Figure 4b , qPCR validation of predicted direct miR379 - 410 targets using RNA from hippocampi of juvenile ( P 22 - 24 ) mice , except of Shank 3 where adult miR - 379 - 410 wt and ko animals were measured . If not indicated , both , male and female samples were used . Cnih2 ( wt n = 12 , ko n = 9 , t19 = 2 . 344 , * p = 0 . 0301 ) , Dlgap3 ( wt n = 12 , ko n = 9 , t19 = 2 . 128 , * p = 0 . 0467 ) , Prr7 ( male wt n = 7 , ko n = 4 , t9 = 2 . 719 , * p = 0 . 0236 ) , Src ( wt n = 12 , ko n = 9 , t19 = 2 . 410 , * p = 0 . 0263 ) , Lzts2 ( male wt n = 7 , ko n = 4 , t9 = 2 . 554 , * p = 0 . 0310 ) , Mpp2 ( male wt n = 7 , ko n = 4 , t9 = 2 . 369 , * p = 0 . 0420 ) , Prr12 ( wt n = 6 , ko n = 4 , t8 = 2 . 375 , * p = 0 . 0449 ) , Shank1 ( wt n = 11 , ko n = 9 , t18 = 2 . 310 , * p = 0 . 0329 ) , Shb ( male wt n = 6 , ko n = 4 , t8 = 3 . 451 , * * p = 0 . 0087 ) and Shank3 ( adult male wt n = 3 , ko n = 5 , t6 = 2 . 538 , * p = 0 . 0442 ) ; unpaired Student ` s t - test . Data are presented as mean ± s . e . m . c , d , Cnih2 , Prr7 and Src are direct miR - 379 - 410 target mRNAs . ( C ) 3 ' - UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3 ' UTR reporter genes ( wt or seed mutant ) together with miRNA mimics . Cnih2 ( n = 3 per group ; wt : * * p = 0 . 0020 , mut : p = 0 . 9267 ; mimic : F1 , 8 = 19 . 92 , p = 0 . 0021 ; utr - construct : F1 , 8 = 4 . 760 , p = 0 . 0607 ; mimic x utr - construct : F1 , 8 = 12 . 99 , p = 0 . 0069 ) , Prr7 ( n = 3 per group ; wt : * * * * p < 0 . 0001 , mut : * p = 0 . 0103 ; wt vs . mut mimic miR329 : * * p = 0 . 0014 ; mimic : F1 , 8 = 153 . 1 , p < 0 . 0001 ; utr - construct : F1 , 8 = 5 . 631 , p = 0 . 0450 ; mimic x utr - construct : F1 , 8 = 38 . 54 , p = 0 . 0003 ) ; Src ( n = 3 per group ; wt : * p = 0 . 0110 , mut : p = 0 . 4724 ; mimic : F1 , 8 = 16 . 99 , p = 0 . 0033 ; utr - construct : F1 , 8 = 0 . 6809 , p = 0 . 4332 ; mimic vs . utr - construct : F1 , 8 = 3 . 920 , p = 0 . 0831 ) ; two - way ANOVA . ( D ) Same as in ( C ) , but in the presence of antimiRs ( pLNAs ) in hippocampal neurons after 48h of PTX stimulation . Cnih2 ( n = 4 per group ; wt : p = 0 . 2688 ; mut : p = 0 . 8668 ; wt ctr vs . mut ctr : p = 0 . 6463 ; anti - miRNA : F1 , 12 = 3 . 629 , p = 0 . 0810 ; utr - construct : F1 , 12 = 0 . 7407 , p = 0 . 4063 ; anti - miRNA x utr - construct : F1 , 12 = 0 . 6679 , p = 0 . 4297 ) , Prr7 ( n = 4 per group ; wt : * p = 0 . 0123 ; mut : p = 0 . 9917 ; wt ctr vs . mut ctr : * p = 0 . 0152 ; anti - miRNA : F1 , 12 = 6 . 082 , p = 0 . 0297 ; utr - construct : F1 , 12 = 5 . 271 ; p = 0 . 0405 ; anti - miRNA x utr - construct : F1 , 12 = 8 . 212 , p = 0 . 0142 ) ; Src ( n = 3 per group ; wt : * p = 0 . 0370 ; mut : p = 0 . 3540 ; wt ctr vs . mut ctr : p = 0 . 9906 ; anti - miRNA : F1 , 12 = 13 . 43 , p = 0 . 0064 ; utr - construct : F1 , 12 = 0 . 5672 , p = 0 . 4730 ; anti - miRNA x utr - construct : F1 , 12 = 1 . 362 , p = 0 . 2768 ) ; two - way ANOVA . Data are presented as mean ± s . d .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4b, qPCR validation of predicted direct miR379-410 targets using RNA from hippocampi of juvenile (P 22-24) mice, except of Shank 3 where adult miR-379-410 wt and ko animals were measured. If not indicated, both, male and female samples were used. Cnih2 (wt n=12, ko n=9, t19=2.344, *p=0.0301), Dlgap3 (wt n=12, ko n=9, t19=2.128, *p=0.0467), Prr7 (male wt n=7, ko n=4, t9=2.719, *p=0.0236), Src (wt n=12, ko n=9, t19=2.410, *p=0.0263), Lzts2 (male wt n=7, ko n=4, t9=2.554, *p=0.0310), Mpp2 (male wt n=7, ko n=4, t9=2.369, *p=0.0420), Prr12 (wt n=6, ko n=4, t8=2.375, *p=0.0449), Shank1 (wt n=11, ko n=9, t18=2.310, *p=0.0329), Shb (male wt n=6, ko n=4, t8=3.451, **p=0.0087) and Shank3 (adult male wt n=3, ko n=5, t6=2.538, *p=0.0442); unpaired Student`s t-test. Data are presented as mean ± s.e.m.c, d, Cnih2, Prr7 and Src are direct miR-379-410 target mRNAs. (C) 3'-UTR luciferase reporter gene assays in cultured rat cortical neurons using the indicated 3'UTR reporter genes (wt or seed mutant) together with miRNA mimics. Cnih2 (n=3 per group; wt: **p=0.0020, mut: p=0.9267; mimic: F1,8=19.92, p=0.0021; utr-construct: F1,8=4.760, p=0.0607; mimic x utr-construct: F1,8=12.99, p=0.0069), Prr7 (n=3 per group; wt: ****p<0.0001, mut: *p=0.0103; wt vs. mut mimic miR329: **p=0.0014; mimic: F1,8=153.1, p<0.0001; utr-construct: F1,8=5.631, p=0.0450; mimic x utr-construct: F1,8=38.54, p=0.0003); Src (n=3 per group; wt: *p=0.0110, mut: p=0.4724; mimic: F1,8=16.99, p=0.0033; utr-construct: F1,8=0.6809, p=0.4332; mimic vs. utr-construct: F1,8=3.920, p=0.0831); two-way ANOVA. (D) Same as in (C), but in the presence of antimiRs (pLNAs) in hippocampal neurons after 48h of PTX stimulation. Cnih2 (n=4 per group; wt: p=0.2688; mut: p=0.8668; wt ctr vs. mut ctr: p=0.6463; anti-miRNA: F1,12=3.629, p=0.0810; utr-construct: F1,12=0.7407, p=0.4063; anti-miRNA x utr-construct: F1,12=0.6679, p=0.4297), Prr7 (n=4 per group; wt: *p=0.0123; mut: p=0.9917; wt ctr vs. mut ctr: *p=0.0152; anti-miRNA: F1,12=6.082, p=0.0297; utr-construct: F1,12=5.271; p=0.0405; anti-miRNA x utr-construct: F1,12=8.212, p=0.0142); Src (n=3 per group; wt: *p=0.0370; mut: p=0.3540; wt ctr vs. mut ctr: p=0.9906; anti-miRNA: F1,12=13.43, p=0.0064; utr-construct: F1,12=0.5672, p=0.4730; anti-miRNA x utr-construct: F1,12=1.362, p=0.2768); two-way ANOVA. Data are presented as mean ± s.d.


