( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 . of GFP - LC3 of p53 − / − HCT116 transiently transfected with p53 mutants is shown in e ( mean ± s . d . , n = 3 experiments , * P 0 . 05 )

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(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells. Quantification of GFP-LC3 puncta of p53−/− HCT116 cells transiently transfected with p53 mutants is shown in e (mean ± s.d., n = 3 experiments, *P 0.05)


( f ) Detection of phosphorylation status of AMPKα , ACCα , TSC2 p70S6K in p53 − / − HCT116 transiently expressing WT or mutant p53 ( n = 5 ) .

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(f) Detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in p53−/− HCT116 cells transiently expressing WT or mutant p53 (n = 5).


p53 inhibition induces autophagy by ER stress ( a - c ) WT HCT116 were transiently transfected with GFP - LC3 , treated with PFT - α for 2 , 4 or 6 h subjected to immunofluorescence to observe colocalization of GFP - LC3 + with ER marker ERp57 ( a ) or a mitochondrial marker III ( b ) . Note that with GFP - LC3 + often contain scarce amounts of ERp57 ( arrows ) after prolonged incubation with PFT - α . of colocalization ( means ± s . d . , n = 3 experiments with 50 per experiment ) was determined by confocal microscopy ( c ) .

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p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c).


( d ) eIF2α phosphorylation in WT or p53 − / − HCT116 treated with PFT - α ( n = 5 ) .

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(d) eIF2α phosphorylation in WT or p53−/− HCT116 cells treated with PFT-α (n = 5).


( e , f ) WT HCT116 ( e , f ) or p53 − / − HCT116 ( f ) were transfected with GFP - LC3 , plus a control siRNA or indicated combination of IRE1α - / or p53 - specific siRNA , treated with PFT - α , thapsigargin , tunicamycin or brefeldin A .

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(e, f) WT HCT116 cells (e, f) or p53−/− HCT116 cells (f) were transfected with GFP-LC3, plus a control siRNA or the indicated combination of IRE1α- and/or p53-specific siRNA, and treated with PFT-α, thapsigargin, tunicamycin or brefeldin A.


( g ) GFP - LC3 - transfected WT or ire1α − / − MEF were treated with PFT - α . Columns in e - g show means ± s . d . of 3 independent experiments ( * P 0 . 05 ) .

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(g) GFP-LC3-transfected WT or ire1α−/− MEF were treated with PFT-α. Columns in e-g show means ± s.d. of 3 independent experiments (*P 0.05).


Kinetics of p53 degradation ( a ) induction of GFP - LC3 ( b ) in WT HCT116 cultured in complete medium in presence of rapamycin ( Rapa ) , tunicamycin ( Tn ) , lithium ( Li ) or in nutrient - free medium ( NF ) .

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Kinetics of p53 degradation (a) and induction of GFP-LC3 puncta (b) in WT HCT116 cells cultured in complete medium in the presence of rapamycin (Rapa), tunicamycin (Tn), lithium (Li) or in nutrient-free medium (NF).


( c ) WT HCT116 transfected with control or HDM2 - targeted siRNAs were cultured in presence or absence of MG132 for 3 h , followed by treatment with autophagy inducers for 6 h ( Tp , thapsigargin ; other abbreviations as in b ) . abundance of p53 HDM2 was determined by immunoblotting of showing accumulation of GFP - LC3 in vacuoles ( GFP - LC3vac ) was determined .

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(c) WT HCT116 cells transfected with control or HDM2-targeted siRNAs were cultured in the presence or absence of MG132 for 3 h, followed by the treatment with autophagy inducers for 6 h (Tp, thapsigargin; other abbreviations as in b). The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP-LC3 in vacuoles (GFP-LC3vac) was determined.


( d ) Effects of HDM2 inhibitors on p53 autophagy . WT HCT116 were left untreated or treated with Nutlin - 3 , RITA / or indicated autophagy inducers , followed by p53 immunoblot or immunofluorescence microscopy to visualize GFP - LC3 redistribution .

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(d) Effects of HDM2 inhibitors on p53 protein levels and autophagy. WT HCT116 were left untreated or treated with Nutlin-3, RITA and/or the indicated autophagy inducers, followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP-LC3 redistribution.


