A . Representative mEPSCs recorded from AII cells in wild - type ( black ) and RIBEYE KO mice ( red ) . The insets in dashed boxes show mEPSCs on expanded scales , while the inset in a continuous box on the right shows the averaged mEPSC waveforms ( average of 100 - 200 isolated mini events ) of the example cells . Calibration bars apply to wild - type and mutant EPSCs . B . RIBEYE KO does not alter fundamental parameters of mEPSCs . Summary graphs show mean mEPSC frequency , amplitude , integrated charge , rise time , and decay time constant . Note that the only excitatory inputs into AII amacrine cells is provided by bipolar cell synapses .

O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE B-SUBCELLULAR O

A. Representative mEPSCs recorded from AII cells in wild-type (black) and RIBEYE KO mice (red). The insets in dashed boxes show mEPSCs on expanded scales, while the inset in a continuous box on the right shows the averaged mEPSC waveforms (average of 100-200 isolated mini events) of the example cells. Calibration bars apply to wild-type and mutant EPSCs.B. RIBEYE KO does not alter fundamental parameters of mEPSCs. Summary graphs show mean mEPSC frequency, amplitude, integrated charge, rise time, and decay time constant. Note that the only excitatory inputs into AII amacrine cells is provided by bipolar cell synapses.


C and D . RIBEYE KO renders mEPSCs sensitive to the slow Ca2 + - buffer EGTA ( C , representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA - AM [ 0 . 2 mM ] ; D , summary graphs of the mean mEPSC frequency ( left ) and amplitude ( right ) after incubation in DMSO or EGTA - AM ) .

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

C and D. RIBEYE KO renders mEPSCs sensitive to the slow Ca2+-buffer EGTA (C, representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA-AM [0.2 mM]; D, summary graphs of the mean mEPSC frequency (left) and amplitude (right) after incubation in DMSO or EGTA-AM).


E and F . mEPSCs in AII amacrine cells are largely triggered by Ca2 + - influx via presynaptic L - type Ca2 + - channels ( E , representative mEPSCs traces recorded in regular [ 2 mM ] or reduced extracellular Ca2 + - concentration [ 0 . 3 mM ] , or in the presence of nimodipine [ 50 µM ] in regular Ca2 + ; F , summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E and F. mEPSCs in AII amacrine cells are largely triggered by Ca2+-influx via presynaptic L-type Ca2+-channels (E, representative mEPSCs traces recorded in regular [2 mM] or reduced extracellular Ca2+-concentration [0.3 mM], or in the presence of nimodipine [50 µM] in regular Ca2+; F, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E).


G and H . mEPSCs in AII amacrine cells are suppressed by the fast Ca2 + - buffer BAPTA ( G , representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA - AM [ 30 µM ] ; H , summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G ) .

O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE 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 O O O

G and H. mEPSCs in AII amacrine cells are suppressed by the fast Ca2+-buffer BAPTA (G, representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA-AM [30 µM]; H, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G).


( A ) Proof - of - principle experiment . Time - lapse images show overexpression of Myc - tagged CPAP - WT ( i ) or CPAPΔT ( ii ) has no effect on two centrosome - containing MCF10A cells ( - Dox , two centrosomes ) . The cells were imaged from interphase to Cytokinesis . Bar graph at right quantifies mitotic duration of cells overexpressing CPAP - WT and CPAPΔT . We define mitotic duration as time consumed from the onset of cell rounding until cytokinesis . Number of cells ( n ) analyzed in each condition is indicated at the top of each bar . Number of independent experiments ( N ) , ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test . CPAP - WT ( iii ) or CPAPΔT ( iv ) expressing extra centrosome - containing MCF10A cells ( + Dox , extra centrosomes ) . Note compared to CPAP - WT expressing centrosomes ( iii ) CPAPΔT expressing centrosomes nucleate enhanced levels of microtubule asters already at prophase - like stage ( iv ) . White arrows indicate centrosomal dots . Red arrows indicate centrosomes nucleating microtubule asters causing multipolar spindles . Scale bar , 2µm . Bar graph at right quantifies mitotic duration and percentage of cells exhibiting prolonged mitosis in cells expressing CPAP - WT and CPAPΔT . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * * P < 0 .