f1 ( a ) Hippocampal tissues obtained from Nrf2 ( − / − ) ( 6 months old , one male and two female ) or wild - type ( WT ) ( 11 months old , two male and one female ) mice were immunoblotted with a Nrf2 antibody , 12E8 , PHF1 , a monoclonal to total tau ( Tau5 ) or a polyclonal to total tau ( Tau ) antibody . The asterisk to the left of the Nrf2 blot designates the position at which a nonspecific band migrated . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( b ) Bar graphs represent the relative optical density of phosphorylated or total tau in the hippocampal tissues . n = 6 ( These data are based on the mouse hippocampi immunoblotted in Supplementary Fig . 1 ) . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * * P0 . 01 ; * * * P0 . 001 ) ( c , d ) Left panels show brain sections stained for tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( c ) and Ser396 / Ser404 ( PHF1 ) ( d ) . The area of delineated by dotted lines is enlarged and shown in the right corner of the image . Right panels shows the same slice stained with DAPI 4 ' , 6 - diamidino - 2 - phenylindole . Scale bar , 50 μm . DG , dentate gyrus ; Hi , dentate hilar area .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a) Hippocampal tissues obtained from Nrf2 (−/−) (6 months old, one male and two female) or wild-type (WT) (11 months old, two male and one female) mice were immunoblotted with a Nrf2 antibody, 12E8, PHF1, a monoclonal to total tau (Tau5) or a polyclonal to total tau (Tau) antibody. The asterisk to the left of the Nrf2 blot designates the position at which a nonspecific band migrated. The relative molecular masses (kDa) are indicated to the left of each blot. (b) Bar graphs represent the relative optical density of phosphorylated or total tau in the hippocampal tissues. n=6 (These data are based on the mouse hippocampi immunoblotted in Supplementary Fig. 1). Data shown are mean±s.e. and were analysed using Student's t-test. (**P0.01; ***P0.001)(c,d) Left panels show brain sections stained for tau phosphorylated at Ser262/Ser356 (12E8) (c) and Ser396/Ser404 (PHF1) (d). The area of delineated by dotted lines is enlarged and shown in the right corner of the image. Right panels shows the same slice stained with DAPI 4',6-diamidino-2-phenylindole. Scale bar, 50 μm. DG, dentate gyrus; Hi, dentate hilar area.


f2Brain cortical tissue obtained from Nrf2 ( − / − ) ( 6 months old , one male and two female ) or wild - type ( 11 months old , two male and one female ) mice were separated into sarkosyl - soluble ( SS ) and insoluble ( SI ) fractions as described in the Methods . The level of total tau was analysed by immunoblotting using a monoclonal antibody to total tau ( Tau5 ) . The relative molecular masses ( kDa ) are indicated to the left of each blot . Bar graph represents the relative optical density of sarkosyl - insoluble tau normalized with that of sarkosyl - soluble tau . n = 3 . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ) .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2Brain cortical tissue obtained from Nrf2 (−/−) (6 months old, one male and two female) or wild-type (11 months old, two male and one female) mice were separated into sarkosyl-soluble (SS) and insoluble (SI) fractions as described in the Methods. The level of total tau was analysed by immunoblotting using a monoclonal antibody to total tau (Tau5). The relative molecular masses (kDa) are indicated to the left of each blot. Bar graph represents the relative optical density of sarkosyl-insoluble tau normalized with that of sarkosyl-soluble tau. n=3. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05).


f3 ( a ) Primary cortical neurons ( DIV 5 ) were treated with SFN ( 10 μM , SFN ) for 12 or 24 h . ( c , d ) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) . ( b ) Stably transfected CN1 . 4 cortical neurons were maintained in the presence of doxycycline ( 1 μg ml − 1 ) to induce the expression of tau for 24 h , and subsequently treated with SFN for 12 or 24 h . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies , respectively . Tau was detected with a polyclonal phospho - independent tau antibody ( Tau ) . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( c , d ) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM, SFN) for 12 or 24 h. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).(b) Stably transfected CN1.4 cortical neurons were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN for 12 or 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau was detected with a polyclonal phospho-independent tau antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (c,d) Bar graphs represent the relative optical density of phosphorylated tau normalized with that of total tau. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01).


f4Mouse brains were obtained from wild - type ( 11 months old , one male ; 5 months old , one female ; 12 months old , two female ) and Nrf2 ( − / − ) ( 10 months old , two male and two female ) mice . ( a ) Mouse brain tissues were homogenized in the lysis buffer , and 10 μg of lysates was incubated with 2 μg of GST - tau protein at 37 ° C for 30 min in the presence or absence of 1 mM ATP . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies , respectively . GST - tau was detected with a monoclonal GST antibody ( GST - tau ; Supplementary Fig . 4 ) . Bar graph of the relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of duplicated independent experiments and were analysed using Student ' s t - test . Mouse brains were obtained from wild - type ( 11 months old , one male ; 5 months old , one female ; 12 months old , two female ) and Nrf2 ( − / − ) ( 10 months old , two male and two female ) mice . ( b ) Mouse brain tissues were homogenized in the phosphatase storage buffer , and phosphatase activity in the lysates was quantified using the serine / threonine phosphatase assay system ( Promega ) by measuring the dephosphorylation of a phosphopeptide , RRA ( pT ) VA in the presence or absence of okadaic acid ( OA , 20 nM ) . PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture . n = 4 . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (a)Mouse brain tissues were homogenized in the lysis buffer, and 10 μg of lysates was incubated with 2 μg of GST-tau protein at 37°C for 30 min in the presence or absence of 1 mM ATP. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies, respectively. GST-tau was detected with a monoclonal GST antibody (GST-tau; Supplementary Fig. 4). Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of duplicated independent experiments and were analysed using Student's t-test.Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (b) Mouse brain tissues were homogenized in the phosphatase storage buffer, and phosphatase activity in the lysates was quantified using the serine/threonine phosphatase assay system (Promega) by measuring the dephosphorylation of a phosphopeptide, RRA(pT)VA in the presence or absence of okadaic acid (OA, 20 nM). PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture. n=4. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test.


f5CN1 . 4mousecortical cells stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . On the next day ( a ) , the cells were washed twice with PBS and the media were replaced with Dox - deficient media with either vehicle , epoxomicin ( 12 . 5 nM , Epox ) , MG132 ( 7 μM ) or 3 - methyladenine ( 10 mM , 3 - MA ) for 24 h . Samples in first lane were from cells maintained in the presence of Dox without medium exchange . CN1 . 4 mouse cortical cells stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . ( b ) Cells were treated with either chloroquine ( 50 μM , CQ ) or bafilomycin A1 ( 10 nM , Baf A1 ) for 24 h . Samples in first lane were from cells maintained in the presence of Dox without medium exchange . ( c ) HEK293 - TN cells were co - transfected with the plasmid expressing tau together either with siRNA of beclin - 1 or scramble RNA as a control , which was kept for 48 h . ( d ) Stably transfected CN1 . 4 cortical cells were maintained in the presence of Dox ( 1 μg ml − 1 ) to induce the expression of tau for 24 h , and subsequently treated with SFN ( 10 μM ) for 12 h . The culture medium was exchanged for fresh media containing Dox ( 1 μg ml − 1 ) but no SFN after washing with PBS . The cells were then incubated for an additional 18 h in the absence or presence of CQ ( 50 μM ) . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies , respectively . Tau , beclin - 1 and LC3 were detected with the appropriate polyclonal antibodies . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( e ) Bar graphs represent the relative optical density of phosphorylated tau , tau or LC3 - II normalized with that of actin . n = 4 . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5CN1.4mousecortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. On the next day (a), the cells were washed twice with PBS and the media were replaced with Dox-deficient media with either vehicle, epoxomicin (12.5 nM, Epox), MG132 (7 μM) or 3-methyladenine (10 mM, 3-MA) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.CN1.4 mouse cortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. (b) Cells were treated with either chloroquine (50 μM, CQ) or bafilomycin A1 (10 nM, Baf A1) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.(c) HEK293-TN cells were co-transfected with the plasmid expressing tau together either with siRNA of beclin-1 or scramble RNA as a control, which was kept for 48 h.(d) Stably transfected CN1.4 cortical cells were maintained in the presence of Dox (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN (10 μM) for 12 h. The culture medium was exchanged for fresh media containing Dox (1 μg ml−1) but no SFN after washing with PBS. The cells were then incubated for an additional 18 h in the absence or presence of CQ (50 μM). The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau, beclin-1 and LC3 were detected with the appropriate polyclonal antibodies. The relative molecular masses (kDa) are indicated to the left of each blot. (e) Bar graphs represent the relative optical density of phosphorylated tau, tau or LC3-II normalized with that of actin. n=4. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01).


