( D ) Antitumor activity of CCB02 in vivo . Left : Subcutaneous xenograft tumor volume measurements of H1975T790M ( 30mg / kg of weight , daily ) in nude mice treated with vehicle or CCB02 . Right : Bar graph shows total tumor volume at day 1 and day 24 of vehicle or CCB02 treated xenograft . Error bars , mean ± SD with n = 8 for control vehicle and n = 8 for CCB02 treatment . Unpaired t - test * P < 0 . 01 .

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 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 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

(D) Antitumor activity of CCB02 in vivo. Left: Subcutaneous xenograft tumor volume measurements of H1975T790M (30mg/kg of weight, daily) in nude mice treated with vehicle or CCB02. Right: Bar graph shows total tumor volume at day 1 and day 24 of vehicle or CCB02 treated xenograft. Error bars, mean ± SD with n = 8 for control vehicle and n = 8 for CCB02 treatment. Unpaired t-test *P < 0.01.


( 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 )

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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 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

(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 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 O O O O O O O O O O O O O O O

(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.


Brain 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 ) .

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 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 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 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

Brain 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).


( 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 ) .

O O O O O O 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 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

(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 O O O O O O 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 B-CONTROLLED_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 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 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 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

(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).


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 . ( 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 .

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

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. (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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_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 O O O O O O O O O O O O O O O O O O O O O O O O

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.


CN1 . 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 .

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

CN1.4mousecortical 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 .

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

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 .

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

(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 ) .

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

(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).


( 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 .

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 B-CONTROLLED_VAR O O O O O

(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 .

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

(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 .

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

(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 .

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

(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 O O O 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

(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).


( 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 .

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

(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 O O O O O O O O O O O O O O O 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 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-CONTROLLED_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 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

(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).


( 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 )

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(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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(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).


( 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 ) .

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(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 .

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

(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.


( 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 .

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(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 .

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

(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.


The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects . The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2 , or by mCherry - tagged PI4Kβ1 expressed from a genomic fragment . Plants shown are one month old . Scale bar , 10 cm .

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The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2, or by mCherry-tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are one month old. Scale bar, 10 cm.


The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants , as shown by propidium iodide ( PI ) staining of five - day - old roots . Arrowhead , oblique cell wall . Insets , magnifications of areas showing cell wall stubs . Scale bars , 50 µm .

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The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of five-day-old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 µm.


Quantifications of cell wall stubs , oblique cell walls and root meristem size ( wild type , n = 15 roots ; pi4kβ1 , n = 15 roots ; pi4kβ2 , n = 14 roots ; pi4kβ1 pi4kβ2 , n = 18 roots ; pPI4Kβ1 : PI4Kβ1 , n = 14 roots , pPI4Kβ2 : PI4Kβ2 , n = 15 roots ) . n . d . , not detected . Data are mean ± SD . Lowercase letters indicate a significant increase of oblique cell walls ( Welch ANOVA with Games - Howell post - hoc test ; p < 0 . 0001 ) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant ( one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 001 ) .

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

Quantifications of cell wall stubs, oblique cell walls and root meristem size (wild type, n = 15 roots; pi4kβ1, n = 15 roots; pi4kβ2, n = 14 roots; pi4kβ1 pi4kβ2, n = 18 roots; pPI4Kβ1:PI4Kβ1, n = 14 roots, pPI4Kβ2:PI4Kβ2, n = 15 roots). n.d., not detected. Data are mean ± SD. Lowercase letters indicate a significant increase of oblique cell walls (Welch ANOVA with Games-Howell post-hoc test; p < 0.0001) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant (one-way ANOVA with post-hoc Tukey HSD; p < 0.001).


Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG - PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters , as indicated , shown for one - month - old plants . Scale bar , 10 cm . Complementation with the pKNOLLE - driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2 / M phase .

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Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG-PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters, as indicated, shown for one-month-old plants. Scale bar, 10 cm. Complementation with the pKNOLLE-driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2/M phase.


PI staining of pKNOLLE : FLAG - PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects . Arrowhead , cell wall stub in the pi4kβ1 pi4kβ2 double mutant . Scale bars , 50 µm .

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PI staining of pKNOLLE:FLAG-PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects. Arrowhead, cell wall stub in the pi4kβ1 pi4kβ2 double mutant. Scale bars, 50 µm.


