( C ) Immunopurification of CPAP complexes from CCB02 - treated extracts . ( i ) CCB02 specifically prevents CPAP - Tubulin interaction . This allows CPAP to bind an enhanced amount of Cep152 γ - tubulin . Control IP experiment is shown below . ( ii ) Plot showing PCM intensities co - immunoprecipitated with CPAP at various concentrations of CCB02 treatment . CPAP amount does not change with CCB02 treatment . Data represent mean ± . ( N ) = 3 .

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

(C) Immunopurification of CPAP complexes from CCB02-treated cell extracts. (i) CCB02 specifically prevents CPAP-Tubulin interaction. This allows CPAP to bind an enhanced amount of Cep152 and γ-tubulin. Control IP experiment is shown below. (ii) Plot showing PCM protein intensities co-immunoprecipitated with CPAP at various concentrations of CCB02 treatment. CPAP amount does not change with CCB02 treatment. Data represent mean ± SEM. (N)=3.


( B - D ) MT - regrowth assays at 0 , 1 . 5 3 . 0 min using MCF10A ( - Dox , two centrosomes ) , MCF10A ( + Dox , extra centrosomes ) MDA - MB - 231 . MT nucleation panel is shown in grey scale ( inset are inverted ) γ - tubulin is shown in red . Note that in contrast to vehicle treatment , CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1 . 5 min after induction of regrowth with simultaneous increase in γ - tubulin recruitment . these were stained with γ - tubulin ( red ) , microtubules ( α - tubulin , grey ) ( DAPI blue ) . Scale bar , 2µm insets , 0 . 5 µm . Bar graphs show MT γ - tubulin intensities at 3 min after induction of MT regrowth . ( N ) = 3 . At least 80 centrosomes were considered to calculate intensities from each . Error bars , mean ± . Unpaired t - * * P < 0 . 001 , * * * P < 0 . 0001 .

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

(B-D) MT-regrowth assays at 0, 1.5 and 3.0 min using MCF10A (-Dox, two centrosomes), MCF10A (+Dox, extra centrosomes) and MDA-MB-231 cells. MT nucleation panel is shown in grey scale (inset images are inverted) and γ-tubulin is shown in red. Note that in contrast to vehicle treatment, CCB02 treatment caused centrosomes to nucleate an enhanced level of microtubules already at 1.5 min after induction of regrowth with simultaneous increase in γ-tubulin recruitment. All these cells were stained with γ-tubulin (red), microtubules (α-tubulin, grey) and DNA (DAPI blue). Scale bar, 2µm and insets, 0.5 µm. Bar graphs show MT and γ-tubulin intensities at 3 min after induction of MT regrowth. (N)=3. At least 80 centrosomes were considered to calculate intensities from each cell line. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001.


( A ) Bar graph quantifies relative of PCM at interphase centrosomes of treated with vehicle , CCB02 , known tubulin binders . Two centrosomes containing MCF10A ( - Dox , two centrosomes ) were used . Elevated PCM intensities were detected only with CCB02 compared to vehicle treated . However , a slight increase in PCNT was observed with Docetaxel treatment . Number of centrosomes tested , n = 100 ( vehicle ) ; n = 100 ( for CCB02 , Taxol , Bacatalin III , Docetaxel Vinblastine ) . ( N ) = 3 . Error bars , mean ± . Ordinary Two - way ANOVA * * P < 0 . 001 , * * * P < 0 .

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

(A) Bar graph quantifies relative intensity of PCM proteins at interphase centrosomes of cells treated with vehicle, CCB02, and known tubulin binders. Two centrosomes containing MCF10A (-Dox, two centrosomes) cells were used. Elevated PCM proteins intensities were detected only with CCB02 compared to vehicle treated cells. However, a slight increase in PCNT was observed with Docetaxel treatment. Number of centrosomes tested, n=100 (vehicle); n=100 (for CCB02, Taxol, Bacatalin III, Docetaxel and Vinblastine). (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001, ***P < 0.0001