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

(A) Proof-of-principle experiment. Time-lapse images show overexpression of Myc-tagged CPAP-WT (i) or CPAPΔT (ii) has no effect on two centrosome-containing MCF10A cells (-Dox, two centrosomes). The cells were imaged from interphase to Cytokinesis. Bar graph at right quantifies mitotic duration of cells overexpressing CPAP-WT and CPAPΔT. We define mitotic duration as time consumed from the onset of cell rounding until cytokinesis. Number of cells (n) analyzed in each condition is indicated at the top of each bar. Number of independent experiments (N), (N)=3. Error bars, mean ± SEM. Unpaired t-test. CPAP-WT (iii) or CPAPΔT (iv) expressing extra centrosome-containing MCF10A cells (+Dox, extra centrosomes). Note compared to CPAP-WT expressing centrosomes (iii) CPAPΔT expressing centrosomes nucleate enhanced levels of microtubule asters already at prophase-like stage (iv). White arrows indicate centrosomal dots. Red arrows indicate centrosomes nucleating microtubule asters causing multipolar spindles. Scale bar, 2µm. Bar graph at right quantifies mitotic duration and percentage of cells exhibiting prolonged mitosis in cells expressing CPAP-WT and CPAPΔT. (N)=3. Error bars, mean ± SEM. Unpaired t-test ***P < 0.0001


( B - D ) Fixed cell images showing the expression of CPAP - WT ( top panel ) and CPAPΔT ( bottom panel ) in extra centrosome containing MCF10A ( + Dox , extra centrosomes ) , MDA - MB - 231 and H1975T790M cells . Note , in CPAP - WT expressing cells , clustered extra centrosomes remain inactive with no or reduced microtubule nucleation . On the other hand , CPAPΔT expression enhances microtubule nucleation in extra centrosomes ( arrows ) and preventing them from clustering at G2 - like interphase and mitosis . Cells were stained with CPAP ( GFP ) , microtubules ( α - tubulin , red ) , Cep152 ( magenta ) . Scale bar ,

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

(B-D) Fixed cell images showing the expression of CPAP-WT (top panel) and CPAPΔT (bottom panel) in extra centrosome containing MCF10A (+Dox, extra centrosomes), MDA-MB-231 and H1975T790M cells. Note, in CPAP-WT expressing cells, clustered extra centrosomes remain inactive with no or reduced microtubule nucleation. On the other hand, CPAPΔT expression enhances microtubule nucleation in extra centrosomes (arrows) and preventing them from clustering at G2-like interphase and mitosis. Cells were stained with CPAP (GFP), microtubules (α-tubulin, red), Cep152 (magenta). Scale bar, 2µm


( E ) Bar graph quantifies percentages of mitotic cells exhibiting multipolar spindles upon CPAP - WT or CPAPΔT expression . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * P < 0 .

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(E) Bar graph quantifies percentages of mitotic cells exhibiting multipolar spindles upon CPAP-WT or CPAPΔT expression. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001


( F ) Subcutaneous xenograft tumor volume measurements of MDA - MB - 231 cells expressing CPAP - WT ( red ) as control or CPAPΔT ( blue ) in nude mice . ( i ) Bar graph at right shows total tumor volume reduction at day 22 . ( ii ) Each condition used 4 mice each containing 4 xenograft tumors . Error bars , mean ± SEM with n = 16 for CPAP - WT and n = 16 for CPAPΔT . Unpaired t - test * * * P < 0 . 0001 .

O O O O B-EXP_ASSAY B-DISEASE B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O B-DISEASE B-EXP_ASSAY O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY B-DISEASE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(F) Subcutaneous xenograft tumor volume measurements of MDA-MB-231 cells expressing CPAP-WT (red) as control or CPAPΔT (blue) in nude mice. (i) Bar graph at right shows total tumor volume reduction at day 22. (ii) Each condition used 4 mice each containing 4 xenograft tumors. Error bars, mean ± SEM with n = 16 for CPAP-WT and n = 16 for CPAPΔT. Unpaired t-test ***P < 0.0001.