( e ) Effect of MG132 HDM2 siRNA on autophagy of WT p53 − / − HCT116 .

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(e) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53−/− HCT116 cells.


( f ) Failure of HDM2 inhibitors to prevent autophagy induced by absence of p53 . Nutlin - 3 RITA were added to p53 − / − HCT116 , followed by of GFP - LC3 .

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(f) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53. Nutlin-3 and RITA were added to p53−/− HCT116 cells, followed by quantification of GFP-LC3 puncta.


( g ) Effect of WT ubiquitination - resistant p53 on autophagy . p53 − / − HCT116 were transfected with GFP - LC3 alone or in combination with different concentrations of pcDNA3 . 1 , WT p53 or p53 lacking ubiquitination site at 13 - 19 ( p53ΔI ) , then treated with rapamycin , tunicamycin or thapsigargin . Thereafter , p53 expression GFP - LC3 were quantified .

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(g) Effect of WT and ubiquitination-resistant p53 on autophagy. p53−/− HCT116 cells were transfected with GFP-LC3 alone or in combination with different concentrations of pcDNA3.1, WT p53 or p53 lacking the ubiquitination site at amino acids 13-19 (p53ΔI), then treated with rapamycin, tunicamycin or thapsigargin. Thereafter, p53 expression and GFP-LC3 puncta were quantified.


( h ) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors . In WT HCT116 transfected with siRNA targeting Atg5 or Beclin 1 , p53 were determined by immunoblot after 6 h of treatment with rapamycin , tunicamycin or thapsigargin .

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(h) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors. In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1, p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin, tunicamycin or thapsigargin.


B . RIBEYE mutations do not alter mouse survival . Data show surviving post - weaning mice ( at > P21 ) derived from matings between mice heterozygous for RBEKI allele ( red ) or RBEKO allele ( bright red ) normalized for total number of mice ( absolute numbers per genotype / absolute number of mice per respective breeding strategy ) expected Mendelian is indicated by dashed . Statistical using 2 - revealed no statistical difference between wild - type mutant mice ( RBEKI allele p = 0 . 33 ; RBEKO allele p = 0 . 11 ) .

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B. RIBEYE mutations do not alter mouse survival. Data show surviving post-weaning mice (at >P21) derived from matings between mice heterozygous for the RBEKI allele (dark red) or the RBEKO allele (bright red) normalized for the total number of mice (absolute numbers per genotype/absolute number of all mice per respective breeding strategy) The expected Mendelian ratio is indicated by the dashed line. Statistical analysis using the 2-test revealed no statistical difference between wild-type and mutant mice (RBEKI allele p=0.33; RBEKO allele p=0.11).


C . Representative immunoblot of retina homogenates from homozygous RBEWT / WT , RBEKI / KI RBEKO / KO mice probed with antibodies to CtBP2 ( to detect B - domain of RIBEYE CtBP2 ) to EGFP ( to detect GCamP3 in RIBEYE A - domain GCamP3 fusion referred to as RIB - G3 ) .

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C. Representative immunoblot of retina homogenates from homozygous RBEWT/WT, RBEKI/KI and RBEKO/KO mice probed with antibodies to CtBP2 (to detect the B-domain of RIBEYE and CtBP2) and to EGFP (to detect GCamP3 in the RIBEYE A-domain GCamP3 fusion protein referred to as RIB-G3).


D . Measurements by RT - PCR of relative abundance of CtBP2 RIBEYE mRNAs using an that simultaneously measures both mRNAs in retina in brain from control wild - type mice ( RBEWT / WT ) , KI - mice expressing RIBEYE - A - domain fusion with GCamP3 ( RBEKI / KI ) , KO mice ( RBEKO / KO ) . These measurements were performed to relative expression of RIBEYE CtBP2 in retina , to examine whether RIBEYE A - domain KO impairs CtBP2 expression in brain , which expresses little RIBEYE . Assays are on a qRT - PCR strategy detecting mRNAs containing spliced connection between exon 2 3 which is common to both transcript isoforms ( RIBEYE CtBP2 ) . Statistical analyses were performed using Student ' s t - ; data are presented as mean ± ( * * * , p < 0 . 001 ; n = 4 mice per genotype ) .