( B ) CCB02 but not conventional tubulin binders ( Taxol , Bacatalin III , Docetaxel Vinblastine ; concentrations used 30nM ) causes activation of extra centrosomes prevents them from clustering . MCF10A with extra centrosomes ( + Dox , extra centrosomes ) were used . In contrast to CCB02 - treatment , interphase centrosomes of tubulin binders - treated consistently display clustered centrosomes . Note , disrupted mitotic spindles were observed in tubulin binders - treated due to their general toxicity to microtubules . were stained with CPAP ( green ) , Cep152 ( magenta ) microtubules ( α - tubulin , red ) . Scale bar , 2µm . Three independent experiments ( N ) = 3 with each at least 100 were examined per

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

(B) CCB02 but not conventional tubulin binders (Taxol, Bacatalin III, Docetaxel and Vinblastine; concentrations used 30nM) causes activation of extra centrosomes and prevents them from clustering. MCF10A cells with extra centrosomes (+Dox, extra centrosomes) were used. In contrast to CCB02-treatment, interphase centrosomes of tubulin binders-treated cells consistently display clustered centrosomes. Note, disrupted mitotic spindles were observed in tubulin binders-treated cells due to their general toxicity to microtubules. Cells were stained with CPAP (green), Cep152 (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm. Three independent experiments (N)=3 with each at least 100 cells were examined per treatment


( C ) Bar graph quantifies percentages of exhibiting multipolar spindles in interphase mitosis of vehicle , CCB02 conventional tubulin binders - treated . At least 150 were analyzed for each independent experiment ( N ) = 3 . Error bars , mean ± . Ordinary Two - way ANOVA * * P < 0 .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Bar graph quantifies percentages of cells exhibiting multipolar spindles in interphase and mitosis of vehicle, CCB02 and conventional tubulin binders-treated cells. At least 150 cells were analyzed for each independent experiment (N)=3. Error bars, mean ± SEM. Ordinary Two-way ANOVA test **P < 0.001


( D ) Kymographs ( i ) averaged microtubule polymerization velocities mediated by vehicle CCB02 . Graph below represents microtubule growth velocities mediated by vehicle ( red ) CCB02 ( blue ) during microtubule end - tracking ( ii ) . CCB02 does not alter microtubule growth although there is a slight increase in growth at 5µM . At least 75 filaments were analyzed for each condition . error bars represent mean ± of three independent experiments ( N ) =

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

(D) Kymographs (i) and averaged microtubule polymerization velocities mediated by vehicle and CCB02. Graph below represents the microtubule growth velocities mediated by vehicle (red) and CCB02 (blue) during microtubule end-tracking assay (ii). CCB02 does not alter the microtubule growth although there is a slight increase in growth at 5µM. At least 75 filaments were analyzed for each condition. The error bars represent mean ± SEM of three independent experiments (N)=3


( E ) Snap - shot show effect of CCB02 on microtubule dynamics in two - centrosome containing MCF10A . microtubule was stained with SiR - tubulin + end tip of microtubule was marked with EB3 - EGFP . A 5µM CCB02 did not alter localization dynamics of EB3 at microtubules indicating that CCB02 does not affect microtubule dynamic At least , 50 were analyzed for each condition . Scale bar ,

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

(E) Snap-shot images show the effect of CCB02 on microtubule dynamics in two-centrosome containing MCF10A cells. The microtubule was stained with SiR-tubulin and +end tip of microtubule was marked with EB3-EGFP. A 5µM CCB02 did not alter localization and dynamics of EB3 at the microtubules indicating that the CCB02 does not affect microtubule dynamic At least, 50 cells were analyzed for each condition. Scale bar, 2µm


( F ) Box plots represent speed ( µm / sec ) ( i ) , lifetime ( sec ) ( ii ) , ( µm ) ( iii ) of growing microtubule tracks in MCF10A treated with 1 , 2 5µM of CCB02 . At least , 60 filaments were analyzed from each from a total of 30 - 40 . ( N ) = 3 . Error bars in boxplots mean ± SD . Ordinary One - way ANOVA was performed . Boxes in plots show growing microtubule parameters such as speed ( µm / sec ) , lifetime ( sec ) ( µm / sec ) . Horizontal lines in boxes show median value . Whiskers at both extremes of boxes indicate minimum maximum values .

