Effects of thioperamide on cholesterol - dependent transcriptional regulation . parental HeLa MZ , NPC1 KO or NPC2 KO were treated or not with thioperamide 10 μM for 18h . Total mRNA was extracted both LDLR mRNA HMG CoA reductase mRNA were quantified by RT - PCR . ( n = 3 independent experiments , error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 )

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Effects of thioperamide on cholesterol-dependent transcriptional regulation. The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h. Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT-PCR. (n=3 independent experiments, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005)


of cholesterol in NPC null mice by enzymatic . WT or NPC1 null mice were treated or not with thioperamide for 6 after weaning sacrificed . total content of unesterified cholesterol in corresponding liver extracts was then quantified using an enzymatic is expressed in nmol per mg tissue . total content of LBPA in same liver extracts was quantified by mass spectrometry ( see Appendix Figure S1 ) , is therefore expressed as a of total phospholipids . total LBPA content of WT mice was only marginally increased by drug , either because doses were low , or because contribution from tissue material unaffected by drug obscured selective changes at level ( despite much effort , we were unable to visualize LBPA in liver samples by immunocytochemistry ) . relative of LBPA vs . cholesterol values is shown .

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Quantification of cholesterol in NPC null mice by enzymatic analysis. WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed. The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue. The total content of LBPA in the same liver extracts was quantified by mass spectrometry (see Appendix Figure S1), and is therefore expressed as a percentage of total phospholipids. The total LBPA content of WT mice was only marginally increased by the drug, either because the doses were low, or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level (despite much effort, we were unable to visualize LBPA in liver samples by immunocytochemistry). The relative ratio of LBPA vs. cholesterol values is shown.


Silver of components in Ago3 immunoisolated from BmN4 . P150 P130 co - immunoprecipitated with Ago3 . n . i . : non - immune IgG .

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Silver staining of protein components in the Ago3 complex immunoisolated from BmN4 cells. P150 and P130 co-immunoprecipitated with Ago3. n.i.: non-immune IgG.


Western blots showing presence of Vret - L ( P150 ) Vret - S ( P130 ) in Ago3 in A . n . i . : non - immune IgG .

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Western blots showing the presence of Vret-L (P150) and Vret-S (P130) in the Ago3 complex in A. n.i.: non-immune IgG.


Top : western blot showing Vret - L Vret - S immunoprecipitated from BmN4 upon UV irradiation . Bottom : CLIP signals showing that Vret - L , but not Vret - S , binds RNAs in vivo .

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Top: western blot showing Vret-L and Vret-S immunoprecipitated from BmN4 cells upon UV irradiation. Bottom: CLIP signals showing that Vret-L, but not Vret-S, binds RNAs in vivo.


Five upper panels : Northern blots showing of RT3 - piRNA , Bmmar6 - piRNA , R2Bm - piRNA , R1Bm - piRNA in normal ( Control ) , Vret - L / S - depleted ( Vret KD ) , Vret - L - depleted ( Vret - L KD ) , Vret - S - depleted ( Vret - S KD ) . Bantam ( miRNA ) was detected as a loading control . Two lower panels : western blots showing efficiency of Vret knockdown . β - Tub was detected as a loading control .

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Five upper panels: Northern blots showing the levels of RT3-piRNA, Bmmar6-piRNA, R2Bm-piRNA, and R1Bm-piRNA in normal (Control), Vret-L/S-depleted (Vret KD), Vret-L-depleted (Vret-L KD), and Vret-S-depleted (Vret-S KD) cells. Bantam (miRNA) was detected as a loading control. Two lower panels: western blots showing the efficiency of the Vret knockdown. β-Tub was detected as a loading control.


Two upper panels : western blots showing abundances of Flag - Vret - L Ago3 in both samples . Note that Flag - Vret - L is present only in Vret - L but not with Ago3 isolated from total lysates ( Fig EV1F ) . Bottom panel : Northern blot showing level of PiggyBac - piRNA associated with Ago3 in total lysate ( Ago3 - IP ) in Vret - L ( Flag - Vret - IP ) . of piRNA signal in two samples is indicated at bottom .

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Two upper panels: western blots showing the abundances of Flag-Vret-L and Ago3 in both samples. Note that Flag-Vret-L is present only in the Vret-L complex but not with Ago3 isolated from total cell lysates (Fig EV1F). Bottom panel: Northern blot showing the level of PiggyBac-piRNA associated with Ago3 in total lysate (Ago3-IP) and in the Vret-L complex (Flag-Vret-IP). The ratio of the piRNA signal intensity in the two samples is indicated at the bottom.


