C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( C ) Histological findings of mouse lung samples at the analysed time points . Left , plots representing the quantitative evaluation ( score : 0 - 5 ) of alveolar infiltrate ( top panel ) and peribronquial / olar infiltrate ( FC ) ( bottom panel ) ( see Materials and Methods ) . Parameters were normalised using the average of the PBS group ( FC = sample value / average PBS ; FC control group , ~ 1 ) . Data are shown as average ± SD of FC ( one - way ANOVA + Tukey post - hoc test ) ( * P < 0 . 05 ) . Right , representative images of lungs stained with hematoxylin - eosin ( the line represents 250 μm ) .

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

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (C) Histological findings of mouse lung samples at the analysed time points. Left, plots representing the quantitative evaluation (score: 0-5) of alveolar infiltrate (top panel) and peribronquial/olar infiltrate (FC) (bottom panel) (see Materials and Methods). Parameters were normalised using the average of the PBS group (FC = sample value/average PBS; FC control group, ~1). Data are shown as average ± SD of FC (one-way ANOVA + Tukey post-hoc test) (*P < 0.05). Right, representative images of lungs stained with hematoxylin-eosin (the line represents 250 μm).


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( D ) IL - 10 secretion by the MPN WT or CV8 strain coding for hIL - 10 WT ( ORF ) , MutSC1 , or MutSC2 . Data are expressed as the mean ± SD of IL - 10 supernatant concentration ( μg / mL ) by biomass ( protein content in μg / mL ) ( n = 3 biological replicas ) .

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

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (D) IL-10 secretion by the MPN WT or CV8 strain coding for hIL-10 WT (ORF), MutSC1, or MutSC2. Data are expressed as the mean ± SD of IL-10 supernatant concentration (μg/mL) by biomass (protein content in μg/mL) (n=3 biological replicas).


( B ) CFUs of PAO1 ( left ) or Mycoplasma strains ( right ) recovered from lung samples . Data are shown as mean ± SD of Log10 CFU / lung homogenate . Two replicates were performed and individual symbols are shown ( experiment 1 , circle ; experiment 2 , square ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CFUs of PAO1 (left) or Mycoplasma strains (right) recovered from lung samples. Data are shown as mean ± SD of Log10 CFU/lung homogenate. Two replicates were performed and individual symbols are shown (experiment 1, circle; experiment 2, square).


( C ) Fold - change ( FC ) of mRNA gene expression of inflammatory markers determined by qPCR . The 2 - ΔΔCt method was used to normalise the values , using gapdh as endogenous control Data are shown as average ± SD of FC . Statistical analyses were performed using unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 * * * ; P < 0 . 001 , * * * * P < 0 . 0001 ) . Two replicates were performed and individual symbols are shown ( Experiment 1 , circle ; Experiment 2 , square ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fold-change (FC) of mRNA gene expression of inflammatory markers determined by qPCR. The 2-ΔΔCt method was used to normalise the values, using gapdh as endogenous control Data are shown as average ± SD of FC. Statistical analyses were performed using unpaired t-test (* P < 0.05; ** P <0.01***; P <0.001, **** P <0.0001). Two replicates were performed and individual symbols are shown (Experiment 1, circle; Experiment 2, square).


( D ) Quantitative analysis of immunochemistry ( IHC ) against neutrophil elastase ( NE ) of lung samples . Data are represented as the mean ± SD of percentage of positive cells , calculated as follows : % = 100 × ( positive cells / positive cells + negative cells ) ( One - Way ANOVA + Tukey post - hoc test , * P < 0 . 05 ) . ( E ) Representative images of IHC staining of lung samples against NE are shown . Arrows indicate NE - positive cells . Scale bar size : 50 µm .

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

(D) Quantitative analysis of immunochemistry (IHC) against neutrophil elastase (NE) of lung samples. Data are represented as the mean ± SD of percentage of positive cells, calculated as follows: % = 100 × (positive cells/positive cells + negative cells) (One-Way ANOVA + Tukey post-hoc test, * P <0.05). (E) Representative images of IHC staining of lung samples against NE are shown. Arrows indicate NE-positive cells. Scale bar size: 50 µm.


( a ) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT - α in HCT116 cells .

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

(a) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT-α in HCT116 cells.