O O O O O O O O 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-SUBCELLULAR O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Box plots represent the speed (µm/sec) (i), lifetime (sec) (ii), and length (µm) (iii) of growing microtubule tracks in MCF10A cells treated with 1, 2 and 5µM of CCB02. At least, 60 filaments were analyzed from each cell from a total of 30-40 cells. (N)=3. Error bars in boxplots mean ± SD. Ordinary One-way ANOVA test was performed. Boxes in all plots show the growing microtubule parameters such as speed (µm/sec), lifetime (sec) and length (µm/sec). Horizontal lines in boxes show the median value. Whiskers at both extremes of boxes indicate minimum and maximum values.


( A ) Real time live imaging of NSCLC ( H1975T790M ) 3D spheroids with vehicle , 5µM erlotinib or 5µM CCB02 . At end of live imaging , spheroids were fixed stained for F - actin ( green ) . Arrow indicates invasive ( vehicle - treated ) non - invasive ( CCB02 - treated ) structures in spheroids . Scale bar ,

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

(A) Real time live imaging of NSCLC (H1975T790M) 3D spheroids with vehicle, 5µM erlotinib or 5µM CCB02. At the end of live imaging, spheroids were fixed and stained for F-actin (green). Arrow indicates invasive (vehicle-treated) and non-invasive (CCB02-treated) structures in spheroids. Scale bar, 100µm


( B ) Time series plot shows spheroid growth rate measurements . CCB02 treatment prevents spheroid growth rate by inhibiting formation of invasive protrusions At least 10 spheroids were measured for each condition . Error bars , mean ± . Unpaired t - * * * P < 0 .

O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

(B) Time series plot shows the spheroid growth rate measurements. CCB02 treatment prevents the spheroid growth rate by inhibiting the formation of invasive protrusions At least 10 spheroids were measured for each condition. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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 two ) or wild - type ( WT ) ( 11 months old , two one ) mice were immunoblotted with a Nrf2 antibody , 12E8 , PHF1 , a monoclonal to total tau ( Tau5 ) or a polyclonal to total tau ( Tau ) antibody . asterisk to left of Nrf2 blot designates position at which a nonspecific band migrated . relative molecular masses ( kDa ) are indicated to left of each blot . ( b ) Bar graphs represent relative optical density of phosphorylated or total tau in hippocampal tissues . n = 6 ( These data are on mouse hippocampi immunoblotted in Supplementary Fig . 1 ) . Data shown are mean ± s . e . were analysed using Student ' s t - . ( * * P0 . 01 ; * * * P0 . 001 )

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

(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 ) Ser396 / Ser404 ( PHF1 ) ( d ) . of delineated by dotted lines is enlarged shown in right corner of image . Right panels shows same slice stained with DAPI 4 ' , 6 - diamidino - 2 - phenylindole . Scale bar , 50 μm . DG , dentate gyrus ; Hi , dentate hilar .

O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE 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 two ) or wild - type ( 11 months old , two one ) mice were separated into sarkosyl - soluble ( SS ) insoluble ( SI ) fractions as described in Methods . level of total tau was analysed by immunoblotting using a monoclonal antibody to total tau ( Tau5 ) . relative molecular masses ( kDa ) are indicated to left of each blot . Bar graph represents relative optical density of sarkosyl - insoluble tau normalized with that of sarkosyl - soluble tau . n = 3 . Data shown are mean ± s . e . were analysed using Student ' s t - . ( * P0 . 05 ) .

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

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 ) cortical neurons ( DIV 5 ) were treated with SFN ( 10 μM , SFN ) for 12 or 24 h . ( c , d ) Bar graphs represent relative optical density of phosphorylated tau normalized with that of total tau . Data shown are mean ± s . e . of three independent experiments were analysed using Student ' s t - . ( * P0 . 05 ; * * P0 . 01 ) .