Two upper panels : western blots showing abundances of Flag - Vret - L Siwi in both samples . Bottom panel : Northern blot showing level of RT3 - piRNA associated with Siwi in total lysate ( Siwi - IP ) in Vret - L ( Flag - Vret - IP ) .

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Two upper panels: western blots showing the abundances of Flag-Vret-L and Siwi in both samples. Bottom panel: Northern blot showing the level of RT3-piRNA associated with Siwi in the total lysate (Siwi-IP) and in the Vret-L complex (Flag-Vret-IP).


Boxplot showing log2 fold - change of Vret - KD ( RPM ) / Control ( RPM ) values of piRNAs in increased , unchanged decreased groups defined by Ago3 - KD ( RPM ) / Control ( RPM ) values shown in Fig EV1I . Boxplot represents median , first third quartile , maximum minimum values of log2 fold - change of piRNAs ( N = 2 ) . Outliers are represented as open circles .

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Boxplot showing log2 fold-change of Vret-KD (RPM)/Control (RPM) values of piRNAs in increased, unchanged and decreased groups defined by Ago3-KD (RPM)/Control (RPM) values shown in Fig EV1I. Boxplot represents median, first and third quartile, and maximum and minimum values of the log2 fold-change of piRNAs (N = 2). Outliers are represented as open circles.


Flag - EGFP - Vret - L WT mutants were immunoprecipitated using anti - Flag antibody then western blotting was performed using anti - Flag , anti - Siwi , anti - Ago3 anti - Vret antibodies . domain structures of Tud1mut ( Y753A / Y757A / N759A ) Tud2mut ( Y973A / Y976A / N978A ) are shown .

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Flag-EGFP and -Vret-L WT and mutants were immunoprecipitated using the anti-Flag antibody and then western blotting was performed using anti-Flag, anti-Siwi, anti-Ago3 and anti-Vret antibodies. The domain structures of Tud1mut (Y753A/Y757A/N759A) and Tud2mut (Y973A/ Y976A/N978A) are shown.


Immunofluorescence shows that Vret signals ( green ) co - localize with Ago3 signals ( red ) . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . three panels on right - side show high magnification of part indicated by dashed box in image on left - side . This panel arrangement applies also for panels B − H . bar chart shows of Vret - positive nuage in Ago3 - positive nuage . Immunofluorescence shows that Vret signals ( green ) partially co - localize with Siwi signals ( red ) . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . bar chart shows of Vret - positive nuage in Siwi - positive nuage .

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Immunofluorescence shows that the Vret signals (green) co-localize with the Ago3 signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the image on the left-hand side. This panel arrangement applies also for panels B−H. The bar chart shows the percentage of Vret-positive nuage in Ago3-positive nuage. Immunofluorescence shows that the Vret signals (green) partially co-localize with the Siwi signals (red). DAPI shows the nuclei (blue). Scale bar: 10 μm. The bar chart shows the percentage of Vret-positive nuage in Siwi-positive nuage.


Immunofluorescence showing Ago3 signals ( green ) in control Vret - depleted BmN4 ( Vret KD ) . Vret is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing Siwi signals ( green ) in control Vret - depleted BmN4 ( Vret KD ) . Vret is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing the Ago3 signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (green) in control and Vret-depleted BmN4 cells (Vret KD). Vret is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing Vret signals ( red ) in control Ago3 - depleted BmN4 ( Ago3 KD ) . Ago3 is shown in green . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing Siwi signals ( red ) in control Ago3 - depleted BmN4 ( Ago3 KD ) . Ago3 is shown in green . DAPI shows nuclei ( blue ) . Scale bar : 10 μm .