( A ) 1H NMR spectra of free CCB02 ( black ) and bound to tubulin in 50 : 1 ratio ( orange ) . Broadening of CCB02 proton peaks indicate its binding ability to

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

(A) 1H NMR spectra of free CCB02 (black) and bound to tubulin in 50:1 ratio (orange). Broadening of CCB02 proton peaks indicate its binding ability to tubulin


( B ) NOESY spectrum recorded for a solution comprising tubulin ( 6 . 5µM ) , CPAP peptide ( 400µM ) and CCB02 . 1 ( 200µM ) with 70ms mixing time . The signals colored in green and ( highlighted by boxes ) correspond to inter - ligand trNOE signals arising due to protein - mediated spin - diffusion ( INPHARMA NOEs ) , while the black peaks represent intra - ligand trNOE signals .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD 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 O O O O O O 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) NOESY spectrum recorded for a solution comprising tubulin (6.5µM), CPAP peptide (400µM) and CCB02.1 (200µM) with 70ms mixing time. The signals colored in green and (highlighted by boxes) correspond to inter-ligand trNOE signals arising due to protein-mediated spin-diffusion (INPHARMA NOEs), while the black peaks represent intra-ligand trNOE signals.


( A ) 14 day CCB02 treatment of MDA - MB - 231 , HCC827 - GR , Calu - 6 and MCF10A ( + Dox , extra centrosomes ) mostly eliminated cells with extra centrosomes . At the end of the treatment , cells were fixed and stained for centrosomes . Bar graph shows the percentage of cells displaying two - and more than two centrosomes . ' Vehicle ' at the bar graph indicates percentages of cells with extra centrosome Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02 - treatment ( open bars ) . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 70 ) . p values were obtained using unpaired t test * * P < 0 . 001 , * * * P < 0 .

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

(A) 14 day CCB02 treatment of MDA-MB-231, HCC827-GR, Calu-6 and MCF10A (+Dox, extra centrosomes) mostly eliminated cells with extra centrosomes. At the end of the treatment, cells were fixed and stained for centrosomes. Bar graph shows the percentage of cells displaying two- and more than two centrosomes. 'Vehicle' at the bar graph indicates percentages of cells with extra centrosome Note that percentages of cells containing more than two centrosomes are significantly reduced upon CCB02-treatment (open bars). Data represent mean ± SEM of three independent experiments. (N)=3, (n=70). p values were obtained using unpaired t test **P < 0.001, ***P < 0.0001


( B ) CCB02 treatment prevents centrosome clustering in HCC827 - GR cells . Activated centrosomes ( arrows ) fail to cluster leading to multipolar mitosis . Cells were stained with Cep152 ( green ) , CPAP ( magenta ) and microtubules ( α - tubulin , red ) . Scale bar , 2µm , Insets , 0 . 5µm .

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

(B) CCB02 treatment prevents centrosome clustering in HCC827-GR cells. Activated centrosomes (arrows) fail to cluster leading to multipolar mitosis. Cells were stained with Cep152 (green), CPAP (magenta) and microtubules (α-tubulin, red). Scale bar, 2µm, Insets, 0.5µm.


Time - lapse images show the effect of CCB02 - mediated centrosome activation in cell cycle progression of MCF10A ( + Dox , extra centrosomes ) ( C cells . White arrows indicate centrosome dots . Red arrows ( in CCB02 - treated cells ) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells . Note , centrosome activation , declustering and prolonged multipolar mitosis occur only with CCB02 treatment . Cartoons of a cell with extra centrosomes below to the panel ( C ) represent various events occurring upon vehicle or CCB02 treatment . Scale bar , 2µm . Bar graphs at right to panel ( C quantifies mitotic duration ( ii ) , quantifies microtubule intensity prior to mitosis ( iii ) and depicts relative events occurring in cells treated with vehicle or CCB02 ( iv ) At least 100 cells were analyzed in each condition . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * P < 0 . 001 , * * * P < 0 .