In vivo localization of mCherry - PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five - day - old complemented pi4kβ1 pi4kβ2 plants . The mCherry - PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole ( VP ) mode with the pinhole set to 2 . Arrowheads , nascent cell plates decorated by mCherry - PI4Kβ1 . Scale bar , 20 µm . Whole - mount immunostaining of five - day - old seedlings expressing mCherry - PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti - tubulin ( red ) and anti - mCherry ( green ) antibodies , and DAPI ( blue ) . ( І ) , ( ІІ ) , magnifications of regions marked in b , representing early and late cytokinetic stages . Scale bars , 20 µm .

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In vivo localization of mCherry-PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five-day-old complemented pi4kβ1 pi4kβ2 plants. The mCherry-PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry-PI4Kβ1. Scale bar, 20 µm. Whole-mount immunostaining of five-day-old seedlings expressing mCherry-PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti-tubulin (red) and anti-mCherry (green) antibodies, and DAPI (blue). (І), (ІІ), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 µm.


Relative localization of mCherry - PI4Kβ1 and the TGN marker , ARF1 , during cytokinesis . Four - day - old seedlings were immunostainned with anti - mCherry ( green ) and anti - ARF1 ( red ) antibodies . Scale bars are 10 µm .

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Relative localization of mCherry-PI4Kβ1 and the TGN marker, ARF1, during cytokinesis. Four-day-old seedlings were immunostainned with anti-mCherry (green) and anti-ARF1 (red) antibodies. Scale bars are 10 µm.


The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five - day - old root tip meristems . Images shown are from 3 roots ( wild type ) and 7 roots ( pi4kβ1 pi4kβ2 double mutant ) . In wild type controls ( A ) , vesicles fused at the cell division plane to give rise to tubular network ( TN ) of a nascent cell plate . White arrowheads , fused vesicles at the division plane . n = 8 cells . In the pi4kβ1 pi4kβ2 double mutant ( B ) , vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse . The nuclei of both wild type and double mutant cells were at telophase or interphase , as judged by the presence of decondensed chromatin and a nuclear envelope . At this stage , failed cell plate formation was never seen in wild type ( A ) . Black arrowheads , unfused vesicles . n = 6 cells . Unfused vesicles clustered around a cell wall stub ( arrow ) . White arrowheads , light vesicles ; black arrowheads , dark vesicles ; black arrow , cell wall stub . n = 8 cells . No cross wall was seen in this cell with two nuclei . n = 6 cells . n , nucleus ; ne , nuclear envelope . Scale bars in magnified figures , 2 μm ; in insets , 1 μm .

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The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five-day-old root tip meristems. Images shown are from 3 roots (wild type) and 7 roots (pi4kβ1 pi4kβ2 double mutant). In wild type controls (A), vesicles fused at the cell division plane to give rise to tubular network (TN) of a nascent cell plate. White arrowheads, fused vesicles at the division plane. n = 8 cells. In the pi4kβ1 pi4kβ2 double mutant (B), vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse. The nuclei of both wild type and double mutant cells were at telophase or interphase, as judged by the presence of decondensed chromatin and a nuclear envelope. At this stage, failed cell plate formation was never seen in wild type (A). Black arrowheads, unfused vesicles. n = 6 cells. Unfused vesicles clustered around a cell wall stub (arrow). White arrowheads, light vesicles; black arrowheads, dark vesicles; black arrow, cell wall stub. n = 8 cells. No cross wall was seen in this cell with two nuclei. n = 6 cells. n, nucleus; ne, nuclear envelope. Scale bars in magnified figures, 2 μm; in insets, 1 μm.


The effects of reduced association of PtdIns ( 4 ) P with the cell plate on the recruitment of CLC2 - GFP were analyzed in root meristem cells of five - day - old plants . Top , Cell plate - associated fluorescence of the PtdIns ( 4 ) P reporter , 2 × mCherryFAPP1 - PH , was monitored at the end of cytokinesis in five - day - old wild type controls or pi4kβ1 pi4kβ2 double mutants , as indicated . Scale bars , 10 µm . Bottom , The intensity of 2 × mCherryFAPP1 - PH signals was quantified at the end of cytokinesis at the cell plate ( left plot ) and at the TGN ( right plot ) . Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane ( wild type , n = 37 cells , 24 roots ; pi4kβ1 pi4kβ2 , n = 55 cells , 41 roots ) . TGN intensities were recorded using the Airyscan VP mode , and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity ( wild type , n = 30 cells , 22 roots ; pi4kβ1 pi4kβ2 , n = 37 cells , 31 roots ) . * * * , significant differences ( p < 0 . 0001 ) according to two - tailed Mann - Whitney U - tests .