O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 presence of doxycycline ( 1 μg ml − 1 ) to induce expression of tau for 24 h , subsequently treated with SFN for 12 or 24 h . of tau phosphorylated at Ser262 / Ser356 Ser396 / Ser404 were analysed by immunoblotting using 12E8 PHF1 antibodies , respectively . Tau was detected with a polyclonal phospho - independent tau antibody ( Tau ) . relative molecular masses ( kDa ) are indicated to left of each blot . ( c , d ) Bar graphs represent relative optical density of phosphorylated tau normalized with that of total tau . Data shown are mean ± s . e . of three independent experiments were analysed using Student ' s t - . ( * P0 . 05 ; * * P0 . 01 ) .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 ; 5 months old , one ; 12 months old , two ) Nrf2 ( − / − ) ( 10 months old , two two ) mice . ( a ) Mouse brain tissues were homogenized in lysis buffer , 10 μg of lysates was incubated with 2 μg of GST - tau at 37 ° C for 30 min in presence or absence of 1 mM ATP . of tau phosphorylated at Ser262 / Ser356 Ser396 / Ser404 were analysed by immunoblotting using a 12E8 PHF1 antibodies , respectively . GST - tau was detected with a monoclonal GST antibody ( GST - tau ; Supplementary Fig . 4 ) . Bar graph of relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of duplicated independent experiments were analysed using Student ' s t - .

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

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 ; 5 months old , one ; 12 months old , two ) Nrf2 ( − / − ) ( 10 months old , two two ) mice . ( b ) Mouse brain tissues were homogenized in phosphatase storage buffer , phosphatase activity in lysates was quantified using serine / threonine phosphatase system ( Promega ) by measuring dephosphorylation of a phosphopeptide , RRA ( pT ) VA in presence or absence of okadaic acid ( OA , 20 nM ) . PP2A activity was defined as activity inhibited by addition of OA to phosphatase reaction mixture . n = 4 . Data shown are mean ± s . e . of three independent experiments were analysed using Student ' s t - .

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

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 stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . On next day ( a ) , were washed twice with PBS 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 maintained in presence of Dox without medium exchange .

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

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 stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . ( b ) were treated with either chloroquine ( 50 μM , CQ ) or bafilomycin A1 ( 10 nM , Baf A1 ) for 24 h . Samples in first lane were from maintained in presence of Dox without medium exchange .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-ORGANISM O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

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 were co - transfected with plasmid expressing tau together either with siRNA of beclin - 1 or scramble as a control , which was kept for 48 h .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 were maintained in presence of Dox ( 1 μg ml − 1 ) to induce expression of tau for 24 h , subsequently treated with SFN ( 10 μM ) for 12 h . culture medium was exchanged for fresh media containing Dox ( 1 μg ml − 1 ) but no SFN after washing with PBS . were then incubated for an additional 18 h in absence or presence of CQ ( 50 μM ) . of tau phosphorylated at Ser262 / Ser356 Ser396 / Ser404 were analysed by immunoblotting using 12E8 PHF1 antibodies , respectively . Tau , beclin - 1 LC3 were detected with appropriate polyclonal antibodies . relative molecular masses ( kDa ) are indicated to left of each blot . ( e ) Bar graphs represent relative optical density of phosphorylated tau , tau or LC3 - II normalized with that of actin . n = 4 . Data shown are mean ± s . e . were analysed using Student ' s t - . ( * P0 . 05 ; * * P0 . 01 ) .

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

(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 ) cortical neurons ( DIV 5 ) were treated with SFN ( 10 μM ) for 12 h , relative mRNA level of each gene was compared with that of not treated with SFN using qRT - PCR .

O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 ; 11 months old , one ; 5 months old , one ; 12 months old , three ) or wild - type ( 10 months old , four two ) mice , relative mRNA level of each gene compared with that of wild - type mice . n = 6 .

O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM 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 ) of p62 / SQSTM1 , Keap1 , mouse NDP52 ( mNDP52 ) Hsp70 in Nrf2 ( − / − ) or wild - type mice were examined by immunoblotting . asterisk on Nrf2 panel indicates a nonspecific band .