f6 ( a ) Primary cortical neurons ( DIV 5 ) were treated with SFN ( 10 μM ) for 12 h , and the relative mRNA level of each gene was compared with that of cells not treated with SFN using qRT - PCR . ( b ) In hippocampal tissues from Nrf2 ( − / − ) ( 5 months old , one male ; 11 months old , one female ; 5 months old , one female ; 12 months old , three female ) or wild - type ( 10 months old , four male and two female ) mice , the relative mRNA level of each gene compared with that of wild - type mice . n = 6 . ( c ) The levels of p62 / SQSTM1 , Keap1 , mouse NDP52 ( mNDP52 ) and Hsp70 in Nrf2 ( − / − ) or wild - type mice were examined by immunoblotting . The asterisk on the Nrf2 panel indicates a nonspecific band . ( d , e : upper panel ) Primary cortical neurons ( DIV 5 ) ( d ) and SH - SY5Y cells ( e ) were treated with SFN for 24 h , and the expression levels of rat and human NDP52 ( rNDP52 and hNDP52 ) examined by immunoblotting using anti - NDP52 antibody . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( d , e : low panel ) Primary cortical neurons ( DIV 5 ) ( d ) and SH - SY5Y cells ( e ) transiently transfected with the pGL4 . 14 / human NDP52 promoter ( 2 , 173 bp ) luciferase reporter plasmid were treated with SFN for 24 h , and assayed for the luciferase activity . ( f ) SH - SY5Y cells were treated with SFN for 12 h , and cell lysates were used for the ChIP assay using anti - Nrf2 antibody as described in the Methods . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f6(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM) for 12 h, and the relative mRNA level of each gene was compared with that of cells not treated with SFN using qRT-PCR.(b) In hippocampal tissues from Nrf2 (−/−) (5 months old, one male; 11 months old, one female; 5 months old, one female; 12 months old, three female) or wild-type (10 months old, four male and two female) mice, the relative mRNA level of each gene compared with that of wild-type mice. n=6.(c) The levels of p62/SQSTM1, Keap1, mouse NDP52 (mNDP52) and Hsp70 in Nrf2 (−/−) or wild-type mice were examined by immunoblotting. The asterisk on the Nrf2 panel indicates a nonspecific band.(d,e: upper panel) Primary cortical neurons (DIV 5) (d) and SH-SY5Y cells (e) were treated with SFN for 24 h, and the expression levels of rat and human NDP52 (rNDP52 and hNDP52) examined by immunoblotting using anti-NDP52 antibody. The relative molecular masses (kDa) are indicated to the left of each blot. (d,e: low panel) Primary cortical neurons (DIV 5) (d) and SH-SY5Y cells (e) transiently transfected with the pGL4.14/human NDP52 promoter (2,173 bp) luciferase reporter plasmid were treated with SFN for 24 h, and assayed for the luciferase activity.(f) SH-SY5Y cells were treated with SFN for 12 h, and cell lysates were used for the ChIP assay using anti-Nrf2 antibody as described in the Methods. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).


f7 ( b ) CN1 . 4 cortical cells and SH - SY5Y cells were transiently transfected with the reporter plasmids with the human NDP52 promoter ( 2 , 317 bp or deleted mutants ) , and luciferase activity was assayed 24 h after transfection . ( c ) SH - SY5Y cells were co - transfected with the reporter plasmids with human NDP52 promoter ( wild or deleted mutants ) along with either the mock plasmid ( pcDNA3 . 1 ) or the plasmid expressing Nrf2 , and the luciferase activity was assayed 24 h after transfection . ( d ) SH - SY5Y cells were transiently co - transfected with the reporter plasmids with human NDP52 promoter or its mutant plasmid in which the putative ARE sequence was site - directly mutated ( X ) , together with the mock plasmid ( pcDNA3 . 1 ) or the plasmid expressing human Nrf2 . The luciferase activity was assayed after 24 h . The induced luciferase activity with co - expression with Nrf2 was not observed in a mutant NDP52 promoter in which all three ARE sites ( − 2 , 057 , − 1 , 023 and − 94 ) were site - directly mutated , indicating these three sites of five ARE cis - acting elements are functional . Data shown are mean ± s . d . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7(b) CN1.4 cortical cells and SH-SY5Y cells were transiently transfected with the reporter plasmids with the human NDP52 promoter (2,317 bp or deleted mutants), and luciferase activity was assayed 24 h after transfection.(c) SH-SY5Y cells were co-transfected with the reporter plasmids with human NDP52 promoter (wild or deleted mutants) along with either the mock plasmid (pcDNA3.1) or the plasmid expressing Nrf2, and the luciferase activity was assayed 24 h after transfection. (d) SH-SY5Y cells were transiently co-transfected with the reporter plasmids with human NDP52 promoter or its mutant plasmid in which the putative ARE sequence was site-directly mutated (X), together with the mock plasmid (pcDNA3.1) or the plasmid expressing human Nrf2. The luciferase activity was assayed after 24 h. The induced luciferase activity with co-expression with Nrf2 was not observed in a mutant NDP52 promoter in which all three ARE sites (−2,057, −1,023 and −94) were site-directly mutated, indicating these three sites of five ARE cis-acting elements are functional. Data shown are mean±s.d. of three independent experiments and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).


f8 ( a ) Primary cortical neurons were transduced with lentivirus expressing sh - RNA for Nrf2 or rat NDP52 ( rNDP52 ) , or a scrambled sh - RNA at DIV 1 , and were maintained until DIV 6 . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using 12E8 - and PHF1 - specific antibodies , respectively . Total tau was detected with a polyclonal tau - specific antibody ( Tau ) . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( b ) Bar graph of the relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test . ( * P0 . 05 ; * * * P0 . 001 ) ( c , d ) Primary cortical neurons were transduced with a control lentivirus ( FIGB ) or with one expressing humanNDP52 ( hNDP52 ) at DIV 1 . To induce autophagy , trehalose ( 150 mM ) was added at DIV 5 and the neurons incubated for 24 h ( DIV 6 ) . Primary cortical neurons were fixed with 4 % paraformaldehyde , and stained with the 12E8 or PHF1 antibodies . Scale bar , 20 μm . The optical density of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) . ( e ) and Ser396 / Ser404 ( PHF1 ) ( f ) in the soma of ~ 30 neurons randomly chosen was analysed with the ImageJ program . Data were analysed using Student ' s t - test ( * * * P0 . 001 ) .

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f8(a) Primary cortical neurons were transduced with lentivirus expressing sh-RNA for Nrf2 or rat NDP52 (rNDP52), or a scrambled sh-RNA at DIV 1, and were maintained until DIV 6. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8- and PHF1-specific antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot. (b) Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test. (*P0.05; ***P0.001)(c,d) Primary cortical neurons were transduced with a control lentivirus (FIGB) or with one expressing humanNDP52 (hNDP52) at DIV 1. To induce autophagy, trehalose (150 mM) was added at DIV 5 and the neurons incubated for 24 h (DIV 6). Primary cortical neurons were fixed with 4% paraformaldehyde, and stained with the 12E8 or PHF1 antibodies. Scale bar, 20 μm. The optical density of tau phosphorylated at Ser262/Ser356 (12E8). (e) and Ser396/Ser404 (PHF1) (f) in the soma of ~30 neurons randomly chosen was analysed with the ImageJ program. Data were analysed using Student's t-test (***P0.001).


f9 ( b ) Naive cortical cells were co - transfected with the plasmids expressing HA - hNDP52 or HA - tagged domain - deleted mutants of human NDP52 together with GFP - tau . Twenty - four hours later , cell lysates were prepared and transfected tau was immunoprecipitated and blots probed for both the HA - hNDP52 constructs and GFP - tau . Homogenates ( input ) used for co - immunoprecipitation were analysed by immunoblotting using anti - HA or a polyclonal tau - specific antibody ( Tau ) . ( c ) Stably transfected CN1 . 4 cortical cells were maintained in the presence of doxycycline ( 1 μg ml − 1 ) to induce the expression of tau for 24 h . Homogenates from CN1 . 4 cortical cells were used for the co - immunoprecipitation of mouse NDP52 ( mNDP52 ) using a Tau5 antibody . Co - precipitation of mNDP52 was examined by immunoblotting using anti - NDP52 antibody . Input of the homogenates used for co - immunoprecipitation was analysed by immunoblotting using anti - NDP52 or Tau antibody . ( d , e ) CN1 . 4 cortical cells were transfected with the mock or HA - mNDP52 plasmid ( d ) or with mNDP52 siRNA or scramble RNA as a control ( e ) . The cells transfected with siRNA or scramble RNA were treated with SFN ( 10 μM ) for 24 h . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies , respectively . Total tau was detected with a polyclonal tau - specific antibody ( Tau ) . The relative molecular masses ( kDa ) are indicated to the left of each blot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f9(b) Naive cortical cells were co-transfected with the plasmids expressing HA-hNDP52 or HA-tagged domain-deleted mutants of human NDP52 together with GFP-tau. Twenty-four hours later, cell lysates were prepared and transfected tau was immunoprecipitated and blots probed for both the HA-hNDP52 constructs and GFP-tau. Homogenates (input) used for co-immunoprecipitation were analysed by immunoblotting using anti-HA or a polyclonal tau-specific antibody (Tau).(c) Stably transfected CN1.4 cortical cells were maintained in the presence of doxycycline (1 μg ml−1) to induce the expression of tau for 24 h. Homogenates from CN1.4 cortical cells were used for the co-immunoprecipitation of mouse NDP52 (mNDP52) using a Tau5 antibody. Co-precipitation of mNDP52 was examined by immunoblotting using anti-NDP52 antibody. Input of the homogenates used for co-immunoprecipitation was analysed by immunoblotting using anti-NDP52 or Tau antibody.(d,e) CN1.4 cortical cells were transfected with the mock or HA-mNDP52 plasmid (d) or with mNDP52 siRNA or scramble RNA as a control (e). The cells transfected with siRNA or scramble RNA were treated with SFN (10 μM) for 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot.


