CD31-positive endothelia in lungs from young and old humans (n = 4-7 individuals per group). Scale bar, 100 µm. a.u. = arbitrary units.


C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 200 µm .

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

C Representative images and quantitative analyses of ACE2 immunohistochemical staining in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 200 µm.


D Double - marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro - SP - C - positive and CD31 - positive endothelia in lungs of age - matched wild type and G3 Terc - / - mice ( n = 3 - 4 mice per group ) . Scale bar , 100 µm . a . u . = arbitrary units .

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

D Double-marker immunofluorescence and quantitative analyses of ACE2 intensity level in type II pneumocytic pro-SP-C-positive and CD31-positive endothelia in lungs of age-matched wild type and G3 Terc-/- mice (n = 3-4 mice per group). Scale bar, 100 µm. a.u. = arbitrary units.


A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F / F at the indicated time points following 4OHT treatment and consequent TRF2 knockout . Scale bar , 25 µm . B RT - qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F / F treated as in A ( n = 3 independent experiments ) .

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

A Immunofluorescence showing gammaH2AX foci in MEFs Trf2F/F at the indicated time points following 4OHT treatment and consequent TRF2 knockout. Scale bar, 25 µm. B RT-qPCR detection of Ace2 mRNA expression levels in MEFs Trf2F/F treated as in A (n = 3 independent experiments).


C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown . Scale bar , 25 µm . D RT - qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C ( n = 6 independent experiments ) .

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

C Immunofluorescence showing gammaH2AX foci in HeLa shTRF2 cells at the indicated time points following doxycycline treatment and consequent TRF2 knockdown. Scale bar, 25 µm. D RT-qPCR detection of ACE2 mRNA expression levels in HeLa shTRF2 cells treated as in C (n = 6 independent experiments).


E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F / F mice treated with tamoxifen ( to induce TRF2 loss and telomere uncapping ) or vehicle . The animals as been injected also with PBS . Scale bar , 10 µm . F RT - qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E ( n = 5 - 7 mice per group ) .

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

E Representative immunofluorescence images of 53BP1 staining in liver from Trf2F/F mice treated with tamoxifen (to induce TRF2 loss and telomere uncapping) or vehicle. The animals as been injected also with PBS. Scale bar, 10 µm. F RT-qPCR detection of Ace2 mRNA expression levels in livers of mice treated as in E (n = 5-7 mice per group).


B Relative luciferase activity in HeLa shTRF2 following ionizing radiation ( 5 Gy ) or TRF2 knockdown upon doxycycline - induced shTRF2 expression ( n = 3 independent experiments ) . Error bars represent the s . e . m . * P < 0 . 05 . Two - way paired ANOVA .

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

B Relative luciferase activity in HeLa shTRF2 following ionizing radiation (5 Gy) or TRF2 knockdown upon doxycycline-induced shTRF2 expression (n=3 independent experiments). Error bars represent the s.e.m. *P<0.05. Two-way paired ANOVA.