O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O B-GENEPROD 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 ) cortical neurons ( DIV 5 ) ( d ) SH - SY5Y ( e ) were treated with SFN for 24 h , expression of rat human NDP52 ( rNDP52 hNDP52 ) examined by immunoblotting using anti - NDP52 antibody . relative molecular masses ( kDa ) are indicated to left of each blot . ( d , e : low panel ) cortical neurons ( DIV 5 ) ( d ) SH - SY5Y ( e ) transiently transfected with pGL4 . 14 / human NDP52 promoter ( 2 , 173 bp ) luciferase reporter plasmid were treated with SFN for 24 h , assayed for luciferase activity .

O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD 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 were treated with SFN for 12 h , lysates were used for ChIP using anti - Nrf2 antibody as described in Methods . Data shown are mean ± s . e . were analysed using Student ' s t - . ( * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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 SH - SY5Y were transiently transfected with reporter plasmids with human NDP52 promoter ( 2 , 317 bp or deleted mutants ) , luciferase activity was assayed 24 h after transfection .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD 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 were co - transfected with reporter plasmids with human NDP52 promoter ( wild or deleted mutants ) along with either mock plasmid ( pcDNA3 . 1 ) or plasmid expressing Nrf2 , luciferase activity was assayed 24 h after transfection . ( d ) SH - SY5Y were transiently co - transfected with reporter plasmids with human NDP52 promoter or its mutant plasmid in which putative ARE was site - directly mutated ( X ) , together with mock plasmid ( pcDNA3 . 1 ) or plasmid expressing human Nrf2 . luciferase activity was assayed after 24 h . induced luciferase activity with co - expression with Nrf2 was not observed in a mutant NDP52 promoter in which three ARE sites ( − 2 , 057 , − 1 , 023 − 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 were analysed using Student ' s t - . ( * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) cortical neurons were transduced with lentivirus expressing sh - for Nrf2 or rat NDP52 ( rNDP52 ) , or a scrambled sh - at DIV 1 , were maintained until DIV 6 . of tau phosphorylated at Ser262 / Ser356 Ser396 / Ser404 were analysed by immunoblotting using 12E8 - PHF1 - specific antibodies , respectively . Total tau was detected with a polyclonal tau - specific antibody ( Tau ) . relative molecular masses ( kDa ) are indicated to left of each blot . ( b ) Bar graph of relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of three independent experiments were analysed using Student ' s t - . ( * P0 . 05 ; * * * P0 . 001 )

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

(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 ) 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 neurons incubated for 24 h ( DIV 6 ) . cortical neurons were fixed with 4 % paraformaldehyde , stained with 12E8 or PHF1 antibodies . Scale bar , 20 μm . optical density of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) . ( e ) Ser396 / Ser404 ( PHF1 ) ( f ) in soma of ~ 30 neurons randomly chosen was analysed with ImageJ program . Data were analysed using Student ' s t - ( * * * P0 . 001 ) .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O 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) 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 were co - transfected with plasmids expressing HA - hNDP52 or HA - tagged domain - deleted mutants of human NDP52 together with GFP - tau . Twenty - four hours later , lysates were prepared transfected tau was immunoprecipitated blots probed for both HA - hNDP52 constructs GFP - tau . Homogenates ( input ) used for co - immunoprecipitation were analysed by immunoblotting using anti - HA or a polyclonal tau - specific antibody ( Tau ) .

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

(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 were maintained in presence of doxycycline ( 1 μg ml − 1 ) to induce expression of tau for 24 h . Homogenates from CN1 . 4 cortical were used for co - immunoprecipitation of mouse NDP52 ( mNDP52 ) using a Tau5 antibody . Co - precipitation of mNDP52 was examined by immunoblotting using anti - NDP52 antibody . Input of homogenates used for co - immunoprecipitation was analysed by immunoblotting using anti - NDP52 or Tau antibody .