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The effects of reduced association of PtdIns(4)P with the cell plate on the recruitment of CLC2-GFP were analyzed in root meristem cells of five-day-old plants. Top, Cell plate-associated fluorescence of the PtdIns(4)P reporter, 2×mCherryFAPP1-PH, was monitored at the end of cytokinesis in five-day-old wild type controls or pi4kβ1 pi4kβ2 double mutants, as indicated. Scale bars, 10 µm. Bottom, The intensity of 2×mCherryFAPP1-PH signals was quantified at the end of cytokinesis at the cell plate (left plot) and at the TGN (right plot). Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane (wild type, n = 37 cells, 24 roots; pi4kβ1 pi4kβ2, n = 55 cells, 41 roots). TGN intensities were recorded using the Airyscan VP mode, and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity (wild type, n=30 cells, 22 roots; pi4kβ1 pi4kβ2, n=37 cells, 31 roots). ***, significant differences (p<0.0001) according to two-tailed Mann-Whitney U-tests.


Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of wild type seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks .

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Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of wild type seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.


Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks .

Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.


Left , quantification of the time of CLC2 - GFP appearance at the cell plate in wild type ( B ) or the pi4kβ1 pi4kβ2 double mutant ( C ) ( wild type , n = 10 cells , 7 roots ; pi4kβ1 pi4kβ2 , n = 24 cells , 19 roots ) . Right , quantification of CLC2 - GFP at the TGN in interphase cells . Five - day - old roots expressing CLC2 - GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity ( wild type , n = 14 cells , 2 roots ; pi4kβ1 pi4kβ2 , n = 15 cells , 2 roots ) .

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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

Left, quantification of the time of CLC2-GFP appearance at the cell plate in wild type (B) or the pi4kβ1 pi4kβ2 double mutant (C) (wild type, n = 10 cells, 7 roots; pi4kβ1 pi4kβ2, n = 24 cells, 19 roots). Right, quantification of CLC2-GFP at the TGN in interphase cells. Five-day-old roots expressing CLC2-GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity (wild type, n = 14 cells, 2 roots; pi4kβ1 pi4kβ2, n = 15 cells, 2 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Five - day - old seedlings were immunostained with anti - KNOLLE ( green ) and counterstained with DAPI ( blue ) . In the pi4kβ1 pi4kβ2 double mutant , diffusion of KNOLLE occurred at the plasma membrane ( arrowheads ) . Scale bars , 10 μm . Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane ( PM ) from ( A ) . Data are mean ± SD from four independent experiments . * * , a significant difference ( p < 0 . 01 ) according to a two - tailed Student ' s t test ( untreated wild type , n = 97 cells , 25 roots ; untreated pi4kβ1 pi4kβ2 , n = 142 cells , 44 roots ; BFA treated wild type , n = 126 cells , 28 roots ; BFA treated pi4kβ1 pi4kβ2 , n = 183 cells , 43 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Five-day-old seedlings were immunostained with anti-KNOLLE (green) and counterstained with DAPI (blue). In the pi4kβ1 pi4kβ2 double mutant, diffusion of KNOLLE occurred at the plasma membrane (arrowheads). Scale bars, 10 μm. Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane (PM) from (A). Data are mean ± SD from four independent experiments. **, a significant difference (p<0.01) according to a two-tailed Student's t test (untreated wild type, n= 97 cells, 25 roots; untreated pi4kβ1 pi4kβ2, n = 142 cells, 44 roots; BFA treated wild type, n = 126 cells, 28 roots; BFA treated pi4kβ1 pi4kβ2, n = 183 cells, 43 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were immunostained with anti - KNOLLE antibodies ( green ) and counterstained with DAPI ( blue ) . Scale bars , 10 μm . Quantification of KNOLLE intensity at the cell plate from ( C ) , normalized to the signal intensity of intracellular compartments . * * * , a significant difference ( p < 0 . 0001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 26 cells , 20 roots ; pi4kβ1 pi4kβ2 , n = 41 cells , 37 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were immunostained with anti-KNOLLE antibodies (green) and counterstained with DAPI (blue). Scale bars, 10 μm. Quantification of KNOLLE intensity at the cell plate from (C), normalized to the signal intensity of intracellular compartments. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 26 cells, 20 roots; pi4kβ1 pi4kβ2, n = 41 cells, 37 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Enhanced accumulation of PIN2 - GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were stained with 2 μM FM 4 - 64 for 3 minutes at room temperature . Scale bars , 10 μm . Quantification of PIN2 - GFP signal at the cell plate from ( E ) , normalized to the intensity at the apical plasma membrane intensity , as indicated by the dashed lines . * * * , a significant difference ( p < 0 . 001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 28 cells , 16 roots ; pi4kβ1 pi4kβ2 , n = 45 cells , 26 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of PIN2-GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were stained with 2 μM FM 4-64 for 3 minutes at room temperature. Scale bars, 10 μm. Quantification of PIN2-GFP signal at the cell plate from (E), normalized to the intensity at the apical plasma membrane intensity, as indicated by the dashed lines. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 28 cells, 16 roots; pi4kβ1 pi4kβ2, n = 45 cells, 26 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . panels g - j display data from the analysis of interphase cells . Internalization of PIN2 - GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min , then washed and incubated with 50 μM CHX and 25 μM BFA . Scale bars , 10 μm . Quantification of punctate signals from ( G ) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants . Data are mean ± SD . * , a significant difference ( p < 0 . 05 ) according to a two - tailed Student ' s t test ; ( wild type , n = 116 cells , 6 roots ; pi4kβ1 pi4kβ2 , n = 110 cells , 7 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Internalization of PIN2-GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min, then washed and incubated with 50 μM CHX and 25 μM BFA. Scale bars, 10 μm. Quantification of punctate signals from (G) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants. Data are mean ± SD. *, a significant difference (p<0.05) according to a two-tailed Student's t test; (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 110 cells, 7 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . panels g - j display data from the analysis of interphase cells . Delayed internalization of FM 4 - 64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant . Four - day - old seedlings were pulsed with 2 μM FM 4 - 64 for 3 minutes on ice and then fluorescence was recorded for 20 min . Scale bars , 10 μm . Quantification of intracellular FM 4 - 64 signal from ( i ) , normalized to apical and basal plasma membrane intensities . * * * , a significant difference ( p < 0 . 0001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 116 cells , 6 roots ; pi4kβ1 pi4kβ2 , n = 66 cells , 9 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Delayed internalization of FM 4-64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant. Four-day-old seedlings were pulsed with 2 μM FM 4-64 for 3 minutes on ice and then fluorescence was recorded for 20 min. Scale bars, 10 μm. Quantification of intracellular FM 4-64 signal from (i), normalized to apical and basal plasma membrane intensities. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 66 cells, 9 roots).


pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Immunostaining of meristem cells of five - day - old plants using anti - tubulin ( green ) counterstained with DAPI ( false colour red ) . Asterisks , multiple nuclei in one cell ; arrowheads , multiple phragmoplasts in one cell . Dashed lines indicate the outlines of exemplary multinucleated cells . Scale bars , 10 µm .

B-CONTROLLED_VAR I-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

pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Immunostaining of meristem cells of five-day-old plants using anti-tubulin (green) counterstained with DAPI (false colour red). Asterisks, multiple nuclei in one cell; arrowheads, multiple phragmoplasts in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.


pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Whole - mount immunostaining of root tips of five - day - old seedlings . Green , anti - KNOLLE ; red , anti - tubulin ; blue , DAPI . ( І ) , ( ІІ ) , magnifications of areas highlighted in dashed boxes . Arrowheads , solid phragmoplasts with thicker cell plates . Scale bars , 20 µm .

B-CONTROLLED_VAR I-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 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

pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Whole-mount immunostaining of root tips of five-day-old seedlings. Green, anti-KNOLLE; red, anti-tubulin; blue, DAPI. (І), (ІІ), magnifications of areas highlighted in dashed boxes. Arrowheads, solid phragmoplasts with thicker cell plates. Scale bars, 20 µm.


pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Quantification of the incidences of multinucleated cells , multiple phragmoplasts and solid phragmoplasts during late cytokinesis ( multinucleated cells : wild type , n = 316 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 673 cells , 44 roots ; multiple phragmoplasts : wild type , n = 64 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 188 cells , 44 roots ; solid phragmoplast in late cytokinesis : wild type , n = 41 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 89 cells , 44 roots ) . Data are mean ± SD from three independent experiments . n . d . not detected .