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D. Measurements by RT-PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild-type mice (RBEWT/WT), KI-mice expressing the RIBEYE-A-domain fusion protein with GCamP3 (RBEKI/KI), and KO mice (RBEKO/KO). These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina, and to examine whether the RIBEYE A-domain KO impairs CtBP2 expression in brain, which expresses little RIBEYE. Assays are based on a qRT-PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms (RIBEYE and CtBP2). Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (***, p<0.001; n=4 mice per genotype).


E . Representative immunofluorescence labeling of cryostat sections from littermate wild - type RBEKI / KI mice for EGFP ( green , to detect GCamP3 in RIB - G3 ) SV2 ( red , to label synapses ) demonstrates expression of A - domain / GCamP3 fusion in knockin mice ( OPL , outer plexiform layer ; INL , inner layer ; IPL , inner plexiform layer ; GCL , granule layer ) . Scale bar = 5 µm .

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E. Representative immunofluorescence labeling of cryostat sections from littermate wild-type and RBEKI/KI mice for EGFP (green, to detect GCamP3 in RIB-G3) and SV2 (red, to label synapses) demonstrates expression of the A-domain/GCamP3 fusion protein in knockin mice (OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, granule cell layer). Scale bar = 5 µm.


F . of CtBP2 of synaptic in retina homogenates from littermate RBEWT / WT RBEKO / KO mice . were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies ; results are normalized to loading controls depending on molecular weights ( actin , fodrin , VCP , GDI ) to wild - type controls . Statistical analyses were performed using Student ' s t - ; data are presented as mean ± ( * , p < 0 . 05 ; n = 4 mice per genotype ) .

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F. Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT/WT and RBEKO/KO mice. Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies; results are normalized to loading controls depending on molecular weights (actin, fodrin, VCP, and GDI) and to wild-type controls. Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (*, p<0.05; n=4 mice per genotype).


G . Immunofluoresence labeling for CtBP2 ( green , to label RIBEYE ) mGluR6 ( red , to label photoreceptorsynapses ) in cryostat sections from littermate RBEWT / WT RBEKO / KOmouseretinas demonstrates that KO abolishes RIBEYE expression ( OPL , outer plexiform layer ) . Scale bars : 5 µm .

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G. Immunofluoresence labeling for CtBP2 (green, to label RIBEYE) and mGluR6 (red, to label photoreceptorsynapses) in cryostat sections from littermate RBEWT/WT and RBEKO/KOmouseretinas demonstrates that the KO abolishes RIBEYE expression (OPL, outer plexiform layer). Scale bars: 5 µm.


A . Overview of synaptic organization of retina in RBEWT / WT RBEKO / KOmice . Cryostat sections from littermate mice were labeled by double immunofluorescence for RIBEYE B - domain ( CtBP2 ; green ) SV2 . Scale bar : 20 µm ; abbreviations : ONL , outer layer ; OPL , outer plexiform layer ; INL , inner layer ; IPL , inner plexiform layer ; GCL , ganglion layer .

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A. Overview of the synaptic organization of the retina in RBEWT/WT and RBEKO/KOmice. Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B-domain (CtBP2; green) and SV2. Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


B . Double immunofluorescence of retinas from RBEWT / WT RBEKO / KO mice for PSD95 ( green , to label postsynaptic specializations ) mGluR6 ( red , to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses ) . Abbreviations are as in A . Scale bars : 20 µm ( left right overview ) , 5 µm ( left panel magnification ) , 2 µm ( right panel magnification ) . C . Same as B , except that retinas were stained for PKCα ( green , to label bipolar neurons ) PSD95 ( red , to label amacrine ) .

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B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice for PSD95 (green, to label postsynaptic specializations) and mGluR6 (red, to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses). Abbreviations are as in A. Scale bars: 20 µm (left and right overview), 5 µm (left panel magnification), 2 µm (right panel magnification).C. Same as B, except that retinas were stained for PKCα (green, to label bipolar neurons) and PSD95 (red, to label amacrine cells).