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

(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 were transfected with mock or HA - mNDP52 plasmid ( d ) or with mNDP52 siRNA or scramble as a control ( e ) . transfected with siRNA or scramble were treated with SFN ( 10 μM ) for 24 h . of tau phosphorylated at Ser262 / Ser356 Ser396 / Ser404 were analysed by immunoblotting using 12E8 PHF1 antibodies , respectively . Total tau was detected with a polyclonal tau - specific antibody ( Tau ) . relative molecular masses ( kDa ) are indicated to left of each blot .

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

(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 co - immunoprecipitation of human NDP52 ( hNDP52 ) using a Tau5 , 12E8 or PHF1 antibody .

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

(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 .

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

(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 insoluble fractions . of total tau tau phosphorylated at Ser262 / Ser356 Ser396 / Ser404 in sarkosyl - insoluble fractions were analysed by immunoblotting using 12E8 PHF1 antibodies , respectively . Samples were also immunoblotted with a polyclonal to total tau ( Tau ) antibody . of hNDP52 , p62 / SQSTM1 LC3 in sarkosyl insoluble fractions also were examined by immunoblotting . relative molecular masses ( kDa ) are indicated to left of each blot . ( d - f ) regression graphs show relative amount of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( d ) , at Ser396 / Ser404 ( PHF1 ) ( e ) or total tau ( f ) compared to hNDP52 present in sarkosyl - insoluble fractions .

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


Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect cytokinetic defects . growth phenotype of 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 . shown are one month old . Scale bar , 10 cm .

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

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.


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

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

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 wall stubs , oblique walls root meristem ( 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 walls ( Welch ANOVA with Games - Howell post - hoc ; p < 0 . 0001 ) or a significant reduction of root meristem in pi4kβ1 pi4kβ2 double mutant ( one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 001 ) .

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

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

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

B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O O O O O O O

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

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

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

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O

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.


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

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

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.


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

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE O 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 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 time lapse of CLC2 - GFP at plate in roots of wild type seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at plate . Times are given relative to instance when plate contacted peripheral plasma , defined as t0 . Scale bars , 10 μm . are from median focal planes chosen from time lapse series recorded as 3D stacks .

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

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

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

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

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

O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE 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 pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for plate formation . Panels a - f display data obtained using cytokinetic Enhanced accumulation of KNOLLE at plate at end of cytokinesis in pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were immunostained with anti - KNOLLE antibodies ( green ) counterstained with DAPI ( blue ) . Scale bars , 10 μm . of KNOLLE at plate from ( C ) , normalized to signal of intracellular compartments . * * * , a significant difference ( p < 0 . 0001 ) according to a two - Mann - Whitney U - ( wild type , n = 26 , 20 roots ; pi4kβ1 pi4kβ2 , n = 41 , 37 roots ) .

O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE 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 pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for plate formation . Panels a - f display data obtained using cytokinetic Enhanced accumulation of PIN2 - GFP at plate at end of cytokinesis in pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were stained with 2 μM FM 4 - 64 for 3 minutes at room . Scale bars , 10 μm . of PIN2 - GFP signal at plate from ( E ) , normalized to at apical plasma , as indicated by dashed lines . * * * , a significant difference ( p < 0 . 001 ) according to a two - Mann - Whitney U - ( wild type , n = 28 , 16 roots ; pi4kβ1 pi4kβ2 , n = 45 , 26 roots ) .

O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE 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 pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for plate formation . panels g - j display data from of interphase . Internalization of PIN2 - GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min , then washed incubated with 50 μM CHX 25 μM BFA . Scale bars , 10 μm . of punctate signals from ( G ) induced by BFA in wild type pi4kβ1 pi4kβ2 double mutants . Data are mean ± SD . * , a significant difference ( p < 0 . 05 ) according to a two - Student ' s t ; ( wild type , n = 116 , 6 roots ; pi4kβ1 pi4kβ2 , n = 110 , 7 roots ) .