B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-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

pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Quantification of the incidences of multinucleated cells, multiple phragmoplasts and solid phragmoplasts during late cytokinesis (multinucleated cells: wild type, n = 316 cells, 29 roots; pi4kβ1 pi4kβ2, n = 673 cells, 44 roots; multiple phragmoplasts: wild type, n = 64 cells, 29 roots; pi4kβ1 pi4kβ2, n = 188 cells, 44 roots; solid phragmoplast in late cytokinesis: wild type, n = 41 cells, 29 roots; pi4kβ1 pi4kβ2, n = 89 cells, 44 roots). Data are mean ± SD from three independent experiments. n.d. not detected.


The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging . Five - day - old seedlings expressing mCherry - TUA5 were imaged at 1 frame per 2 min . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Cells are outlined by dashed lines in the first and last frames of each series . White arrowhead , ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant . Scale bars , 10 µm . Plot profiles obtained from dashed lines marked in ( A ) . Black arrowhead , ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant .

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

The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging. Five-day-old seedlings expressing mCherry-TUA5 were imaged at 1 frame per 2 min. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Cells are outlined by dashed lines in the first and last frames of each series. White arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. Scale bars, 10 µm. Plot profiles obtained from dashed lines marked in (A). Black arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant.


The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging . 3D projections from time points selected from ( A ) , when the transition to a ring phragmoplast has occurred in wild type , but solid phragmoplast persisted in the double mutant . Scale bars , 10 µm . Duration of phragmoplast persistence during cytokinesis , determined from initiation to disbanding . * * * , a significant difference ( p < 0 . 001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 12 cells , 9 roots ; pi4kβ1 pi4kβ2 , n = 21 cells , 16 roots ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O O O O O O O O O O

The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging. 3D projections from time points selected from (A), when the transition to a ring phragmoplast has occurred in wild type, but solid phragmoplast persisted in the double mutant. Scale bars, 10 µm. Duration of phragmoplast persistence during cytokinesis, determined from initiation to disbanding. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 12 cells, 9 roots; pi4kβ1 pi4kβ2, n = 21 cells, 16 roots).


The dynamics of the microtubule bundling protein , MAP65 - 3 , were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants . Image series of 3D projections of GFP - MAP65 - 3 costained with FM 4 - 64 in root tips of four - day - old wild type seedlings . The best angle for visualization was obtained by rotation of the x - axis . Images are representative for 8 cells from 5 roots . Scale bar , 10 µm . Image series of 3D projections of GFP - MAP65 - 3 costained with FM 4 - 64 in root tips of four - day - old pi4kβ1 pi4kβ2 seedlings . The best angle for visualization was obtained by rotation of the x - axis . Images are representative for 11 cells from 10 roots . Arrowheads , persisting signal of GFP - MAP65 - 3 in the center of the cell division plane . Scale bars , 10 µm . Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts , marked by dashed lines in ( A ) and ( B ) . Arrowhead , persisting signal of GFP - MAP65 - 3 in the center of the cell division plane . Times in ( A - C ) are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 .

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-CONTROLLED_VAR I-CONTROLLED_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 O O O O 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 O O O O O O O O O O B-CONTROLLED_VAR I-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 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 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

The dynamics of the microtubule bundling protein, MAP65-3, were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old wild type seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 8 cells from 5 roots. Scale bar, 10 µm. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old pi4kβ1 pi4kβ2 seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 11 cells from 10 roots. Arrowheads, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Scale bars, 10 µm. Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts, marked by dashed lines in (A) and (B). Arrowhead, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Times in (A-C) are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0.


Medial confocal planes from z - stacks from ( a ) and ( b ) demonstrating attachment of the FM 4 - 64 - stained cell plate with the peripheral plasma membrane , defining t0 . Scale bars , 10 µm .

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Medial confocal planes from z-stacks from (a) and (b) demonstrating attachment of the FM 4-64-stained cell plate with the peripheral plasma membrane, defining t0. Scale bars, 10 µm.


3D reconstruction of ring and disc - shaped immunofluorescence patterns for GFP - MAP65 - 3 and microtubules , based on super resolution structured illumination microscopy ( SIM ) , costained with DAPI . Top panels , wild type ; bottom panels , pi4kβ1 pi4kβ2 double mutants . Scale bars , 10 µm .

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

3D reconstruction of ring and disc-shaped immunofluorescence patterns for GFP-MAP65-3 and microtubules, based on super resolution structured illumination microscopy (SIM), costained with DAPI. Top panels, wild type; bottom panels, pi4kβ1 pi4kβ2 double mutants. Scale bars, 10 µm.