A B . Double immunofluorescence of retinas from RBEWT / WT RBEKO / KO mice , respectively , for PSD95 ( green , to label presynaptic photoreceptor terminals ) Cav1 . 4 ( red , to label presynaptic Ca2 + - channel ) . Scale bar : 20 µm ; abbreviations : ONL , outer layer ; OPL , outer plexiform layer ; INL , inner layer . ( A1 to A4 ) Magnification of Cav1 . 4 in outer plexiform layer ( OPL ) in retina of RBEWT / WT ( B1 to B4 ) RBEKO / KO mice . arrow heads indicate Ca2 + - channel distribution at selected synapses . represent 2 µm z - stack projections . Scale bar : 2 µm .

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A and B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice, respectively, for PSD95 (green, to label presynaptic photoreceptor terminals) and Cav1.4 (red, to label presynaptic Ca2+-channel). Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (A1 to A4) Magnification of Cav1.4 staining in the outer plexiform layer (OPL) in the retina of RBEWT/WT and (B1 to B4) RBEKO/KO mice. White arrow heads indicate Ca2+-channel distribution at selected synapses. Images represent 2 µm z-stack projections. Scale bar: 2 µm.


A . Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in terminals of RBEWT / WTmice . Asterisks in A2 A3 denote photoreceptor active zones of RBEKO / KOmice in which synaptic ribbon is absent . Except for absence of synaptic ribbons , ultrastructural appearance of presynaptic terminals is comparable to that of RBEWT / WTmice . Abbreviations : sr , synaptic ribbon ; pre , presynaptic ; post , postsynaptic . Scale bars : 500 nm .

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A. Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO/KOmice in which the synaptic ribbon is absent. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT/WTmice. Abbreviations: sr, synaptic ribbon; pre, presynaptic; post, postsynaptic. Scale bars: 500 nm.


B . Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in terminals of RBEWT / WTmice . Except for absence of synaptic ribbons , ultrastructural appearance of presynaptic terminals is comparable to RBEWT / WTmice . Abbreviations are same as in A . Scale bars : 1 µm ( B1 , B2 , B4 , B5 ) ; 300 nm ( B3 , B6 ) .

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B. Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT/WTmice. Abbreviations are the same as in A. Scale bars: 1 µm (B1, B2, B4, B5); 300 nm (B3, B6).


A B . Representative transmission EM pictures of RBEWT / WT ( A ) RBEKO / KO ( B ) photoreceptor ribbon synapses ( blue rectangles , adjacent to synaptic junction used for measuring junction - associated vesicle density [ = 200 nm x 300 nm ] ; black squares , are in presynaptic cytosol used to measure synaptic vesicles [ = 200 nm x 200 nm ] ) . C . Average vesicle ( expressed as vesicle numbers in adjacent to synaptic junctions ( blue rectangle ) or cytosol ( black square ) normalized to RBEWT / WT . D . Average number of docked vesicles within a 150 nm range in vicinity of active zone .

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A and B. Representative transmission EM pictures of RBEWT/WT (A) and RBEKO/KO (B) photoreceptor ribbon synapses (blue rectangles, area adjacent to synaptic junction used for measuring junction-associated vesicle density [size = 200 nm x 300 nm]; black squares, are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [size = 200 nm x 200 nm]).C. Average vesicle ratio (expressed as vesicle numbers in the area adjacent to synaptic junctions (blue rectangle) or the cytosol (black square) normalized to RBEWT/WT.D. Average number of docked vesicles within a 150 nm range in the vicinity of the active zone.


