( C ) CD4 T were treated with VbP or ABT737 / S63845 . Lysate supernatant samples were collected at indicated time points . Samples were analyzed by immunoblotting . αSS αLS indicate use of a small subunit or large subunit - specific caspase - 1 antibody respectively . ( Lys = lysate , SN = supernatant , FL = full ) . One representative experiment out of three is shown .

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

(C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.


( A ) Gene expression profile of 15 inflammasome - related genes in human T monocyte subsets . TPM values were retrieved from a public RNASeq dataset log2 transformed . Both , rows columns are clustered using euclidean complete linkage . ( Tfh , T follicular helper ; Tregs , T regulatory ; Th , T helper ; CM , central memory T ; EM , effector memory T ; TE , terminal effector T ; CD14 + C16 - , classical monocytes ; CD14 + CD16 + , intermediate monocytes ; CD14 - CD16 + , non - classical monocytes ) .

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

(A) Gene expression profile of 15 inflammasome-related genes in human T cell and monocyte subsets. TPM values were retrieved from a public RNASeq dataset and log2 transformed. Both, rows and columns are clustered using euclidean distance and complete linkage. (Tfh, T follicular helper cells; Tregs, T regulatory cells; Th, T helper cells; CM, central memory T cells; EM, effector memory T cells; TE, terminal effector T cells; CD14+ C16-, classical monocytes; CD14+ CD16+, intermediate monocytes; CD14- CD16+, non-classical monocytes).


( B ) Immunoblotting of CD4 T MDMs treated with Pam3CSK4 for 6 hours . * indicates an unspecific band . One representative experiment out of three is shown .

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

(B) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours. * indicates an unspecific band. One representative experiment out of three is shown.


( C ) Immunoblotting of polyclonal CD4 T targeted with two gRNAs for each indicated target gene . One representative experiment out of three is shown .

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

(C) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene. One representative experiment out of three is shown.


( D ) CD4 T from indicated polyclonal gene targeting approach were treated with indicated stimuli for 18 hours . data points ± from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


( B ) MiSeq of indicated gene targeting approaches from samples collected at indicated time points post editing . DPP9 DPP8 were targeted with one gRNA / target gene , CARD8 was targeted with two gRNAs . On day of nucleofection , T were activated with human T activator CD3 / CD28 beads expanded in presence of IL - 2 . On day 10 , were switched to IL - 7 / IL - 15 containing medium . ( C ) Fraction of wildtype in - frame reads are depicted . One representative experiment out of three is shown .

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

(B) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing. DPP9 and DPP8 were targeted with one gRNA/target gene, CARD8 was targeted with two gRNAs. On the day of nucleofection, T cells were activated with human T activator CD3/CD28 beads and expanded in the presence of IL-2. On day 10, cells were switched to IL-7/IL-15 containing medium. (C) Fraction of wildtype and in-frame reads are depicted. One representative experiment out of three is shown.


( A ) CD4 T were activated with human T activator CD3 / CD28 beads ( bead : 1 : 5 ) for 4 days . Controls were kept resting in presence of IL - 7 IL - 15 . On day 4 were stimulated as indicated for 18 hours cytotoxicity was assessed by LDH - activity in supernatant . data points ± from four independent donors . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) .

O O O B-GENEPROD B-CELL_TYPE O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak).


( B ) Immunoblotting of CD4 T lysates treated as in ( A ) for 3 to 4 days . * indicates an unspecific band . ( C ) of immunoblots shown in ( B ) additional donors ( n = 9 for but GSDMD , GSDMD n = 8 , for one donor of GSDMD band was considered unreliable due to uneven substrate distribution ) . data points ± are shown . Statistics indicate significance by one - way ANOVA of log2 - transformed fold change values of band intensities ( activated / non - activated T ) normalized to β - Actin : * * * p ≤ 0 . 001 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) . band ratios were tested against β - Actin . CARD8 full was additionally tested against CARD8 .

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

(B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band. (C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.