The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants . Crosses of pi4kβ1 pi4kβ2 and mpk4 - 2 mutants resulted in combined genotypes with increased growth defects , as indicated . 14 - day - old seedlings are shown . Scale bars , 1 cm . Top , The severe growth defects of the combined genotypes were accompanied by increased tissue distortion , as shown by 3D projections after propidium iodide staining of root tips of 14 - day - old seedlings . Scale bars , 100 µm . Bottom , Multinucleation of cells was increased in the combined genotypes , as evident from immunofluorescence of microtubules ( red ) counterstained with DAPI ( blue ) . Asterisks , multiple nuclei in one cell . Dashed lines indicate the outlines of exemplary multinucleated cells . Scale bars , 10 µm .

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

The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants. Crosses of pi4kβ1 pi4kβ2 and mpk4-2 mutants resulted in combined genotypes with increased growth defects, as indicated. 14-day-old seedlings are shown. Scale bars, 1 cm. Top, The severe growth defects of the combined genotypes were accompanied by increased tissue distortion, as shown by 3D projections after propidium iodide staining of root tips of 14-day-old seedlings. Scale bars, 100 µm. Bottom, Multinucleation of cells was increased in the combined genotypes, as evident from immunofluorescence of microtubules (red) counterstained with DAPI (blue). Asterisks, multiple nuclei in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.


Quantification of cytokinetic defects , as shownin the bottom panels of ( B ) . Cytokinetic defects were enhanced in pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) ( for multinucleated cells , including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells , both according to a Welch ANOVA with Games - Howell post - hoc test ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ( wild type , n = 1650 cells , 13 roots ; pi4kβ1 pi4kβ2 , n = 1441 cells , 19 roots ; mpk4 - 2 , n = 1429 cells , 16 roots ; pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) , n = 574 cells , 16 roots ) ; and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 05 , lowercase letters ( wild type , n = 1981 cells , 13 roots ; pi4kβ1 pi4kβ2 , n = 1758 cells , 19 roots ; mpk4 - 2 , n = 1721 cells , 16 roots , pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) , n = 660 cells , 16 roots ) .

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Quantification of cytokinetic defects, as shownin the bottom panels of (B). Cytokinetic defects were enhanced in pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-) (for multinucleated cells, including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells, both according to a Welch ANOVA with Games-Howell post-hoc test; ***, p < 0.001; **,p < 0.01 (wild type, n = 1650 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1441 cells, 19 roots; mpk4-2, n = 1429 cells, 16 roots; pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 574 cells, 16 roots); and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one-way ANOVA with post-hoc Tukey HSD; p < 0.05, lowercase letters (wild type, n = 1981 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1758 cells, 19 roots; mpk4-2, n = 1721 cells, 16 roots, pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 660 cells, 16 roots).


Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . PI4Kβ1 interacts with MPK4 in yeast two hybrid tests . MPK4 was used as a bait , while PI4Kβ1 was divided into two fragments to be used as prey . The results are representative for three independent experiments .

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Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. PI4Kβ1 interacts with MPK4 in yeast two hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments.


Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . In vivo co - immunoprecipitation of MPK4 and PI4Kβ1 from 14 - day - old plants . PI4Kβ1 was specifically co - precipitated with MPK4 - myc using anti - myc antibodies . Arrowhead , Migration of PI4Kβ1 . The results are representative for three independent experiments .

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Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. In vivo co-immunoprecipitation of MPK4 and PI4Kβ1 from 14-day-old plants. PI4Kβ1 was specifically co-precipitated with MPK4-myc using anti-myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments.


Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . Coordination of MPK4 - YFP and mCherry - PI4Kβ1 during cytokinesis in five - day - old root meristem cells . MPK4 co - localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates . Images were obtained with an LSM880 in Airyscan super - resolution ( SR ) mode . Scale bars , 10 µm .

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Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. Coordination of MPK4-YFP and mCherry-PI4Kβ1 during cytokinesis in five-day-old root meristem cells. MPK4 co-localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super-resolution (SR) mode. Scale bars, 10 µm.


Live cell time lapse of MPK4 - YFP and mRFPFAPP1 - PH during cytokinesis , obtained with an LSM880 in Airyscan resolution versus sensitivity ( R - S ) mode . The time series is representative for 6 cells from 5 roots . Arrowheads indicate the initiation and leading edges of the growing cell plate . Scale bar , 10 µm .

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Live cell time lapse of MPK4-YFP and mRFPFAPP1-PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R-S) mode. The time series is representative for 6 cells from 5 roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 µm.