f10 ( a ) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co - immunoprecipitation of human NDP52 ( hNDP52 ) using a Tau5 , 12E8 or PHF1 antibody . ( b ) Homogenates used for co - immunoprecipitation in a were analysed by immunoblotting using anti - NDP52 , Tau5 , 12E8 or PHF1 antibody . ( c ) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl - soluble and insoluble fractions . The levels of total tau and tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 in the sarkosyl - insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies , respectively . Samples were also immunoblotted with a polyclonal to total tau ( Tau ) antibody . The levels of hNDP52 , p62 / SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( d - f ) The regression graphs show the relative amount of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( d ) , at Ser396 / Ser404 ( PHF1 ) ( e ) or total tau ( f ) compared to hNDP52 present in the sarkosyl - insoluble fractions .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f10(a) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co-immunoprecipitation of human NDP52 (hNDP52) using a Tau5, 12E8 or PHF1 antibody.(b) Homogenates used for co-immunoprecipitation in a were analysed by immunoblotting using anti-NDP52, Tau5, 12E8 or PHF1 antibody.(c) Cerebral cortical tissues from diagnosed AD cases were separated into sarkosyl-soluble and insoluble fractions. The levels of total tau and tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 in the sarkosyl-insoluble fractions were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Samples were also immunoblotted with a polyclonal to total tau (Tau) antibody. The levels of hNDP52, p62/SQSTM1 and LC3 in the sarkosyl insoluble fractions also were examined by immunoblotting. The relative molecular masses (kDa) are indicated to the left of each blot. (d-f) The regression graphs show the relative amount of tau phosphorylated at Ser262/Ser356 (12E8) (d), at Ser396/Ser404 (PHF1) (e) or total tau (f) compared to hNDP52 present in the sarkosyl-insoluble fractions.


Figure 1 ( C ) Competition of sybodies and ACE2 for spike protein binding , investigated by GCI . Biotinylated spike protein was immobilized on the GCI sensor and then Sb # 15 ( 200 nM , left ) , or Sb # 68 ( 200 nM , right ) were injected alone or premixed with human ACE2 ( 100 nM ) . Sb # 0 represents a non - randomized control sybody .

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Figure 1(C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.


Figure 2Neutralization assays using VSVΔG pseudotyped with wildtype SARS - CoV - 2 spike protein . Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs . ( A ) Sb # 15 , Sb # 68 , or an equimolar mixure of both sybodies . ( B ) Sb # 14 , Sb # 16 , Sb # 45 , or , as a positive control , the previously - described sybody MR3 . ( C ) Neutralization by bivalent sybody - Fc fusions . Error bars correspond to standard deviations of three biological replicates .

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Figure 2Neutralization assays using VSVΔG pseudotyped with wildtype SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. (A) Sb#15, Sb#68, or an equimolar mixure of both sybodies. (B) Sb#14, Sb#16, Sb#45, or, as a positive control, the previously-described sybody MR3. (C) Neutralization by bivalent sybody-Fc fusions. Error bars correspond to standard deviations of three biological replicates.


Figure 4 ( C ) Neutralization assay using VSVΔG pseudotyped with wildtype Spike - protein . Relative infectivity was determined in response to increasing GS4 or Tripod - GS4r concentration . The corresponding neutralization data for isolated Sb # 15 and Sb # 68 are provided as reference . ( D ) Control experiment using VSVΔG pseudotyped with VSV - G . Infectivity was not affected by Tripod - GS4r .

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Figure 4(C) Neutralization assay using VSVΔG pseudotyped with wildtype Spike-protein. Relative infectivity was determined in response to increasing GS4 or Tripod-GS4r concentration. The corresponding neutralization data for isolated Sb#15 and Sb#68 are provided as reference. (D) Control experiment using VSVΔG pseudotyped with VSV-G. Infectivity was not affected by Tripod-GS4r.


Figure 5 ( C ) Neutralization assay using VSVΔG pseudotyped with Spike - protein containing the Q493R or P384H mutation , respectively . Relative infectivity in response to increasing binder concentrations was determined .

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Figure 5(C) Neutralization assay using VSVΔG pseudotyped with Spike-protein containing the Q493R or P384H mutation, respectively. Relative infectivity in response to increasing binder concentrations was determined.


Figure 6 ( A ) GCI - based kinetic analysis of interactions between immobilized sybodies ( indicated above each plot ) and wild - type ( WT ) or mutant RBDs carrying the individual K417N , E484K or N501Y mutations ( left panels ) , the combined triple KEN ( Beta ) mutations ( middle panels ) , or the double L452R / T478K ( Delta ) mutations ( right panels ) . Sb # 15 , Sb # 68 and GS4 were immobilized on independent flow - cells via biotinylated Avi - tags , and the RBD variants were sequentially injected at a concentration of 200 nM .

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Figure 6(A) GCI-based kinetic analysis of interactions between immobilized sybodies (indicated above each plot) and wild-type (WT) or mutant RBDs carrying the individual K417N, E484K or N501Y mutations (left panels), the combined triple KEN (Beta) mutations (middle panels), or the double L452R/T478K (Delta) mutations (right panels). Sb#15, Sb#68 and GS4 were immobilized on independent flow-cells via biotinylated Avi-tags, and the RBD variants were sequentially injected at a concentration of 200 nM.


Figure 1B Silver stain of the GST ‐ PTB RRM2pull ‐ down of the wild ‐ type RRM2 and the Y247Q mutant . Five microlitre of the pull ‐ down was run on a 15 % SDS - PAGE and silver ‐ stained . Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull ‐ down . The region encompassing 50 kDa to the top of the gel was sliced and subjected to in ‐ gel digestion and mass spectrometry . The two strongest bands visible in this region are labelled as Matrin3 and Raver1 . C Proteins identified in the GST ‐ PTB RRM2 pull ‐ down ranked by their unique peptide number . The table shows the different proteins we found binding to RRM2 , as well as when present , the sequence of the PRI motif . The indicated function is only a guideline as many have more functions than shown . D Western blot of the GSTpull ‐ down using antibodies against Matrin3 and Raver1 . Lanes 1 and 2 show 5 and 10 % of input , respectively , lanes 3 and 4 show GST ‐ PTB full ‐ length pull ‐ down of wild ‐ type and Y247Q mutant , respectively , and lanes 5 and 6 show GST ‐ PTB RRM2 pull ‐ down of wild ‐ type and Y247Q mutant , respectively , and lane 7 with pull ‐ down using GST alone .

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Figure 1B Silver stain of the GST‐PTB RRM2pull‐down of the wild‐type RRM2 and the Y247Q mutant. Five microlitre of the pull‐down was run on a 15% SDS-PAGE and silver‐stained. Three strong bands can be seen which are due to the recombinant protein and the beads used for the pull‐down. The region encompassing 50 kDa to the top of the gel was sliced and subjected to in‐gel digestion and mass spectrometry. The two strongest bands visible in this region are labelled as Matrin3 and Raver1.C Proteins identified in the GST‐PTB RRM2 pull‐down ranked by their unique peptide number. The table shows the different proteins we found binding to RRM2, as well as when present, the sequence of the PRI motif. The indicated function is only a guideline as many have more functions than shown.D Western blot of the GSTpull‐down using antibodies against Matrin3 and Raver1. Lanes 1 and 2 show 5 and 10% of input, respectively, lanes 3 and 4 show GST‐PTB full‐length pull‐down of wild‐type and Y247Q mutant, respectively, and lanes 5 and 6 show GST‐PTB RRM2 pull‐down of wild‐type and Y247Q mutant, respectively, and lane 7 with pull‐down using GST alone.