A , B Data were normalised using control group are represented as fold - change ( FC ) of mean ( MFI ) . Macrophage were obtained from human donors ( n = 4 biological replicas ) incubated with medium ( CON ) or recombinant human IL - 10 ( hIL - 10r ) , MPN wild - type ( WT ) or MPN expressing IL - 10 ORF ( WT _ IL10 ) . Data are represented as mean ± Standard Error of Mean ( ) . Statistical was performed using one - way ANOVA + post - hoc Tukey multiple comparison ( * P < 0 . 05 ; * * 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 B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Data were normalised using the control group and are represented as fold-change (FC) of the mean fluorescence intensity (MFI). Macrophage cells were obtained from human donors (n = 4 biological replicas) and incubated with medium (CON) or recombinant human IL-10 (hIL-10r), MPN wild-type (WT) or MPN expressing IL-10 ORF (WT_IL10). Data are represented as mean ± Standard Error of the Mean (SEM). Statistical analysis was performed using one-way ANOVA + post-hoc Tukey multiple comparison test (*P < 0.05; **P < 0.001, ***P < 0.0001).


( A ) EC - 50 ( molar , M ) for human IL - 10 recombinant ( hIL - 10r ) IL - 10 WT ( IL - 10 ORF ) different variants ( MutM , hIL - 10r , Mut1 , Mut2 , Mut3 , MutSC1 , MutSC2 ) expressed by M . pneumoniae . Data are represented as mean ± SD . Statistical comparison was done by One - way ANOVA + post - hoc Bonferroni multiple comparison , using IL - 10 ORF condition as a reference ( * P < 0 . 05 ) .

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

(A) EC-50 (molar, M) for human IL-10 recombinant protein (hIL-10r) and IL-10 WT (IL-10 ORF) and different variants (MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1, and MutSC2) expressed by M. pneumoniae. Data are represented as mean ± SD. Statistical comparison was done by One-way ANOVA + post-hoc Bonferroni multiple comparison test, using the IL-10 ORF condition as a reference (*P < 0.05).


( B ) Fold - change ( FC ) in expression ( fg / CFU ) of IL - 10 variants Mut3 MutSC1 secreted to medium normalised by expression level of IL - 10 ORF ( ORF ) . Statistical comparison of mean ± SD was performed by One - way ANOVA + Tukey post - hoc ( * P < 0 . 05 ) .

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

(B) Fold-change (FC) in expression levels (fg/CFU) of IL-10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL-10 ORF (ORF). Statistical comparison of mean ± SD was performed by One-way ANOVA + Tukey post-hoc test (*P < 0.05).


( C , D ) Average SD values for FC in relative EC - 50 values determined by flow cytometry of phosphorylated STAT3 after a 20 - min exposure of BlaER1 ( C ) or HAFTL ( D ) lines to IL - 10 ORF ( reference ) or mutant MutSC1 or MutSC2 Numbers indicate average FC ± SD .

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

(C, D) Average and SD values for the FC in the relative EC-50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20-min exposure of the BlaER1 (C) or HAFTL (D) cell lines to IL-10 ORF (reference) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD.


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( A ) Average ± SD loads recovered of WT ( circle ) CV8 ( square ) strains in mouse lungs at 2 - dpi or 4 - dpi . Data are shown as log10 CFU / lung homogenate .

O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE 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). (A) Average ± SD bacterial loads recovered of the WT (circle) and CV8 (square) strains in mouse lungs at 2-dpi or 4-dpi. Data are shown as log10 CFU/ lung homogenate.


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains ( B ) Inflammatory profile of mouse lungs inoculated with WT ( blue ) , CV8 ( blue ) , or PBS ( grey ) . Gene expression was analysed by RT - qPCR using gapdh as an endogenous control ( see Materials Methods ) . Data are shown as average ± SD of fold - change ( FC ) in mRNA expression ( One - way ANOVA + Tukey post - hoc ; * P < 0 . 05 ) .

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

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (B) Inflammatory profile of mouse lungs inoculated with WT (dark blue), CV8 (light blue), or PBS (grey). Gene expression was analysed by RT-qPCR using gapdh as an endogenous control (see Materials and Methods). Data are shown as average ± SD of fold-change (FC) in mRNA expression (One-way ANOVA + Tukey post-hoc test; *P < 0.05).


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( C ) Histological findings of mouse lung samples at analysed time points . Left , plots representing quantitative evaluation ( score : 0 - 5 ) of alveolar infiltrate ( top panel ) peribronquial / olar infiltrate ( FC ) ( bottom panel ) ( see Materials Methods ) . Parameters were normalised using average of 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 ) ( * P < 0 . 05 ) . Right , representative of lungs stained with hematoxylin - eosin ( represents 250 μm ) .