A . Experimental design representative traces for of synaptic transmission in retinas from wild - type ( black ) RIBEYE KO mice ( red ) . Presynaptic bipolar neurons were depolarized in voltage - clamp mode from - 70 mV to - 10 mV for 0 . 5 s as indicated on top to open Ca2 + - channels trigger release . Presynaptic Ca2 + - currents postsynaptic EPSCs were measured simultaneously as indicated in traces ; dotted box displays an expansion of initial Ca2 + - currents EPSCs . B . RIBEYE KO does not alter presynaptic Ca2 + - currents . Summary graphs show average peak Ca2 + - currents Ca2 + - current . C . RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization . Plot shows integrated EPSC charge as a function of time ; both initial sustained neurotransmitter release are impaired . D . RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by summary graph of peak EPSC amplitude . E . RIBEYE KO suppresses both initial synchronous sustained phase of release induced by presynaptic depolarization . Summary graphs display integrated EPSC charges for initial phase ( 0 - 50 ms ) sustained phase ( 50 - 500 ms ) . F . RIBEYE KO does not alter kinetics of EPSCs . Summary graphs depict mean synaptic delays ( left ) , rise times ( center ) decay time constants ( right ) of EPSCs .

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A. Experimental design and representative traces for analysis of synaptic transmission in retinas from wild-type (black) and RIBEYE KO mice (red). Presynaptic bipolar neurons were depolarized in voltage-clamp mode from -70 mV to -10 mV for 0.5 s as indicated on top to open Ca2+-channels and trigger release. Presynaptic Ca2+-currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces; dotted box displays an expansion of the initial Ca2+-currents and EPSCs.B. RIBEYE KO does not alter presynaptic Ca2+-currents. Summary graphs show average peak Ca2+-currents and Ca2+-current.C. RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization. Plot shows integrated EPSC charge as a function of time; both initial and sustained neurotransmitter release are impaired.D. RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude.E. RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization. Summary graphs display integrated EPSC charges for the initial phase (0-50 ms) and the sustained phase (50-500 ms).F. RIBEYE KO does not alter the kinetics of EPSCs. Summary graphs depict the mean synaptic delays (left), rise times (center) and decay time constants (right) of EPSCs.


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

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

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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 F . mEPSCs in AII amacrine 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 + - [ 0 . 3 mM ] , or in presence of nimodipine [ 50 µM ] in regular Ca2 + ; F , summary graphs of mean mEPSC frequency amplitude under conditions described for E ) .

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

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

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

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(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 exhibiting multipolar spindles upon CPAP - WT or CPAPΔT expression . ( N ) = 3 . Error bars , mean ± . Unpaired t - * * 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 volume measurements of MDA - MB - 231 expressing CPAP - WT ( red ) as control or CPAPΔT ( blue ) in nude mice . ( i ) Bar graph at right shows total volume reduction at day 22 . ( ii ) Each condition used 4 mice each containing 4 xenograft tumors . Error bars , mean ± with n = 16 for CPAP - WT n = 16 for CPAPΔT . Unpaired t - * * * P < 0 . 0001 .

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(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 ) bound to tubulin in 50 : 1 ( orange ) . Broadening of CCB02 proton peaks indicate its binding ability to

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(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 ) CCB02 . 1 ( 200µM ) with 70ms mixing time . signals colored in green ( highlighted by boxes ) correspond to inter - ligand trNOE signals arising due to - mediated spin - diffusion ( INPHARMA NOEs ) , while black peaks represent intra - ligand trNOE signals .

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

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(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 . Activated centrosomes ( arrows ) fail to cluster leading to multipolar mitosis . were stained with Cep152 ( green ) , CPAP ( magenta ) microtubules ( α - tubulin , red ) . Scale bar , 2µm , Insets , 0 . 5µm .

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(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 show effect of CCB02 - mediated centrosome activation in cycle progression of MCF10A ( + Dox , extra centrosomes ) ( C . arrows indicate centrosome dots . Red arrows ( in CCB02 - treated ) indicate activated centrosomes with enhanced microtubule compared to vehicle treated . Note , centrosome activation , declustering prolonged multipolar mitosis occur only with CCB02 treatment . Cartoons of a with extra centrosomes below to 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 prior to mitosis ( iii ) depicts relative events occurring in treated with vehicle or CCB02 ( iv ) At least 100 were analyzed in each condition . ( N ) = 3 . Error bars , mean ± . Unpaired t - * * P < 0 . 001 , * * * P < 0 .