Immunofluorescence showing the Vret signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing the Siwi signals (red) in control and Ago3-depleted BmN4 cells (Ago3 KD). Ago3 is shown in green. DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing Flag - Ago3 WT , KA mutant DDH mutant signals ( green ) expressed in naïve BmN4 ( Control ) . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . Immunofluorescence showing Flag - Ago3 WT , KA mutant DDH mutant signals ( green ) in Ago3 - depleted ( Ago3 KD ) . DAPI shows nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) expressed in naïve BmN4 cells (Control). DAPI shows the nuclei (blue). Scale bar: 10 μm. Immunofluorescence showing Flag-Ago3 WT, KA mutant and DDH mutant signals (green) in Ago3-depleted cells (Ago3 KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing Ago3 signals ( green ) in control Siwi - depleted BmN4 ( Siwi KD ) . Siwi is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . three panels on right - side show high magnification of part indicated by dashed box in on left - side . This panel arrangement applies also for panels B , H I . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 106 ; Small : IntDen value < 106 ) . Immunofluorescence showing Vret signals ( green ) in control Siwi - depleted BmN4 ( Siwi KD ) . Siwi is shown in red . DAPI shows nuclei ( blue ) . Scale bar : 10 μm . Bar chart indicates dot sizes of > 100 Ago3 particles in each case ( Large : IntDen value > 105 ; Small : IntDen value < 105 ) .

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Immunofluorescence showing the Ago3 signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. The three panels on the right-hand side show high magnification images of the part indicated by the white dashed line box in the images on the left-hand side. This panel arrangement applies also for panels B, H and I. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 106; Small: IntDen value < 106). Immunofluorescence showing the Vret signals (green) in control and Siwi-depleted BmN4 cells (Siwi KD). Siwi is shown in red. DAPI shows the nuclei (blue). Scale bar: 10 μm. Bar chart indicates dot sizes of > 100 Ago3 particles in each case (Large: IntDen value > 105; Small: IntDen value < 105).


Western blotting was performed before after Siwi KD using anti - Ago3 antibody . Total BmN4 lysate ( Total ) , supernatant ( Sup ) pellet ( Pellet ) fractions upon centrifugation were used .

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Western blotting was performed before and after Siwi KD using the anti-Ago3 antibody. Total BmN4 lysate (Total), supernatant (Sup) and pellet (Pellet) fractions upon centrifugation were used.


Western blotting was performed using anti - Ago3 antibody on Ago3 immunoisolated from Siwi - depleted . Ago3 immunoprecipitated was untreated ( − ) or treated with phosphatase ( + ) prior to western botting .

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Western blotting was performed using the anti-Ago3 antibody on Ago3 immunoisolated from Siwi-depleted cells. Ago3 immunoprecipitated was untreated (−) or treated with phosphatase (+) prior to western botting.


Western blot showing degree of phosphorylation of Flag - Ago3 WT 8SA mutant in Siwi - depleted ( Siwi KD ) .

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Western blot showing the degree of phosphorylation of Flag-Ago3 WT and the 8SA mutant in Siwi-depleted cells (Siwi KD).


Western blots showing degree of phosphorylation of Flag - Ago3 WT its DDH KA mutants in Siwi - depleted ( Siwi KD ) . second panel from top corresponds to top panel with a longer exposure time . Siwi β - Tub as a loading control were also detected also by western blotting .

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Western blots showing the degree of phosphorylation of Flag-Ago3 WT and its DDH and KA mutants in Siwi-depleted cells (Siwi KD). The second panel from the top corresponds to the top panel with a longer exposure time. Siwi and β-Tub as a loading control were also detected also by western blotting.


Western blotting was performed using anti - Ago3 , anti - Vret anti - Siwi antibodies on lysates of BmN4 before ( Control ) after Siwi ( Siwi KD ) Siwi / Vret ( Siwi / Vret KD ) depletion . β - Tub was detected as a loading control .

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Western blotting was performed using anti-Ago3, anti-Vret and anti-Siwi antibodies on lysates of BmN4 cells before (Control) and after Siwi (Siwi KD) and Siwi/Vret (Siwi/Vret KD) depletion. β-Tub was detected as a loading control.


Immunofluorescence showing Flag - Ago3 signals ( red ) in normal ( Control ) Siwi - depleted BmN4 ( Siwi KD ) . DAPI shows nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing the Flag-Ago3 signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Immunofluorescence showing Flag - Ago3 8SA 8SE mutant signals ( red ) in normal ( Control ) Siwi - depleted BmN4 ( Siwi KD ) . DAPI shows nuclei ( blue ) . Scale bar : 10 μm .

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Immunofluorescence showing the Flag-Ago3 8SA and 8SE mutant signals (red) in normal (Control) and Siwi-depleted BmN4 cells (Siwi KD). DAPI shows the nuclei (blue). Scale bar: 10 μm.


Western blot showing Ago3 immunopurified from BmN4 before ( Control ) after Siwi knockdown ( Siwi KD ) . 32P - labeling of RNAs isolated from Ago3 ( a mixture of unphosphorylated phosphorylated ) immunopurified from Siwi - depleted . 15 - 20 - nt RNAs ( indicated by a black ) were isolated sequenced .