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

Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression of MCF10A (+Dox, extra centrosomes) (C cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment. Cartoons of a cell with extra centrosomes below to the panel (C) represent various events occurring upon vehicle or CCB02 treatment. Scale bar, 2µm. Bar graphs at right to panel (C quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv) At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001


Time - lapse images show the effect of CCB02 - mediated centrosome activation in cell cycle progression o HCC827 - GR ( D ) cells . White arrows indicate centrosome dots . Red arrows ( in CCB02 - treated cells ) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells . Note , centrosome activation , declustering and prolonged multipolar mitosis occur only with CCB02 treatment Bar graphs at right to pane ( D ) quantifies mitotic duration ( ii ) , quantifies microtubule intensity prior to mitosis ( iii ) and depicts relative events occurring in cells treated with vehicle or CCB02 ( iv ) . At least 100 cells were analyzed in each condition . ( N ) = 3 . Error bars , mean ± SEM . Unpaired t - test * * P < 0 . 001 , * * * P < 0 .

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

Time-lapse images show the effect of CCB02-mediated centrosome activation in cell cycle progression o HCC827-GR (D) cells. White arrows indicate centrosome dots. Red arrows (in CCB02-treated cells) indicate activated centrosomes with enhanced microtubule intensity compared to vehicle treated cells. Note, centrosome activation, declustering and prolonged multipolar mitosis occur only with CCB02 treatment Bar graphs at right to pane (D) quantifies mitotic duration (ii), quantifies microtubule intensity prior to mitosis (iii) and depicts relative events occurring in cells treated with vehicle or CCB02 (iv). At least 100 cells were analyzed in each condition. (N)=3. Error bars, mean ± SEM. Unpaired t-test **P < 0.001, ***P < 0.0001


( i ) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes ( + Dox , extra centrosomes ) ( A Cells were treated with CPAP siRNA and control ( scramble ) siRNA for 48 hours . ( ii ) Western blots show depletion of CPAP when cells were treated with siRNA specific for

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

(i) Experimental strategy to test if the effect of CCB02 is CPAP dependent in MCF10A cells containing extra centrosomes (+Dox, extra centrosomes) (A Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP


Experimental strategy to test if the effect of CCB02 is CPAP dependent i MDA - MB - 23 cell ( B ) . Cells were treated with CPAP siRNA and control ( scramble ) siRNA for 48 hours . ( ii ) Western blots show depletion of CPAP when cells were treated with siRNA specific for

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

Experimental strategy to test if the effect of CCB02 is CPAP dependent i MDA-MB-23 cell (B). Cells were treated with CPAP siRNA and control (scramble) siRNA for 48 hours. (ii) Western blots show depletion of CPAP when cells were treated with siRNA specific for CPAP


( C ) Fractions of cells containing centrosomes after siRNA treatment . 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments . Bar graph shows the percentage of cells with extra centrosomes in MCF10A ( + Dox , extra centrosomes ) in the presence of control siRNA or CPAP siRNA . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 300 cells per condition ) . p values were obtained using Ordinary One - way ANOVA test * P < 0 . 01 , * * P < 0 .

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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_LINE O O B-SMALL_MOLECULE O O B-SUBCELLULAR 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 O O O O O 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) Fractions of cells containing centrosomes after siRNA treatment. 48 hours of siRNA treatment still contained a fraction of cells with extra centrosomes that were analyzed in the following experiments. Bar graph shows the percentage of cells with extra centrosomes in MCF10A (+Dox, extra centrosomes) in the presence of control siRNA or CPAP siRNA. Data represent mean ± SEM of three independent experiments. (N)=3, (n=300 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01, **P < 0.001


( D ) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A ( i ) MDA - MB - 231 cells ( ii ) CPAP depletion does not prevent centrosomes from clustering ( second rows of i and ii ) . Note , in CPAP depleted cultures , centrosomes are marked with Cep152 ( green ) where CPAP ( magenta ) is detected faintly . In CPAP depleted cells , CCB02 does not prevent extra centrosomes from clustering ( fourth rows of i and ii ) . Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA ( third rows of i and ii ) . Cells are stained for Cep152 ( green ) , CPAP ( magenta ) , microtubules ( α - tubulin , red ) and DNA ( DAPI , blue ) . Scale bar ,

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

(D) Effect of CCB02 in the presence of control siRNA and CPAP siRNA in MCF10A (i) MDA-MB-231 cells (ii) CPAP depletion does not prevent centrosomes from clustering (second rows of i and ii). Note, in CPAP depleted cultures, centrosomes are marked with Cep152 (green) where CPAP (magenta) is detected faintly. In CPAP depleted cells, CCB02 does not prevent extra centrosomes from clustering (fourth rows of i and ii). Note CCB02 does prevent extra centrosomes from clustering in cultures treated with control siRNA (third rows of i and ii). Cells are stained for Cep152 (green), CPAP (magenta), microtubules (α-tubulin, red) and DNA (DAPI, blue). Scale bar, 2µm