( b ) Autophagy induced by PFT - α , p53 knockdown , or p53 knockout in HCT116 cells . The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments ( mean ± s . e . m . ; * P 0 . 05 ) . Culture in nutrient - free ( NF ) conditions was used as a positive control .

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

(b) Autophagy induced by PFT-α, p53 knockdown, or p53 knockout in HCT116 cells. The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments (mean ± s.e.m.; *P 0.05). Culture in nutrient-free (NF) conditions was used as a positive control.


( c ) Effect of p53 on the maturation of LC3 . Immunoblots are shown for wild - type ( WT ) or p53 − / − HCT116 ( n = 3 ) .

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

(c) Effect of p53 on the maturation of LC3. Immunoblots are shown for wild-type (WT) or p53−/− HCT116 (n = 3).


( d ) GFP - LC3 puncta induced by PFT - α , p53 knockdown or knockout . WT and p53 − / − HCT116 cells were transfected with control or p53 - specific siRNAs , re - transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT - α . The percentage of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) is reported ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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

(d) GFP-LC3 puncta induced by PFT-α, p53 knockdown or knockout. WT and p53−/− HCT116 cells were transfected with control or p53-specific siRNAs, re-transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT-α. The percentage of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (mean ± s.d., n = 3; *P 0.05).


( e ) GFP - LC3 puncta in p53 − / − MEF , compared with WT MEF transfected with GFP - LC3 ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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

(e) GFP-LC3 puncta in p53−/− MEF, compared with WT MEF transfected with GFP-LC3 (mean ± s.d., n = 3; *P 0.05).


( f ) GFP - LC3 dots in tissues from p53 + / + or p53 − / − mice expressing a GFP - LC3 transgene ( mean ± s . d . ; * P 0 . 05 ) , without or after 24 h of starvation . The number of GFP - LC3 dots per area was determined for a minimum of 4 fields ( at × 400 magnification for 4 slides per animal , 3 animals per condition ) .

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

(f) GFP-LC3 dots in tissues from p53+/+ or p53−/− mice expressing a GFP-LC3 transgene (mean ± s.d.; *P 0.05), without or after 24 h of starvation. The number of GFP-LC3 dots per area was determined for a minimum of 4 fields (at ×400 magnification for 4 slides per animal, 3 animals per condition).


( g ) TEM pictures of p53 − / − and p53 + / − mouse livers . p53 − / − hepatocytes carry approximately 3 autophagosomes , p53 + / − controls approximately 1 per cell .

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

(g) TEM pictures of p53−/− and p53+/− mouse livers. p53−/− hepatocytes carry approximately 3 autophagosomes, p53+/− controls approximately 1 per cell.


( h ) Modulation of DsRed : : LGG - 1 levels by the p53 orthologue CEP - 1 in C . elegans . Representative pictures are shown for WT animals and for nematodes carrying a cep - 1 deletion allele ( gk138 ) . The count of puncta per cell was 6 . 9 ± 3 . 3 for WT , 52 . 2 ± 9 . 4 for cep - 1 ( gk138 ) embryos ( mean ± s . e . m . for 20 individuals , 5 cells per embryo ) . The quantification of DsRed : : LGG - 1 puncta is shown for the indicated genetic backgrounds and conditions ( dots represent pixel intensity for individual embryos ) .

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

(h) Modulation of DsRed::LGG-1 levels by the p53 orthologue CEP-1 in C. elegans. Representative pictures are shown for WT animals and for nematodes carrying a cep-1 deletion allele (gk138). The count of puncta per cell was 6.9 ± 3.3 for WT, 52.2 ± 9.4 for cep-1 (gk138) embryos (mean ± s.e.m. for 20 individuals, 5 cells per embryo). The quantification of DsRed::LGG-1 puncta is shown for the indicated genetic backgrounds and conditions (dots represent pixel intensity for individual embryos).


( a ) PFT - α or p53 siRNA - induced GFP - LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5 , Atg10 , Atg12 , Beclin 1 or hVps34 . Culture in nutrient - free ( NF ) conditions was used as a control of autophagy induction .

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

(a) PFT-α or p53 siRNA-induced GFP-LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5, Atg10, Atg12, Beclin 1 or hVps34. Culture in nutrient-free (NF) conditions was used as a control of autophagy induction.