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Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). 32P-labeling of RNAs isolated from Ago3 (a mixture of unphosphorylated and phosphorylated) immunopurified from Siwi-depleted cells. 15-20-nt RNAs (indicated by a black line) were isolated and sequenced.


measurement of distances between 3 ′ - ends of Target - S ( 15 - 20 - nt RNAs in A ) 5 ′ - ends of Ago3 - bound piRNAs .

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The measurement of distances between the 3′-ends of Target-S (15-20-nt RNAs in A) and the 5′-ends of Ago3-bound piRNAs.


32P - labeling of RNAs isolated from Ago3 . Target - Ss are isolated from Siwi - depleted BmN4 but are not isolated from Siwi / Vret double knockdown .

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32P-labeling of RNAs isolated from Ago3. Target-Ss are isolated from Siwi-depleted BmN4 cells but are not isolated from Siwi/Vret double knockdown cells.


Western blot showing Ago3 immunopurified from BmN4 before ( Control ) after Siwi knockdown ( Siwi KD ) . Northern blot showing Bmmar6 - piRNA precursors in Ago3 . 50 - 100 - nt RNAs ( indicated by a black ) were isolated sequenced .

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Western blot showing Ago3 immunopurified from BmN4 cells before (Control) and after Siwi knockdown (Siwi KD). Northern blot showing Bmmar6-piRNA precursors in the Ago3 complex. 50-100-nt RNAs (indicated by a black line) were isolated and sequenced.


measurement of distances between 5 ′ - ends of Target - L 5 ′ - ends of Ago3 - bound piRNAs .

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The measurement of distances between the 5′-ends of Target-L and the 5′-ends of Ago3-bound piRNAs.


measurement of distances between 5 ′ - 3 ′ - ends of Target - L fragments on same genomic strand . Heat map displays 5 ′ - ends of Target - L in a window around most abundant 1 , 000 3 ′ - ends of Target - L . measurement of distances between 5 ′ - ends 3 ′ - ends of Target - S fragments on same genomic strand . heat map displays 5 ′ - ends of Target - S in a window around most abundant 1 , 000 3 ′ - ends of Target - S . measurement of distances between 5 ′ - ends of Target - L 3 ′ - ends of Target - S on same genomic strand . heat map displays 5 ′ - ends of Target - L in a window around most abundant 1 , 000 3 ′ - ends of Target - S . measurement of distances between 5 ′ - ends of Target - S 3 ′ - ends of Target - L on same genomic strand . heat map displays 5 ′ - ends of Target - S in a window around most abundant 1 , 000 3 ′ - ends of Target - L .

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

The measurement of distances between the 5′- and 3′-ends of Target-L fragments on the same genomic strand. Heat map displays 5′-ends of Target-L in a window around the most abundant 1,000 3′-ends of Target-L. The measurement of distances between the 5′-ends and 3′-ends of Target-S fragments on the same genomic strand. The heat map displays 5′-ends of Target-S in a window around the most abundant 1,000 3′-ends of Target-S. The measurement of distances between the 5′-ends of Target-L and the 3′-ends of Target-S on the same genomic strand. The heat map displays 5′-ends of Target-L in a window around the most abundant 1,000 3′-ends of Target-S. The measurement of distances between the 5′-ends of Target-S and the 3′-ends of Target-L on the same genomic strand. The heat map displays 5′-ends of Target-S in a window around the most abundant 1,000 3′-ends of Target-L.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


( F ) Representative widefield 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 O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

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

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


( A - D ) Representative widefield 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 .

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(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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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 .

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


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 .

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


Survival of HME , 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 - .

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

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 .

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


Western 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 .

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


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

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 .

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

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 .

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

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

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 - .

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

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 .

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

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


HME control ( vec ) 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 .

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

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 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O 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 ) 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 .

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

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


HME vec , KO1 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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 .

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

O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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

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


( I ) Overexpression of hTau increased STAT1 activity in HEK293 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 .

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

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


( C , D ) 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 .

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

(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 .

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

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


( H ) STAT1 mRNA was analyzed by qPCR in AAV - hTau or AAV - eGFP infected hippocampal tissues ( n = 6 , Mann - Whitney ) . 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

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

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


( C ) Downregulation of STAT1 ameliorated hTau - induced spatial learning deficit shown by 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 .

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

(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 .

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

(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 .

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

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