( E ) Bar graph shows the percentage of cells with multipolar mitosis . Higher percentage of multipolar mitotic cells is observed in control siRNA - treated cultures . Bar graph on right shows the percentage of cells with multipolar mitosis . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 680 cells ) . p values were obtained using unpaired t test . * * * P < 0 .

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

(E) Bar graph shows the percentage of cells with multipolar mitosis. Higher percentage of multipolar mitotic cells is observed in control siRNA-treated cultures. Bar graph on right shows the percentage of cells with multipolar mitosis. Data represent mean ± SEM of three independent experiments. (N)=3, (n=680 cells). p values were obtained using unpaired t test. ***P < 0.0001


( F ) Bar graph shows the percentage of living ( TUNEL - negative ) and dyeing ( TUNEL - positive ) cells under various conditions tested . Higher percentage of TUNEL - positive cells is observed in CCB02 - treated cultures . Data represent mean ± SEM of three independent experiments . ( N ) = 3 , ( n = 500 cells per condition ) . p values were obtained using Ordinary One - way ANOVA test * P < 0 . 01 .

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

(F) Bar graph shows the percentage of living (TUNEL-negative) and dyeing (TUNEL-positive) cells under various conditions tested. Higher percentage of TUNEL-positive cells is observed in CCB02-treated cultures. Data represent mean ± SEM of three independent experiments. (N)=3, (n=500 cells per condition). p values were obtained using Ordinary One-way ANOVA test *P < 0.01.


( A ) Interphase centrosomes of CCB02 - treated two - centrosome containing MCF10A cells display an enhanced level of PCM proteins . CPAP ( green ) intensity remains same . Cells are stained for CPAP ( green ) , PCM proteins ( magenta ) Cep152 ( i ) , PCNT ( ii ) and CDK5RAP2 ( iii ) and microtubules ( α - tubulin , red ) . Scale bars , 2µm for left panels and 0 . 2µm for right panels . Heat map images show intensity saturation ( blue ) for CPAP , Cep152 , PCNT and CDK5RAP2 upon CCB02 treatment . Line graphs at right to each panels indicate the heat map intensity of single centrosomes of cells ( given in boxes at heat map panels ) treated with vehicle ( blue line ) or CCB02 ( red line ) . Images were recorded with low laser excitation and high zoom

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

(A) Interphase centrosomes of CCB02-treated two-centrosome containing MCF10A cells display an enhanced level of PCM proteins. CPAP (green) intensity remains same. Cells are stained for CPAP (green), PCM proteins (magenta) Cep152 (i), PCNT (ii) and CDK5RAP2 (iii) and microtubules (α-tubulin, red). Scale bars, 2µm for left panels and 0.2µm for right panels. Heat map images show intensity saturation (blue) for CPAP, Cep152, PCNT and CDK5RAP2 upon CCB02 treatment. Line graphs at right to each panels indicate the heat map intensity of single centrosomes of cells (given in boxes at heat map panels) treated with vehicle (blue line) or CCB02 (red line). Images were recorded with low laser excitation and high zoom factor


( B ) Bar graph quantifies relative intensity of CPAP - interacting proteins in interphase centrosomes compared to vehicle treatment . At least 150 centrosomes ( for vehicle ) ; 130 centrosomes ( for CCB02 ) were analyzed in each condition . Error bars , mean ± SEM . ( N ) = 3 . Ordinary Two - way ANOVA test * * * P < 0 .

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

(B) Bar graph quantifies relative intensity of CPAP-interacting proteins in interphase centrosomes compared to vehicle treatment. At least 150 centrosomes (for vehicle); 130 centrosomes (for CCB02) were analyzed in each condition. Error bars, mean ± SEM. (N)=3. Ordinary Two-way ANOVA test ***P < 0.0001


( 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 .

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

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

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

(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 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

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

O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR 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 O O O O O O O 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 ) 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 ) =

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

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

(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 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 .