O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE 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 pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for plate formation . panels g - j display data from of interphase . Delayed internalization of FM 4 - 64 from plasma in pi4kβ1 pi4kβ2 double mutant . Four - day - old seedlings were pulsed with 2 μM FM 4 - 64 for 3 minutes on ice then was recorded for 20 min . Scale bars , 10 μm . of intracellular FM 4 - 64 signal from ( i ) , normalized to apical basal plasma intensities . * * * , a significant difference ( p < 0 . 0001 ) according to a two - Mann - Whitney U - ( wild type , n = 116 , 6 roots ; pi4kβ1 pi4kβ2 , n = 66 , 9 roots ) .

O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE 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 of five - day - old using anti - tubulin ( green ) counterstained with DAPI ( false colour red ) . Asterisks , multiple nuclei in one ; arrowheads , multiple phragmoplasts in one . Dashed lines indicate outlines of exemplary multinucleated . Scale bars , 10 µm .

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

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 plates . Scale bars , 20 µm .

B-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR 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 . of incidences of multinucleated , multiple phragmoplasts solid phragmoplasts during late cytokinesis ( multinucleated : wild type , n = 316 , 29 roots ; pi4kβ1 pi4kβ2 , n = 673 , 44 roots ; multiple phragmoplasts : wild type , n = 64 , 29 roots ; pi4kβ1 pi4kβ2 , n = 188 , 44 roots ; solid phragmoplast in late cytokinesis : wild type , n = 41 , 29 roots ; pi4kβ1 pi4kβ2 , n = 89 , 44 roots ) . Data are mean ± SD from three independent experiments . n . d . not detected .

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


dynamic transitions of phragmoplast microtubules were recorded by live time lapse imaging . Five - day - old seedlings expressing mCherry - TUA5 were imaged at 1 frame per 2 min . Times are given relative to instance when plate contacted peripheral plasma , defined as t0 . are outlined by dashed lines in first last frames of each series . arrowhead , ectopic stabilization of microtubules in central phragmoplasts of 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 pi4kβ1 pi4kβ2 double mutant .

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


dynamic transitions of phragmoplast microtubules were recorded by live time lapse imaging . 3D projections from time points selected from ( A ) , when transition to a ring phragmoplast has occurred in wild type , but solid phragmoplast persisted in 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 - Mann - Whitney U - ( wild type , n = 12 , 9 roots ; pi4kβ1 pi4kβ2 , n = 21 , 16 roots ) .

O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD 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).


dynamics of microtubule bundling , MAP65 - 3 , were recorded in live time lapse analyses of cytokinetic from root tips of wild type controls 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 . best angle for visualization was obtained by rotation of x - axis . are representative for 8 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 . best angle for visualization was obtained by rotation of x - axis . are representative for 11 from 10 roots . Arrowheads , persisting signal of GFP - MAP65 - 3 in center of division plane . Scale bars , 10 µm . Plot profiles were obtained from medial confocal planes at time points displaying transition from disc to ring phragmoplasts , marked by dashed lines in ( A ) ( B ) . Arrowhead , persisting signal of GFP - MAP65 - 3 in center of division plane . Times in ( A - C ) are given relative to instance when plate contacted peripheral plasma , defined as t0 .

O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O O B-GENEPROD I-GENEPROD B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR 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 ) ( b ) demonstrating attachment of FM 4 - 64 - stained plate with peripheral plasma , defining t0 . Scale bars , 10 µm .

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

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 disc - shaped immunofluorescence patterns for GFP - MAP65 - 3 microtubules , 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 B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD 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.


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

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


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

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-TISSUE O O

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 plate , functional interplay of PI4Kβ1 MPK4 . PI4Kβ1 interacts with MPK4 in yeast two hybrid . MPK4 was used as a bait , while PI4Kβ1 was divided into two fragments to be used as prey . results are representative for three independent experiments .

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

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 plate , functional interplay of PI4Kβ1 MPK4 . In vivo co - immunoprecipitation of MPK4 PI4Kβ1 from 14 - day - old . PI4Kβ1 was specifically co - precipitated with MPK4 - myc using anti - myc antibodies . Arrowhead , Migration of PI4Kβ1 . results are representative for three independent experiments .

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O

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

O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

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

O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 in peripheral blood from healthy donors indicated metastatic cancer .

O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O

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


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

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

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


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

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

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

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

(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).