Left panels , The distribution of mRFPFAPP1 - PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls ( top ) and mpk4 - 2 mutants ( bottom ) . Intensity profiles were recorded as indicated by the dashed lines . Scale bars , 10 µm . Right top , intensity of mRFPFAPP1 - PH at the cell plate normalized vs . the intensity at the apical plasma membrane ( wild type , n = 43 cells , 34 roots ; mpk4 - 2 , n = 32 cells , 28 roots ) . Right bottom , intensity of mRFPFAPP1 - PH at the TGN normalized vs . the intensity at the apical plasma membrane ( wild type , n = 40 cells , 29 roots ; mpk4 - 2 , n = 26 cells , 23 roots ) . * , a significant difference ( p < 0 . 05 ) according to a two - tailed Student ' s test .

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Left panels, The distribution of mRFPFAPP1-PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls (top) and mpk4-2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 µm. Right top, intensity of mRFPFAPP1-PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4-2, n = 32 cells, 28 roots). Right bottom, intensity of mRFPFAPP1-PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots ; mpk4-2, n = 26 cells, 23 roots). *, a significant difference (p<0.05) according to a two-tailed Student's test.


( A ) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients .

(A) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients.


( B ) RT - PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples .

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(B) RT-PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples.


( C ) RT - PCR analysis of the expression of IFN - γ and TNF - α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients . ( D ) RT - PCR analysis of the expression of miR - 125b - 5p and miR - 99a - 5p in γδ T cells isolated from indicated samples . ( E ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from indicated samples .

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(C) RT-PCR analysis of the expression of IFN-γ and TNF-α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients. (D) RT-PCR analysis of the expression of miR-125b-5p and miR-99a-5p in γδ T cells isolated from indicated samples. (E) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from indicated samples.


( A ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from thymic biopsies , cultured for 11 days with IL - 7 or IL - 2 ( n = 12 - 15 independent biological samples ) . ( B ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells freshly isolated from thymic biopsies ( Thymus ) and PBLs ( n = 6 - 14 independent biological samples ) .

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(A) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymic biopsies, cultured for 11 days with IL-7 or IL-2 (n=12-15 independent biological samples). (B) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells freshly isolated from thymic biopsies (Thymus) and PBLs (n=6-14 independent biological samples).


( C - D ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) in γδ T cells isolated from thymus ( C ) or from PBLs ( D ) and cultured with the indicated cytokines , respectively for 11 days ( C ) or for 4 - 6 days ( D ) . When indicated , γδ PBLs were also co - cultured with an anti - TGF - β blocking antibody . Data are normalized to the value obtained in IL - 7 cultures ( n = 4 - 10 independent biological samples ) .

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(C-D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4-6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n=4-10 independent biological samples).


( E - F ) RT - PCR analysis of the expression of miR - 181a ( - 5p and - 2 - 3p ) versus FACS analysis of the expression of TNF - α ( left panel ) , IFN - γ ( middle panel ) and NKGD2 ( right panel ) in γδ T cells isolated from thymus ( E ) and PBLs ( F ) . Samples were either freshly isolated or cultured for 11 days ( γδ thymocytes ) or for 6 days ( γδ PBLs ) with the indicated cytokines ( n = 5 independent biological samples ) .

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(E-F) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel) and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n=5 independent biological samples).


( D ) FACS analysis of the expression of Annexin V in miR - 181a versus empty transduced ( GFP + ) and untransduced ( GFP - ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) .

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(D) FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).


( E ) RT - PCR analysis of the expression of Bcl2 and Bcl - xL ( normalized to the values obtained with the empty virus ) in transduced ( GFP + ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) .

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(E) RT-PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).


( F ) FACS - derived expression of indicated surface and intracellular markers in miR - 181a versus empty transduced ( GFP + ) and untransduced ( GFP - ) γδ T thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 7 - 11 independent biological samples ) .

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(F) FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=7-11 independent biological samples).


( B ) RT - PCR analysis of the expression of the indicated genes associated with Mapk , Notch , Irf4 or Stat1 signaling in sorted GFP + γδ thymocytes transduced with empty or miR - 181a viral vector . Data are normalized to the value obtained with the empty virus . Samples were cultured with IL - 7 plus IL - 2 for 11 days ( n = 3 - 5 independent biological samples ) .