( b ) PFT - α or p53 siRNA - mediated GFP - LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1 .

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

(b) PFT-α or p53 siRNA-mediated GFP-LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1.


( c ) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells . MCF7 cells stably transfected with a tetracycline - repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression , transfected with GFP - LC3 and subjected to p53 knockdown before quantification of GFP - LC3 puncta . Data in a - c are mean ± s . d . of 3 independent experiments .

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

(c) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells. MCF7 cells stably transfected with a tetracycline-repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression, transfected with GFP-LC3 and subjected to p53 knockdown before quantification of GFP-LC3 puncta. Data in a-c are mean ± s.d. of 3 independent experiments.


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - α and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-α and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - alpha and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-alpha and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( g ) Immunoblot detection of LC3 - I / II in cells treated with PFT - α and / or leupeptin in WT HCT116 cells ( n = 3 ) .

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

(g) Immunoblot detection of LC3-I/II in cells treated with PFT-α and/or leupeptin in WT HCT116 cells (n = 3).


( a ) Immunoblot detection of the phosphorylation status of AMPKα , ACCα , TSC2 and p70S6K in HCT116 cells . Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα , ACCα and TSC2 was detected in p53 − / − HCT116 , compared with WT HCT116 cells after culture in complete medium ( n = 5 ) .

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

(a) Immunoblot detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in HCT116 cells. Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα, ACCα and TSC2 was detected in p53−/− HCT116, compared with WT HCT116 cells after culture in complete medium (n = 5).


( b ) Quantification of GFP - LC3vac cells in WT and p53 − / − HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK ( mean ± s . d . of 3 independent experiments ; * P 0 . 05 ) . The inset in b demonstrates the efficiency of siRNA - mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis ( n = 3 ) . Note that the antibody recognizes both AMPKα1 and AMPKα2 .

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

(b) Quantification of GFP-LC3vac cells in WT and p53−/− HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK (mean ± s.d. of 3 independent experiments; *P 0.05). The inset in b demonstrates the efficiency of siRNA-mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis (n = 3). Note that the antibody recognizes both AMPKα1 and AMPKα2.


( c ) Immunoblot detection of ACCα phosphorylation in WT and p53 − / − HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 ( n = 5 ) ; Co , control .

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

(c) Immunoblot detection of ACCα phosphorylation in WT and p53−/− HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 (n = 5); Co, control.


( d ) Quantification of GFP - LC3vac cells ( means ± s . d . , n = 3 separate experiments ) in WT HCT116 and p53 − / − HCT116 treated with PFT - α and / or rapamycin .

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

(d) Quantification of GFP-LC3vac cells (means ± s.d., n = 3 separate experiments) in WT HCT116 and p53−/− HCT116 treated with PFT-α and/or rapamycin.


( a ) Cytosolic ATP levels in WT and p53 − / − HCT116 cells in the absence of glucose . Measurements were performed on luciferase - expressing HCT116 , after perfusion of the cells with medium first containing then missing glucose .

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

(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose.


( b ) Effect of methylpyruvate ( MetPyr ) on ATP levels in WT p53 − / − HCT116 cells depleted of glucose . ATP levels were measured as in a , in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr .

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

(b) Effect of methylpyruvate (MetPyr) on ATP levels in WT p53−/− HCT116 cells depleted of glucose. ATP levels were measured as in a, in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr.


( c ) Effect of autophagy on the ATP levels of WT and p53 − / − HCT116 cells depleted of glucose . Cells were transfected with siRNAs specific for Beclin 1 and AMPKα , or they were treated with rapamycin for 6 h , followed by measurement of ATP levels as in a , before or after glucose withdrawal ( means ± s . d . of triplicates , 3 independent experiments ) .

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

(c) Effect of autophagy on the ATP levels of WT and p53−/− HCT116 cells depleted of glucose. Cells were transfected with siRNAs specific for Beclin 1 and AMPKα, or they were treated with rapamycin for 6 h, followed by measurement of ATP levels as in a, before or after glucose withdrawal (means ± s.d. of triplicates, 3 independent experiments).