O O O O O O O O O O O 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 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 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

(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 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 ,

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 O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O 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 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 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 .

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

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

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

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


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

O O O 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 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 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 B-GENEPROD O O B-TISSUE O O 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 O O

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


( c , d ) Left panels show brain sections stained for tau phosphorylated at Ser262 / Ser356 ( 12E8 ) ( c ) and Ser396 / Ser404 ( PHF1 ) ( d ) . The area of delineated by dotted lines is enlarged and shown in the right corner of the image . Right panels shows the same slice stained with DAPI 4 ' , 6 - diamidino - 2 - phenylindole . Scale bar , 50 μm . DG , dentate gyrus ; Hi , dentate hilar area .

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

B-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 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 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 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 O O

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


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

O O O B-CELL_TYPE I-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 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 O O

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


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

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

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


Mouse brains were obtained from wild - type ( 11 months old , one male ; 5 months old , one female ; 12 months old , two female ) and Nrf2 ( − / − ) ( 10 months old , two male and two female ) mice . ( a ) Mouse brain tissues were homogenized in the lysis buffer , and 10 μg of lysates was incubated with 2 μg of GST - tau protein at 37 ° C for 30 min in the presence or absence of 1 mM ATP . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies , respectively . GST - tau was detected with a monoclonal GST antibody ( GST - tau ; Supplementary Fig . 4 ) . Bar graph of the relative optical density of phosphorylated tau normalized to actin . Data shown are mean ± s . e . of duplicated independent experiments and were analysed using Student ' s t - test .

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

Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (a)Mouse brain tissues were homogenized in the lysis buffer, and 10 μg of lysates was incubated with 2 μg of GST-tau protein at 37°C for 30 min in the presence or absence of 1 mM ATP. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using a 12E8 and PHF1 antibodies, respectively. GST-tau was detected with a monoclonal GST antibody (GST-tau; Supplementary Fig. 4). Bar graph of the relative optical density of phosphorylated tau normalized to actin. Data shown are mean±s.e. of duplicated independent experiments and were analysed using Student's t-test.


Mouse brains were obtained from wild - type ( 11 months old , one male ; 5 months old , one female ; 12 months old , two female ) and Nrf2 ( − / − ) ( 10 months old , two male and two female ) mice . ( b ) Mouse brain tissues were homogenized in the phosphatase storage buffer , and phosphatase activity in the lysates was quantified using the serine / threonine phosphatase assay system ( Promega ) by measuring the dephosphorylation of a phosphopeptide , RRA ( pT ) VA in the presence or absence of okadaic acid ( OA , 20 nM ) . PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture . n = 4 . Data shown are mean ± s . e . of three independent experiments and were analysed using Student ' s t - test .

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

Mouse brains were obtained from wild-type (11 months old, one male; 5 months old, one female; 12 months old, two female) and Nrf2 (−/−) (10 months old, two male and two female) mice. (b) Mouse brain tissues were homogenized in the phosphatase storage buffer, and phosphatase activity in the lysates was quantified using the serine/threonine phosphatase assay system (Promega) by measuring the dephosphorylation of a phosphopeptide, RRA(pT)VA in the presence or absence of okadaic acid (OA, 20 nM). PP2A activity was defined as the activity inhibited by the addition of OA to the phosphatase reaction mixture. n=4. Data shown are mean±s.e. of three independent experiments and were analysed using Student's t-test.


CN1 . 4mousecortical cells stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . On the next day ( a ) , the cells were washed twice with PBS and the media were replaced with Dox - deficient media with either vehicle , epoxomicin ( 12 . 5 nM , Epox ) , MG132 ( 7 μM ) or 3 - methyladenine ( 10 mM , 3 - MA ) for 24 h . Samples in first lane were from cells maintained in the presence of Dox without medium exchange .

B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 O 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 O O

CN1.4mousecortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. On the next day (a), the cells were washed twice with PBS and the media were replaced with Dox-deficient media with either vehicle, epoxomicin (12.5 nM, Epox), MG132 (7 μM) or 3-methyladenine (10 mM, 3-MA) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.