Figure 2B FLAGimmunoprecipitation of Matrin3 and Raver1 , both with wild ‐ type and with PRI mutated , and FLAG ‐ MS2 as a negative control . The immunoprecipitated complex was separated in a SDS - PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild ‐ type Matrin3 ( lane 2 ) and Raver1 ( lane 4 ) . The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins . C The 20 ‐ residue Raver1 491 - 511 ( lane 1 ) and the Matrin3 346 - 365 ( lane 3 ) peptide fused to MS2 were transcribed and translated in vitro ( Input ) and then pulled down with GST ‐ PTB or with GST ‐ SXL as a control . Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested ( lane 4 ) .

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Figure 2B FLAGimmunoprecipitation of Matrin3 and Raver1, both with wild‐type and with PRI mutated, and FLAG‐MS2 as a negative control. The immunoprecipitated complex was separated in a SDS-PAGE and subjected to Western blot using antibody against PTB which showed interaction to wild‐type Matrin3 (lane 2) and Raver1 (lane 4). The input was also analysed by Western blot with antibodies against PTB as a loading control and against the FLAG tag to ensure equal expression of proteins.C The 20‐residue Raver1 491-511 (lane 1) and the Matrin3 346-365 (lane 3) peptide fused to MS2 were transcribed and translated in vitro (Input) and then pulled down with GST‐PTB or with GST‐SXL as a control. Effects of single mutation of the Matrin3 PRI GILPPP to GIAPPP were also tested (lane 4).


Figure 3A Western blot probed for Matrin3 ( top panel ) , PTB ( middle panel ) and actin ( lower panel ) . Lanes 1 - 4 contain a twofold dilution of the control C2 sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % , lane 4 - 100 % ) . Lanes 4 - 7 contain equal amount of proteins , as can be confirmed by the anti ‐ actin ( lower panel ) , of control sample ( lane 4 ) , double ‐ PTB and nPTB siRNA ‐ treated sample ( lane 5 ) , Matrin3 siRNA ‐ treated sample ( lane 6 ) and triple knockdown of Matrin3 , PTB and nPTB ( lane 7 ) . Lane 7 is from the same gel and exposure , but some lanes present in the original gel were cropped for clarity , and a black line to indicate cropping was placed . F RT - PCR validation of Matrin3 ‐ regulated alternative spicing events in the ST7 , ACSL3 , PLEKHA3 , TCF12 , VWA5A , PTBP2 , PTBP3 , C3orf17 , ZMYND8 , VEZT , PIGX and DMD genes . In each case , triplicates for each condition ( C - control , M - Matrin3 , PTB / nPTB and Matrin3 / PTB / nPTB siRNA transfection samples ) were analysed and exon inclusion ( EI ) percentage is shown beneath the corresponding lane , along with the standard deviation ( s . d . ) .

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Figure 3A Western blot probed for Matrin3 (top panel), PTB (middle panel) and actin (lower panel). Lanes 1-4 contain a twofold dilution of the control C2 sample (lane 1-12.5%, lane 2-25%, lane 3-50%, lane 4-100%). Lanes 4-7 contain equal amount of proteins, as can be confirmed by the anti‐actin (lower panel), of control sample (lane 4), double‐PTB and nPTB siRNA‐treated sample (lane 5), Matrin3 siRNA‐treated sample (lane 6) and triple knockdown of Matrin3, PTB and nPTB (lane 7). Lane 7 is from the same gel and exposure, but some lanes present in the original gel were cropped for clarity, and a black line to indicate cropping was placed.F RT-PCR validation of Matrin3‐regulated alternative spicing events in the ST7, ACSL3, PLEKHA3, TCF12, VWA5A, PTBP2, PTBP3, C3orf17, ZMYND8, VEZT, PIGX and DMD genes. In each case, triplicates for each condition (C-control, M-Matrin3, PTB/nPTB and Matrin3/PTB/nPTB siRNA transfection samples) were analysed and exon inclusion (EI) percentage is shown beneath the corresponding lane, along with the standard deviation (s.d.).


Figure 5A Matrin3 crosslinking in Matrin3 ‐ regulated pre ‐ mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3 ′ ss and 5 ′ ss , respectively , the upstream flanking exon and 500 nucleotides downstream of its 5 ′ ss and the downstream flanking exon with 500 nucleotides upstream of its 5 ′ ss . The iCLIP tags were mapped onto silenced ASE ( blue , n = 421 ) , enhanced ASE ( red , n = 187 ) and to control ASE ( grey , n = 16 , 242 ) , and percentage of occupancy is plotted . B Same as in ( A ) but using iCLIP tags obtained from PTBiCLIP and mapped onto PTB / nPTB ‐ regulated ASE , silenced ASE ( blue , n = 729 ) , enhanced ( red , n = 820 ) and control ASE ( grey , n = 14 , 599 ) . C Matrin3 crosslinked nucleotides mapped onto PTB ‐ regulated ASE . D PTB crosslinked nucleotides mapped onto Matrin3 ‐ regulated ASE .

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Figure 5A Matrin3 crosslinking in Matrin3‐regulated pre‐mRNAs where position of crosslinked nucleotides was mapped onto the regulated exon and the 500 nucleotides upstream and downstream of its 3′ss and 5′ss, respectively, the upstream flanking exon and 500 nucleotides downstream of its 5′ss and the downstream flanking exon with 500 nucleotides upstream of its 5′ss. The iCLIP tags were mapped onto silenced ASE (blue, n = 421), enhanced ASE (red, n = 187) and to control ASE (grey, n = 16,242), and percentage of occupancy is plotted.B Same as in (A) but using iCLIP tags obtained from PTBiCLIP and mapped onto PTB/nPTB‐regulated ASE, silenced ASE (blue, n = 729), enhanced (red, n = 820) and control ASE (grey, n = 14,599).C Matrin3 crosslinked nucleotides mapped onto PTB‐regulated ASE.D PTB crosslinked nucleotides mapped onto Matrin3‐regulated ASE.


Figure 6B Western blot of the overexpressed Matrin3 and its mutants probed with anti ‐ FLAG and anti ‐ actin antibodies . C , D RT - PCR analysis using primers specific to the ST7 exon 11 ( C ) and ABI2 exon 8 ( D ) splicing reporters , respectively , of samples where FLAG ‐ tagged Matrin3 and its mutants have been overexpressed . Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion . * P 0 . 01 compared with control sample ( − ) . E Western blot of overexpressed FLAG ‐ tagged Matrin3 and PTB , in control siRNA and PTB / nPTB siRNA ‐ transfected cells , probed with anti ‐ FLAG to detect overexpressed proteins , anti ‐ PTB to detect the knockdown levels and anti ‐ actin to confirm protein loading . A titration of control sample ( lane 1 - 12 . 5 % , lane 2 - 25 % , lane 3 - 50 % and lane 4 - 100 % ) is also included to assess the levels of knockdown . The band present above the PTB doublet in lane 6 is FLAG ‐ tagged PTB . F , G RT - PCR analysis using primers specific to the ST7 exon 11 ( F ) and ABI2 exon 8 ( G ) splicing reporters , respectively , of samples where Matrin3 or PTB expression vectors were co ‐ transfected , in control siRNA and PTB / nPTB siRNA ‐ transfected cells . Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion . * P 0 . 01 when compared with control sample in each condition ( ‐ ) .

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Figure 6B Western blot of the overexpressed Matrin3 and its mutants probed with anti‐FLAG and anti‐actin antibodies.C, D RT-PCR analysis using primers specific to the ST7 exon 11 (C) and ABI2 exon 8 (D) splicing reporters, respectively, of samples where FLAG‐tagged Matrin3 and its mutants have been overexpressed. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 compared with control sample (−).E Western blot of overexpressed FLAG‐tagged Matrin3 and PTB, in control siRNA and PTB/nPTB siRNA‐transfected cells, probed with anti‐FLAG to detect overexpressed proteins, anti‐PTB to detect the knockdown levels and anti‐actin to confirm protein loading. A titration of control sample (lane 1-12.5%, lane 2-25%, lane 3-50% and lane 4-100%) is also included to assess the levels of knockdown. The band present above the PTB doublet in lane 6 is FLAG‐tagged PTB.F, G RT-PCR analysis using primers specific to the ST7 exon 11 (F) and ABI2 exon 8 (G) splicing reporters, respectively, of samples where Matrin3 or PTB expression vectors were co‐transfected, in control siRNA and PTB/nPTB siRNA‐transfected cells. Quantification of at least three replicates for each condition is shown as a histogram of the percentage of exon inclusion. *P 0.01 when compared with control sample in each condition (‐).