( d ) Metabolic stress - induced cell death is attenuated in the absence of p53 . HCT116 cells were transfected with control , Atg5 - , Atg10 - , or AMPKα - specific siRNAs and were subjected 48 h later to metabolic stress ( cultured for 48 h in nutrient - free , hypoxic conditions ) and stained with DiOC6 ( 3 ) and PI . The black and white portions of the columns refer to the DiOC6 ( 3 ) low PI − ( dying ) and DiOC6 ( 3 ) low PI + ( dead ) population , respectively .

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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Metabolic stress-induced cell death is attenuated in the absence of p53. HCT116 cells were transfected with control, Atg5-, Atg10-, or AMPKα-specific siRNAs and were subjected 48 h later to metabolic stress (cultured for 48 h in nutrient-free, hypoxic conditions) and stained with DiOC6(3) and PI. The black and white portions of the columns refer to the DiOC6(3)low PI− (dying) and DiOC6(3)low PI+(dead) population, respectively.


( e ) Metabolic stress - induced decrease of clonogenic survival was attenuated in the absence of p53 . HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival .

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

(e) Metabolic stress-induced decrease of clonogenic survival was attenuated in the absence of p53. HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival.


( f ) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion . MCF7 cells that carry a tetracycline - repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it , transfected with a control siRNA or a p53 - specific siRNA , subjected to metabolic stress , and finally stained with DiOC6 ( 3 ) / PI .

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

(f) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion. MCF7 cells that carry a tetracycline-repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it, transfected with a control siRNA or a p53-specific siRNA, subjected to metabolic stress, and finally stained with DiOC6(3)/PI.


( g ) Effect of p53 and autophagy on the survival of metabolically stressed MEFs . WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays . Results in d - g are mean ± s . d . of 3 separate experiments ( * P 0 . 05 ) ; Co , control .

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

(g) Effect of p53 and autophagy on the survival of metabolically stressed MEFs. WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays. Results in d-g are mean ± s.d. of 3 separate experiments (*P 0.05); Co, control.


( a , b ) PFT - α - mediated stimulation of autophagy in cytoplasts . GFP - LC3 - transfected HeLa cells were enucleated by density - gradient centrifugation after cytochalasin B treatment and cultured on polylysine - coated coverslips . Mixtures of cells and cytoplasts ( arrows , identified by the lack of Hoechst 33342 staining ) were then exposed to three different autophagy inducers : PFT - α , nutrient starvation and rapamycin . Results are means ± s . d . n = 3 experiments ) .

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

(a, b) PFT-α-mediated stimulation of autophagy in cytoplasts. GFP-LC3-transfected HeLa cells were enucleated by density-gradient centrifugation after cytochalasin B treatment and cultured on polylysine-coated coverslips. Mixtures of cells and cytoplasts (arrows, identified by the lack of Hoechst 33342 staining) were then exposed to three different autophagy inducers: PFT-α, nutrient starvation and rapamycin. Results are means ± s.d. n = 3 experiments).


( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 cells . Representative micrographs of cells transfected with plasmids expressing WT , nuclear and cytoplasmic p53 are shown in

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

(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells.Representative micrographs of cells transfected with plasmids expressing WT, nuclear and cytoplasmic p53 are shown in d


( 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 )

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

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

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

(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 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 ) .

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

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 cells treated with PFT - α ( n = 5 ) .

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

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


( 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 .

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

(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 ) .

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

(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 ) 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 ) .

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

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 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 .

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

(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 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 .

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

(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 and HDM2 siRNA on autophagy of WT and p53 − / − HCT116 cells .

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

(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 the absence of p53 . Nutlin - 3 and RITA were added to p53 − / − HCT116 cells , followed by quantification of GFP - LC3 puncta .

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

(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 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 .

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

(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 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 .

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

(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 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 ) .

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

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 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 ) .

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

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 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 ) .

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

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 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 .

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

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 . 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 ) .

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

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 ) 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 .

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

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 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 .

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

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 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 ) .

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

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 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 .

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

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 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 .

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

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 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 ) .

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

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 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 .

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

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 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 .

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

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

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

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

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

(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 O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR 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 .

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

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

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

(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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

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

(B) 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 O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-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 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

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 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-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 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 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

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-CONTROLLED_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 B-CONTROLLED_VAR 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

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

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

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

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

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

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

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

(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 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

(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 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 B-CONTROLLED_VAR 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

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

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

(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