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(B) RT-PCR analysis of the expression of the indicated genes associated with Mapk, Notch, Irf4 or Stat1 signaling in sorted GFP+ γδ thymocytes transduced with empty or miR-181a viral vector. Data are normalized to the value obtained with the empty virus. Samples were cultured with IL-7 plus IL-2 for 11 days (n=3-5 independent biological samples).


( C ) Dual luciferase reporter assay performed to verify binding between miR - 181a and Map3k2 , Notch2 , Irf4 or Stat1 mRNA . HEK293 T cells were co - transfected with a pmirGLO Dual - Luciferase miRNA Target Expression Vector containing either the WT or mutated ( Mut ) 3 ′ UTR target sites plus miR - 181a . A negative construct ( without miR - 181a binding sites ) was included . Firefly / Renilla ratios were normalized to those obtained for the empty vector ( n = 4 independent biological samples ) .

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(C) Dual luciferase reporter assay performed to verify binding between miR-181a and Map3k2, Notch2, Irf4 or Stat1 mRNA. HEK293 T cells were co-transfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing either the WT or mutated (Mut) 3′ UTR target sites plus miR-181a. A negative construct (without miR-181a binding sites) was included. Firefly/Renilla ratios were normalized to those obtained for the empty vector (n=4 independent biological samples).


( E - G ) RT - PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression ( E ) , Nkg2d or Tnfa expression ( F ) , and NKG2D or TNF - α protein expression ( G ) after siRNA - mediated knockdown of Map3k2 or Notch2 . In each experiment , a control condition was also used where a nontargeting negative control siRNA was transfected . γδ were cultured with IL - 7 for 4 days ( n = 6 independent biological samples ) .

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(E-G) RT-PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression (E), Nkg2d or Tnfa expression (F), and NKG2D or TNF-α protein expression (G) after siRNA-mediated knockdown of Map3k2 or Notch2. In each experiment, a control condition was also used where a nontargeting negative control siRNA was transfected. γδ were cultured with IL-7 for 4 days (n=6 independent biological samples).


( B ) Cytoplasmic TDP - 43 immunostaining is elevated not only in poly - GA transduced neurons , but also in the non - transduced receiver cells . White and red arrows indicate cells with cytoplasmic TDP - 43 in GFP positive and negative cells , respectively .

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(B) Cytoplasmic TDP-43 immunostaining is elevated not only in poly-GA transduced neurons, but also in the non-transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively.


( C ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . Cells with and without GFP signal were counted separately ( indicated by + / − ) . Two groups ( GFP negative donor , GFP positive receiver ) were excluded due to very high GFP transduction rate and very low GFP transmission rate . n = 4 biological replicates . In total 283 donor GFP , 273 donor GA175 - GFP , 284 receiver GFP and 266 receiver GA175 - GFP cells were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 .

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(C) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP negative donor, GFP positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n=4 biological replicates. In total 283 donor GFP, 273 donor GA175-GFP, 284 receiver GFP and 266 receiver GA175-GFP cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.


Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . Immunofluorescence staining of TDP - 43 ∆ NLS aggregate number per cell

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Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. Immunofluorescence staining of TDP-43∆NLS aggregate number per cell


Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . Immunofluorescence staining of TDP - 43 ∆ NLS aggregate number per

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Co - culture model in HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and TDP - 43ΔNLS - GFP in donor and receiver compartment . automatic quantification of TDP - 43 ∆ NLS aggregate number per cell In ( E ) n = 3 biological replicates with 368 donor iRFP , 251 donor GA175 - iRFP , 430 receiver iRFP and 328 GA175 - iRFP cells were analyzed . Cells with and without GFP signal were analyzed separately ( indicated by + / − ) . White and red arrows indicate cells with cytoplasmic TDP - 43 in GFP positive and negative cells , respectively . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. automatic quantification of TDP-43∆NLS aggregate number per cell In (E) n=3 biological replicates with 368 donor iRFP, 251 donor GA175-iRFP, 430 receiver iRFP and 328 GA175-iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/−). White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( F ) in addition to filter trap assay of SDS - insoluble TDP - 43ΔNLS - GFP aggregates compared in iRFP or GA175 - iRFP transfected cells .

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 B-CONTROLLED_VAR O O O O O

(F) in addition to filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates compared in iRFP or GA175-iRFP transfected cells.


( G ) GFP mRNA expression levels were measured by qPCR . RNA levels were normalized to GAPDH , β - actin and β2 - microglobulin mRNA . Bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction . * denotes p < 0 . 05 , and * * denotes p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction. * denotes p<0.05, and ** denotes p<0.01.