Figure 1C . Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP - Cwh43 , GFP - Cwh43 - k696fs or GFP - Cwh43 - Leu533ter fusion proteins . To label the ER , HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP - calreticulin fusion protein containing an ER retention signal ( RFP - calreticulin - KDEL ) . To label the Golgi apparatus , cells were immunostained for golgin - 97 ( red ) . When compared with wild type GFP - Cwh43 , GFP - Cwh43 - k696fs and GFP - Cwh43 - Leu533 showed decreased association with the Golgi apparatus ( but not the ER ) and were diffusely distributed throughout the cytoplasm . Scale bar is approximately 5 µm . D . Western blot analysis of total membrane , aqueous and lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout ( KO ) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced . Cells were transfected to overexpress a control GFP plasmid , a plasmid encoding human wild type Cwh43 with GFP fused to the N - terminus , or a plasmid encoding human CWH43 harboring the iNPH - associated mutation ( Lys696AsnfsTer23 ) with GFP fused to the N - terminus . The Western blot was stained using an antibody directed against CD59 , a GPI - anchored protein .

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Figure 1C. Fluorescence micrographs of HeLa cells transfected with plasmids encoding human GFP-Cwh43, GFP-Cwh43-k696fs or GFP-Cwh43-Leu533ter fusion proteins. To label the ER, HeLa cells were transduced with a baculovirus encoding a fluorescently labeled RFP-calreticulin fusion protein containing an ER retention signal (RFP-calreticulin-KDEL). To label the Golgi apparatus, cells were immunostained for golgin-97 (red). When compared with wild type GFP-Cwh43, GFP-Cwh43-k696fs and GFP-Cwh43-Leu533 showed decreased association with the Golgi apparatus (but not the ER) and were diffusely distributed throughout the cytoplasm. Scale bar is approximately 5 µm.D. Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type HeLa cells and two independent CRISPR CWH43 knockout (KO) HeLa cell lines in which a mutated CWH43 gene encodes a protein that is truncated near Leu533 and CWH43 mRNA and protein are markedly reduced. Cells were transfected to overexpress a control GFP plasmid, a plasmid encoding human wild type Cwh43 with GFP fused to the N-terminus, or a plasmid encoding human CWH43 harboring the iNPH-associated mutation (Lys696AsnfsTer23) with GFP fused to the N-terminus. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.


Figure 2Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell . Cilia were visualized using an antibody for acetylated alpha tubulin ( green ) . Cwh43 immunoreactivity was visualized using a specific anti - Cwh43 antibody ( red ) . Scale bar ( left column ) is approximately 4 µm . Images in the column on the right represent a Z - stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell . Scale bar ( right column ) is approximately 5 µm .

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Figure 2Confocal fluorescence immunocytochemistry images of a single cultured mouse ciliated ependymal cell. Cilia were visualized using an antibody for acetylated alpha tubulin (green). Cwh43 immunoreactivity was visualized using a specific anti-Cwh43 antibody (red). Scale bar (left column) is approximately 4 µm. Images in the column on the right represent a Z-stack reconstruction of confocal images showing localization of Cwh43 immunoreactivity in cilia of a mouse ependymal cell. Scale bar (right column) is approximately 5 µm.


Figure 3A . Scatter plot comparing ventricular volume from CWH43WT / WT ( wild type , WT ) , heterozygous CWH43WT / M533 , homozygous CWH43M533 / M533 and CWH43M533 / A530 mice at 6 months . Ventricular volume was calculated from T2 - weighted MR images of the mouse brain using a custom automated computer algorithm . Horizontal bars indicate the mean of the measurements in each column . Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t - test ( P = 0 . 0015 for CWH43WT / M533 ; P = 0 . 0014 for CWH43M533 / M533 ; P = 0 . 006 for CWH43M533 / A530 ) . D . Box plot showing rotarod performance data for CWH43WT / WT , CWH43WT / M533 , CWH43M533 / M533 and CWH43M533 / A530 mice at 7 months of age . Data shown are the mean , 1st quartile , 3rd quartile , minimum and maximum for each group of mice . Statistical significance was determined using the unpaired t - test . When compared to wild type CWH43WT / WT mice ( n = 10 ) , balance time on the rotarod was decreased significantly among heterozygous CWH43WT / M533 mice ( * P = 0 . 04 , n = 8 ) , homozygous CWH43M533 / M533 mice ( * P = 0 . 03 , n = 7 ) , and compound heterozygous CWH43M533 / A530 mice ( * P = 0 . 03 , n = 4 ) .

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Figure 3A. Scatter plot comparing ventricular volume from CWH43WT/WT (wild type, WT), heterozygous CWH43WT/M533, homozygous CWH43M533/M533 and CWH43M533/A530 mice at 6 months. Ventricular volume was calculated from T2-weighted MR images of the mouse brain using a custom automated computer algorithm. Horizontal bars indicate the mean of the measurements in each column. Statistical significance for each mutant mouse line compared to WT was determined using the unpaired t-test (P=0.0015 for CWH43WT/M533; P=0.0014 for CWH43M533/M533; P=0.006 for CWH43M533/A530).D. Box plot showing rotarod performance data for CWH43WT/WT, CWH43WT/M533, CWH43M533/M533 and CWH43M533/A530 mice at 7 months of age. Data shown are the mean, 1st quartile, 3rd quartile, minimum and maximum for each group of mice. Statistical significance was determined using the unpaired t-test. When compared to wild type CWH43WT/WT mice (n=10), balance time on the rotarod was decreased significantly among heterozygous CWH43WT/M533 mice (*P=0.04, n=8), homozygous CWH43M533/M533 mice (*P=0.03, n=7), and compound heterozygous CWH43M533/A530 mice (*P=0.03, n=4).


Figure 4Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533 / M533 mice . Scale bar is approximately 40 µm . The graph on the right quantifies the data from the electron micrographs on the left . Data shown are the mean ± SEM . * = P = 0 . 004 , n = 4 , unpaired t - test . Western blot analysis of total membrane , aqueous and lipid ( GPI - anchor - containing ) Triton X - 114 extracts derived from wild type , CWH43M533 / M533 and CWH43WT / M533mouse brain or kidney . The Western blot was stained using an antibody directed against CD59 , a GPI - anchored protein . Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT / WT , CWH43M533 / M533 and CWH43M533 / A530 mice . Arrowheads point to apical surfaces of ependymal and choroid plexus cells . Nuclei are counterstained using DAPI ( blue ) . Scale bar is approximately 5 µm .

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Figure 4Scanning electron micrographs of the ependymal surface of the lateral ventricle of CWH43 WT and CWH43M533/M533 mice. Scale bar is approximately 40 µm. The graph on the right quantifies the data from the electron micrographs on the left. Data shown are the mean ± SEM. * = P=0.004, n = 4, unpaired t-test.Western blot analysis of total membrane, aqueous and lipid (GPI-anchor-containing) Triton X-114 extracts derived from wild type, CWH43M533/M533 and CWH43WT/M533mouse brain or kidney. The Western blot was stained using an antibody directed against CD59, a GPI-anchored protein.Fluorescence immunohistochemistry for CD59 in the ependymal layer and choroid plexus of the lateral ventricle from CWH43WT/WT, CWH43M533/M533 and CWH43M533/A530 mice. Arrowheads point to apical surfaces of ependymal and choroid plexus cells. Nuclei are counterstained using DAPI (blue). Scale bar is approximately 5 µm.


Figure 2D , E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation ( D ) , and for molecules involved in V ( D ) J recombination and TCR signaling ( E ) . In D and E , dot color represents the scaled average expression of the specified gene across the various clusters , and dot size indicates the percentage of cells expressing the specified gene .

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Figure 2D, E Dot plots showing the expression level of transcripts coding for established markers of intrathymic T cell differentiation (D), and for molecules involved in V(D)J recombination and TCR signaling (E). In D and E, dot color represents the scaled average expression of the specified gene across the various clusters, and dot size indicates the percentage of cells expressing the specified gene.


Figure 3C UMAP plots colored according to the expression level of the specified genes .