CN1 . 4 mouse cortical cells stably expressing inducible tau were treated with doxycycline ( Dox , 1 μg ml − 1 ) for 24 h . ( b ) Cells were treated with either chloroquine ( 50 μM , CQ ) or bafilomycin A1 ( 10 nM , Baf A1 ) for 24 h . Samples in first lane were from cells maintained in the presence of Dox without medium exchange .

B-CELL_LINE I-CELL_LINE I-CELL_LINE B-ORGANISM 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 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 O O

CN1.4 mouse cortical cells stably expressing inducible tau were treated with doxycycline (Dox, 1 μg ml−1) for 24 h. (b) Cells were treated with either chloroquine (50 μM, CQ) or bafilomycin A1 (10 nM, Baf A1) for 24 h. Samples in first lane were from cells maintained in the presence of Dox without medium exchange.


( c ) HEK293 - TN cells were co - transfected with the plasmid expressing tau together either with siRNA of beclin - 1 or scramble RNA as a control , which was kept for 48 h .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O 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 O

(c) HEK293-TN cells were co-transfected with the plasmid expressing tau together either with siRNA of beclin-1 or scramble RNA as a control, which was kept for 48 h.


( d ) Stably transfected CN1 . 4 cortical cells were maintained in the presence of Dox ( 1 μg ml − 1 ) to induce the expression of tau for 24 h , and subsequently treated with SFN ( 10 μM ) for 12 h . The culture medium was exchanged for fresh media containing Dox ( 1 μg ml − 1 ) but no SFN after washing with PBS . The cells were then incubated for an additional 18 h in the absence or presence of CQ ( 50 μM ) . The levels of tau phosphorylated at Ser262 / Ser356 and Ser396 / Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies , respectively . Tau , beclin - 1 and LC3 were detected with the appropriate polyclonal antibodies . The relative molecular masses ( kDa ) are indicated to the left of each blot . ( e ) Bar graphs represent the relative optical density of phosphorylated tau , tau or LC3 - II normalized with that of actin . n = 4 . Data shown are mean ± s . e . and were analysed using Student ' s t - test . ( * P0 . 05 ; * * P0 . 01 ) .

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

(d) Stably transfected CN1.4 cortical cells were maintained in the presence of Dox (1 μg ml−1) to induce the expression of tau for 24 h, and subsequently treated with SFN (10 μM) for 12 h. The culture medium was exchanged for fresh media containing Dox (1 μg ml−1) but no SFN after washing with PBS. The cells were then incubated for an additional 18 h in the absence or presence of CQ (50 μM). The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively. Tau, beclin-1 and LC3 were detected with the appropriate polyclonal antibodies. The relative molecular masses (kDa) are indicated to the left of each blot. (e) Bar graphs represent the relative optical density of phosphorylated tau, tau or LC3-II normalized with that of actin. n=4. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01).


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

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

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

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

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

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

(f) SH-SY5Y cells were treated with SFN for 12 h, and cell lysates were used for the ChIP assay using anti-Nrf2 antibody as described in the Methods. Data shown are mean±s.e. and were analysed using Student's t-test. (*P0.05; **P0.01; ***P0.001).


( b ) CN1 . 4 cortical cells and SH - SY5Y cells were transiently transfected with the reporter plasmids with the human NDP52 promoter ( 2 , 317 bp or deleted mutants ) , and luciferase activity was assayed 24 h after transfection .

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

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

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


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

O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O 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 O O O O B-GENEPROD 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 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 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 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 ) 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 ) .

O O O O O B-CELL_TYPE I-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 O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE I-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 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-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 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 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 ) .

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

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

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

(d,e) CN1.4 cortical cells were transfected with the mock or HA-mNDP52 plasmid (d) or with mNDP52 siRNA or scramble RNA as a control (e). The cells transfected with siRNA or scramble RNA were treated with SFN (10 μM) for 24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8 and PHF1 antibodies, respectively. Total tau was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses (kDa) are indicated to the left of each blot.


( a ) Homogenates from cerebral cortical tissue of a diagnosed AD case were used for the co - immunoprecipitation of human NDP52 ( hNDP52 ) using a Tau5 , 12E8 or PHF1 antibody .

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

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


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 .

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


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 .

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

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

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


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

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

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

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

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


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

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


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

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


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 .

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

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

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


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

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

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


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

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