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

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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 if effect of CCB02 is CPAP dependent in MCF10A containing extra centrosomes ( + Dox , extra centrosomes ) ( A were treated with CPAP siRNA control ( scramble ) siRNA for 48 hours . ( ii ) Western blots show depletion of CPAP when were treated with siRNA specific for

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(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 if effect of CCB02 is CPAP dependent i MDA - MB - 23 ( B ) . were treated with CPAP siRNA control ( scramble ) siRNA for 48 hours . ( ii ) Western blots show depletion of CPAP when were treated with siRNA specific for

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

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

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

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

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(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 display an enhanced level of PCM . CPAP ( green ) remains same . are stained for CPAP ( green ) , PCM ( magenta ) Cep152 ( i ) , PCNT ( ii ) CDK5RAP2 ( iii ) microtubules ( α - tubulin , red ) . Scale bars , 2µm for left panels 0 . 2µm for right panels . Heat map show saturation ( blue ) for CPAP , Cep152 , PCNT CDK5RAP2 upon CCB02 treatment . graphs at right to each panels indicate heat map of single centrosomes of ( given in boxes at heat map panels ) treated with vehicle ( blue ) or CCB02 ( red ) . were recorded with low excitation high zoom

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(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 of CPAP - interacting 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 ± . ( N ) = 3 . Ordinary Two - way ANOVA * * * P < 0 .

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O O O O O O O O O O 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) for 12 h, and the relative mRNA level of each gene was compared with that of cells not treated with SFN using qRT-PCR.


( b ) In hippocampal tissues from Nrf2 ( − / − ) ( 5 months old , one ; 11 months old , one ; 5 months old , one ; 12 months old , three ) or wild - type ( 10 months old , four two ) mice , relative mRNA level of each gene compared with that of wild - type mice . n = 6 .

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(b) In hippocampal tissues from Nrf2 (−/−) (5 months old, one male; 11 months old, one female; 5 months old, one female; 12 months old, three female) or wild-type (10 months old, four male and two female) mice, the relative mRNA level of each gene compared with that of wild-type mice. n=6.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


( c , d ) cortical neurons were transduced with a control lentivirus ( FIGB ) or with one expressing humanNDP52 ( hNDP52 ) at DIV 1 . To induce autophagy , trehalose ( 150 mM ) was added at DIV 5 neurons incubated for 24 h ( DIV 6 ) . cortical neurons were fixed with 4 % paraformaldehyde , stained with 12E8 or PHF1 antibodies . Scale bar , 20 μm . optical density of tau phosphorylated at Ser262 / Ser356 ( 12E8 ) . ( e ) Ser396 / Ser404 ( PHF1 ) ( f ) in soma of ~ 30 neurons randomly chosen was analysed with ImageJ program . Data were analysed using Student ' s t - ( * * * P0 . 001 ) .

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

(c,d) Primary cortical neurons were transduced with a control lentivirus (FIGB) or with one expressing humanNDP52 (hNDP52) at DIV 1. To induce autophagy, trehalose (150 mM) was added at DIV 5 and the neurons incubated for 24 h (DIV 6). Primary cortical neurons were fixed with 4% paraformaldehyde, and stained with the 12E8 or PHF1 antibodies. Scale bar, 20 μm. The optical density of tau phosphorylated at Ser262/Ser356 (12E8). (e) and Ser396/Ser404 (PHF1) (f) in the soma of ~30 neurons randomly chosen was analysed with the ImageJ program. Data were analysed using Student's t-test (***P0.001).


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

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

(b) Naive cortical cells were co-transfected with the plasmids expressing HA-hNDP52 or HA-tagged domain-deleted mutants of human NDP52 together with GFP-tau. Twenty-four hours later, cell lysates were prepared and transfected tau was immunoprecipitated and blots probed for both the HA-hNDP52 constructs and GFP-tau. Homogenates (input) used for co-immunoprecipitation were analysed by immunoblotting using anti-HA or a polyclonal tau-specific antibody (Tau).


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

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

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


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

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

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


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

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

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

(b) Homogenates used for co-immunoprecipitation in a were analysed by immunoblotting using anti-NDP52, Tau5, 12E8 or PHF1 antibody.


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

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

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


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

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

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


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

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