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Figure 3C UMAP plots colored according to the expression level of the specified genes.


Figure 4D Dot plot showing the expression level of genes belonging to the γδ TCR - induced signature across the cell subsets corresponding to Dataset 2 . Based on the gene set that was specifically upregulated during the DPsmall → DPCD69 + / SP transition ( see Fig EV3B ) , it allowed to subdivide the γδ TCR - induced signature into a ' γδ TCR only ' signature and a ' αβ and γδ TCR shared '

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Figure 4D Dot plot showing the expression level of genes belonging to the γδ TCR-induced signature across the cell subsets corresponding to Dataset 2. Based on the gene set that was specifically upregulated during the DPsmall → DPCD69+/SP transition (see Fig EV3B), it allowed to subdivide the γδ TCR-induced signature into a 'γδ TCR only' signature and a 'αβ and γδ TCR shared' signature


Figure 5A , B UMAP plots of Dataset 3 which contains five WT ( DN3a , DN3b γδ + , DN4 γδ + , DN3b γδ - and DN4 γδ - ) and two Lat - / - ( DN3 γδ - and DN3 γδint ) scRNAseq datasets . The UMAP plot is colored according to sorted samples ( A ) , and to expression levels of the specified genes ( B ) .

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Figure 5A, B UMAP plots of Dataset 3 which contains five WT (DN3a, DN3b γδ+, DN4 γδ+, DN3b γδ- and DN4 γδ-) and two Lat-/- (DN3 γδ- and DN3 γδint) scRNAseq datasets. The UMAP plot is colored according to sorted samples (A), and to expression levels of the specified genes (B).


Figure 6A , B , C AUCell scores for the γδ TCR - induced signature ( A ) , γδ TCR only signature ( B ) and αβ and γδ TCR shared signature ( C ) are displayed on UMAP ( left ) and violin ( right ) plots of the 7 sorted subsets corresponding to Dataset 3 . As expected , AUCell scores for the 3 signatures were very low for DN3a cells , low for WT DN3b γδ - and WT DN4 γδ - cells , and high for WT DN3b γδ + and WT DN4 γδ + cells .

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Figure 6A,B,C AUCell scores for the γδ TCR-induced signature (A), γδ TCR only signature (B) and αβ and γδ TCR shared signature (C) are displayed on UMAP (left) and violin (right) plots of the 7 sorted subsets corresponding to Dataset 3. As expected, AUCell scores for the 3 signatures were very low for DN3a cells, low for WT DN3b γδ- and WT DN4 γδ- cells, and high for WT DN3b γδ+ and WT DN4 γδ+ cells.


Figure 7B Bar plot showing the percentage of cells in the G0 / G1 ( red bars ) , S ( blue bars ) and G2 / M ( green bars ) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3 .

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Figure 7B Bar plot showing the percentage of cells in the G0/G1 (red bars), S (blue bars) and G2/M (green bars) phases of the cell cycle among each of the 7 sorted cell subsets of Dataset 3.


Figure 1 , C . siRNA - mediated knockdown of EXOSC10 ( EXO10 ) , but not of DIS3 and DIS3L , significantly increased generation of poly - GA in HeLa cells expressing ( G4C2 ) 80 repeats . ctrl " indicates a control vector which lacks the G4C2 repeat but still contains the 5 ' flanking region and 3xTAG . . Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT - qPCR . E , F . Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing ( G4C2 ) 80 repeats visualized by in situ hybridization targeting sense RNA foci . In total 25 ( Ct ) and 24 ( EXO10 # 5 ) images were analyzed in 3 independent experiments . G . Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing ( G4C2 ) 80 repeats . Each dot represents average cellular RNA foci intensity ( Arbitrary unit ) of RNA foci - positive cells in each randomly taken image . In total 25 ( Ct ) and 24 ( EXO10 # 5 ) images were analyzed in 3 independent experiments .

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Figure 1, C. siRNA-mediated knockdown of EXOSC10 (EXO10), but not of DIS3 and DIS3L, significantly increased generation of poly-GA in HeLa cells expressing (G4C2) 80 repeats. ctrl" indicates a control vector which lacks the G4C2 repeat but still contains the 5' flanking region and 3xTAG.. Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT-qPCR.E, F. Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing (G4C2) 80 repeats visualized by in situ hybridization targeting sense RNA foci. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments. G. Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing (G4C2) 80 repeats. Each dot represents average cellular RNA foci intensity (Arbitrary unit) of RNA foci-positive cells in each randomly taken image. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments.


Figure 2A , B . Dose - dependent suppression of poly - GA by exogenous EXOSC10 upon EXOSC10 knockdown . C . Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown . Concentration of transfected EXOSC10 plasmid was 0 . 2 ng / μl in 1 ml medium . D . Overexpression of EXOSC10 decreases G4C2 repeat RNA .

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Figure 2A, B. Dose-dependent suppression of poly-GA by exogenous EXOSC10 upon EXOSC10 knockdown.C. Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown. Concentration of transfected EXOSC10 plasmid was 0.2 ng/μl in 1 ml medium.D. Overexpression of EXOSC10 decreases G4C2 repeat RNA.


Figure 3A , B . Under transcription inhibition with Actinomycin D , cells with EXOSC10 knockdown ( EXO10 KD ) showed more stable expression of the repeat RNA compared to cells with control knockdown with non - targeting siRNA ( Ct KD ) . C , D . Degradation of FAM - labeled synthetic ( G4C2 ) 8 repeat RNA with recombinant human EXOSC10 ( hEXO10 ) ( Upper left ) . 3 ' blocked FAM - labeled G4C2 repeat RNA resisting 3 ' exoribonuclease activity is not degraded by recombinant EXOSC10 ( Lower left ) . Lack of recombinant EXOSC10 ( Upper right ) or Mg2 + ion which is essential co - factor of EXOSC10 ( Lower right ) abolished the RNA degradation activity . 3 or 4 independent experiments . Quantification of the top bands are shown in ( D ) . E , F . Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 ( Left ) . Without recombinant EXOSC10 , no substrate degradation was observed ( Right ) . 5 or 6 independent experiments . Quantification is shown in ( F ) .

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Figure 3A, B. Under transcription inhibition with Actinomycin D, cells with EXOSC10 knockdown (EXO10 KD) showed more stable expression of the repeat RNA compared to cells with control knockdown with non-targeting siRNA (Ct KD).C, D. Degradation of FAM-labeled synthetic (G4C2)8 repeat RNA with recombinant human EXOSC10 (hEXO10) (Upper left). 3' blocked FAM-labeled G4C2 repeat RNA resisting 3' exoribonuclease activity is not degraded by recombinant EXOSC10 (Lower left). Lack of recombinant EXOSC10 (Upper right) or Mg2+ ion which is essential co-factor of EXOSC10 (Lower right) abolished the RNA degradation activity. 3 or 4 independent experiments. Quantification of the top bands are shown in (D).E, F. Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 (Left). Without recombinant EXOSC10, no substrate degradation was observed (Right). 5 or 6 independent experiments. Quantification is shown in (F).


Figure 4A . In situ hybridization . G4C2 RNA foci were detected in a C9 patient - derived fibroblast but not in a control - derived fibroblast with non - targeting siRNA treatment ( left panels , control / si Ct . middle panels , C9 / si Ct ) . Knockdown of EXSOC10 increased RNA foci ( right panels , C9 / siEXOSC10 # 5 ) . Nuclei were stained with DAPI ( blue ) . White arrows indicate nuclear RNA foci . The green arrows show cytoplasmic RNA foci . B . Quantification of % cell with G4C2 RNA foci . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . C . Quantification of the number of G4C2 RNA foci / cell . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . D . Quantification of % cell with C4G2 RNA foci . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . E . Quantification of the number of C4G2 RNA foci / cell . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . F , G . Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT - qPCR . 10 independent knockdown experiments ( Case1 N = 4 , Case2 N = 4 , Case3 N = 2 ) were performed .

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Figure 4A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci.B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. F, G. Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT-qPCR. 10 independent knockdown experiments (Case1 N=4, Case2 N=4, Case3 N=2) were performed.


Figure 5A , B , C . Double knockdown of EXOSC10 and DIS3 significantly increased poly - GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing ( G4C2 ) 80 repeats . D . Overexpression of DIS3 compensated EXOSC10 knockdown - induced repeat RNA accumulation . E , F . Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown - induced GA increase .

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Figure 5A, B, C. Double knockdown of EXOSC10 and DIS3 significantly increased poly-GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing (G4C2) 80 repeats.D. Overexpression of DIS3 compensated EXOSC10 knockdown-induced repeat RNA accumulation.E, F. Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown-induced GA increase.


Figure 6C - F . HA - tagged poly - GA , poly - GP , poly - GR or poly - PR ( red ) and EXOSC10 ( green ) were immunostained with anti - HA and anti - EXOSC10 antibodies . Nuclei were stained with DAPI ( blue ) . G . EXOSC10 ( green ) were immunostained with anti - EXOSC10 antibody in cells transfected with control mock plasmid . Nuclei were stained with DAPI ( blue ) . H , I . Quantification of the frequency of cells with mislocalized EXOSC10 . Only DPR - positive cells were quantified ( except mock transfected cells expressing no DPR . ( Total cell counts : ( figure H ) mock 300 cells , GA 112 cells , GP 69 cells , GR 53 cells , ( figure I ) mock 445 cells , PR 159 cells ) .

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Figure 6C-F. HA-tagged poly-GA, poly-GP, poly-GR or poly-PR (red) and EXOSC10 (green) were immunostained with anti-HA and anti-EXOSC10 antibodies. Nuclei were stained with DAPI (blue).G. EXOSC10 (green) were immunostained with anti-EXOSC10 antibody in cells transfected with control mock plasmid. Nuclei were stained with DAPI (blue).H, I. Quantification of the frequency of cells with mislocalized EXOSC10. Only DPR-positive cells were quantified (except mock transfected cells expressing no DPR. (Total cell counts: (figure H) mock 300 cells, GA 112 cells, GP 69 cells, GR 53 cells, (figure I) mock 445 cells, PR 159 cells).


Figure 7B . Immunoblot validation of successful fractionation using antibodies against HDAC1 ( nuclear marker ) and GAPDH ( cytoplasmic marker ) . C . Co - immunoprecipitation of GFP - DPR interacting proteins in cytoplasmic fraction using GFP - trap beads . Immunoprecipitants were served for immunoblot with anti - EXOSC10 antibody . The same membrane was reprobed with anti - GFP antibody . D . Quantification of the relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " . E . Co - immunoprecipitation of GFP - DPR interacting proteins in nuclear fraction using GFP - trap beads followed by immunoblot using anti - EXOSC10 antibody . The same membrane was reprobed with anti - GFP antibody . F . Quantification of the relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " .

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Figure 7B. Immunoblot validation of successful fractionation using antibodies against HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker).C. Co-immunoprecipitation of GFP-DPR interacting proteins in cytoplasmic fraction using GFP-trap beads. Immunoprecipitants were served for immunoblot with anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. D. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". E. Co-immunoprecipitation of GFP-DPR interacting proteins in nuclear fraction using GFP-trap beads followed by immunoblot using anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. F. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input".


Figure 8B , C . Increased expression of 3 ' extended snoRA48 precursor upon EXOSC10 knockdown ( B ) or GFP - poly - GR177 / GFP - poly - PR166 expression ( C ) on RT - qPCR . D , E . Increased expression of 3 ' extended snoRA68 precursor upon EXOSC10 knockdown ( D ) or GFP - poly - GR177 / GFP - poly - PR166 expressions ( E ) on RT - qPCR . F . RT - qPCR analysis showed increased cellular G4C2 repeat RNA expression from co - transfected EF1 ( G4C2 ) 80 repeat construct in the presence of GFP , GFP - poly - GR177 or GFP - poly - PR166 .

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Figure 8B, C. Increased expression of 3'extended snoRA48 precursor upon EXOSC10 knockdown (B) or GFP-poly-GR177/ GFP-poly-PR166 expression (C) on RT-qPCR. D, E. Increased expression of 3'extended snoRA68 precursor upon EXOSC10 knockdown (D) or GFP-poly-GR177 /GFP-poly-PR166 expressions (E) on RT-qPCR.F. RT-qPCR analysis showed increased cellular G4C2 repeat RNA expression from co-transfected EF1 (G4C2)80 repeat construct in the presence of GFP, GFP-poly-GR177 or GFP-poly-PR166.


f1 ( a - f ) HepG2 cells were treated with bovine serum albumin ( Con ) , 500 μM PA or 100 nM rapamycin ( Rap ) for 9 h and subjected to following analyses . ( a , c - f ) Cells were stained with ubiquitin ( Ub ) , p62 , LC3 and LAMP1 antibodies and DAPI ( blue ) . Boxed areas are magnified in right - most panels ( c , d ) . Co - localization between LAMP1 and LC3 staining ( e ) was quantified ( f ; n = 3 ) . ( b ) Cells were subjected to serial protein extraction ( solubility fractionation ) with indicated concentration of Triton X - 100 ( TX100 ) or sodium dodecyl sulphate ( SDS ) and analysed by immunoblotting with indicated antibodies . ( a - f ) HepG2 cells were treated with bovine serum albumin ( Con ) , 500 μM PA or 100 nM rapamycin ( Rap ) for 9 h and subjected to following analyses . ( a , c - f ) Cells were stained with ubiquitin ( Ub ) , p62 , LC3 and LAMP1 antibodies and DAPI ( blue ) . Boxed areas are magnified in right - most panels ( c , d ) . Co - localization between LAMP1 and LC3 staining ( e ) was quantified ( f ; n = 3 ) . ( a - f ) HepG2 cells were treated with bovine serum albumin ( Con ) , 500 μM PA or 100 nM rapamycin ( Rap ) for 9 h and subjected to following analyses . ( a , c - f ) Cells were stained with ubiquitin ( Ub ) , p62 , LC3 and LAMP1 antibodies and DAPI ( blue ) . Boxed areas are magnified in right - most panels ( c , d ) . Co - localization between LAMP1 and LC3 staining ( e ) was quantified ( f ; n = 3 ) . ( g , h ) HepG2 cells stably transduced with mCherry ( mCh ) - GFP - LC3 - expressing retroviruses were treated with Con , Rap or PA for 9 h and examined under a live confocal microscope ( g ) . Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out . Number of autolysosomes was quantified ( h ; n = 7 ) . Scale bar , 5 μm . All data are shown as mean ± s . e . m . * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

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f1(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).(b) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulphate (SDS) and analysed by immunoblotting with indicated antibodies.(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).(g,h) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 h and examined under a live confocal microscope (g). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified (h; n=7). Scale bar, 5 μm. All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


f2HepG2 cells were treated with bovine serum albumin ( Con or ( − ) ) , PA ( 500 μM ) , PA + verapamil ( Ver , 50 μM ) or PA + nicardipine ( Nic , 100 μM ) for 9 h . ( a - d ) After each treatment , cells were loaded with a calcium indicator X - Rhod - 1 - AM ( a , b ) or Fura - 2 - AM ( c , d ) . Calcium levels were visualized by laser confocal microscopy ( a ) or by dual fluorescent microscopy ( c , 340 / 380 nm ratio image ) and quantified ( b , d ; n = 8 and 30 , respectively ) . ( e - h ) Cells with indicated treatments were subjected to solubility fractionation . 1 % Triton X - 100 - insoluble fractions were dissolved in 2 % SDS , analysed by immunoblotting ( e , f ) and quantified ( g , h ; n = 3 ) . ( i - l ) Cells with indicated treatments were subjected to immunostaining with indicated antibodies ( i , k ) . DNA was stained with DAPI ( blue ) . Amount of aggregated proteins was quantified ( j ; n = 10 ) . Co - localization between LAMP1 and LC3 was quantified ( l ; n = 4 ) . Boxed areas in fluorescence images are magnified in right - most panels ( i , k ) . Scale bar , 10 μm ( a ) , 20 μm ( c ) , 5 μm ( i , k ) . All data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - test ) . Molecular weight markers are indicated in kDa .

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f2HepG2 cells were treated with bovine serum albumin (Con or (−)), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 9 h. (a-d) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM (a,b) or Fura-2-AM (c,d). Calcium levels were visualized by laser confocal microscopy (a) or by dual fluorescent microscopy (c, 340/380 nm ratio image) and quantified (b,d; n=8 and 30, respectively).(e-h) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analysed by immunoblotting (e,f) and quantified (g,h; n=3).(i-l) Cells with indicated treatments were subjected to immunostaining with indicated antibodies (i,k). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified (j; n=10). Co-localization between LAMP1 and LC3 was quantified (l; n=4). Boxed areas in fluorescence images are magnified in right-most panels (i,k). Scale bar, 10 μm (a), 20 μm (c), 5 μm (i,k). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.