O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 ( D ) IL - 10 secretion by MPN WT or CV8 coding for hIL - 10 WT ( ORF ) , MutSC1 , or MutSC2 . Data are expressed as mean ± SD of IL - 10 supernatant ( μg / mL ) by biomass ( content in μg / mL ) ( n = 3 biological replicas ) .

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

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 symbols are shown ( experiment 1 , circle ; experiment 2 , square ) .

O O O B-EXP_ASSAY O B-ORGANISM O O O O B-ORGANISM O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE 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 . 2 - ΔΔCt method was used to normalise values , using gapdh as endogenous control Data are shown as average ± SD of FC . Statistical analyses were performed using unpaired t - ( * P < 0 . 05 ; * * P < 0 . 01 * * * ; P < 0 . 001 , * * * * P < 0 . 0001 ) . Two replicates were performed symbols are shown ( Experiment 1 , circle ; Experiment 2 , square ) .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of immunochemistry ( ) against neutrophil elastase ( NE ) of lung samples . Data are represented as mean ± SD of of positive , calculated as follows : % = 100 × ( positive / positive + negative ) ( One - Way ANOVA + Tukey post - hoc , * P < 0 . 05 ) . ( E ) Representative of of lung samples against NE are shown . Arrows indicate NE - positive . Scale bar : 50 µm .

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

O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE 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 . number of autophagosomes autolysosomes was determined for at least 50 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 maturation of LC3 . Immunoblots are shown for wild - type ( WT ) or p53 − / − HCT116 ( n = 3 ) .

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

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

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

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(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 . number of GFP - LC3 dots per was determined for a minimum of 4 fields ( at × 400 magnification for 4 slides per , 3 per condition ) .

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

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(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 by p53 orthologue CEP - 1 in C . elegans . Representative pictures are shown for WT for nematodes carrying a cep - 1 deletion allele ( gk138 ) . of per was 6 . 9 ± 3 . 3 for WT , 52 . 2 ± 9 . 4 for cep - 1 ( gk138 ) ( mean ± s . e . m . for 20 individuals , 5 per ) . of DsRed : : LGG - 1 is shown for indicated genetic backgrounds conditions ( dots represent pixel for ) .

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(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 in WT HCT116 transfected with siRNAs to deplete 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 .

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(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 in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1 .

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(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 . MCF7 stably transfected with a tetracycline - repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression , transfected with GFP - LC3 subjected to p53 knockdown before of GFP - LC3 . Data in a - c are mean ± s . d . of 3 independent experiments .

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

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(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 . GFP - LC3 - expressing HeLa were treated with PFT - alpha / or BafA1 then immunostained for Lamp - 2 to observe colocalization between GFP - LC3 Lamp - 2 . Representative confocal microphotographs are shown together with profiles of colocalization ( d ) within of interest , indicated by orientation of arrow . overlap was plotted for control cultured in presence of PFT - α / or BafA1 after 6 h ( d , e ) . Columns represent of colocalization of GFP - LC3 Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 for each condition . kinetics of GFP - LC3 redistribution was quantified by conventional 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 treated with PFT - α / or leupeptin in WT HCT116 ( n = 3 ) .

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

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

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(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 p53 − / − HCT116 subjected to knockdown of AMPKα1 AMPKα2 ( n = 5 ) ; Co , control .

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


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

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

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(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 in WT p53 − / − HCT116 depleted of glucose . ATP were measured as in a , in presence of glucose 15 min after its withdrawal or replacement by MetPyr .

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

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

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(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 absence of p53 . HCT116 subjected to metabolic stress as in d were monitored for clonogenic survival .

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(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 restores survival advantage conferred by p53 depletion . MCF7 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 , finally stained with DiOC6 ( 3 ) / PI .

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(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 autophagy on survival of metabolically stressed MEFs . WT MEFs were transfected with indicated siRNAs 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 .

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(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 were enucleated by density - gradient centrifugation after cytochalasin B treatment cultured on polylysine - coated coverslips . Mixtures of cytoplasts ( arrows , identified by lack of Hoechst 33342 ) were then exposed to three different autophagy inducers : PFT - α , nutrient starvation rapamycin . Results are means ± s . d . n = 3 experiments ) .

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(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 . Representative of transfected with plasmids expressing WT , p53 are shown in

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD 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 autophagy . WT HCT116 were left untreated or treated with Nutlin - 3 , RITA / or indicated autophagy inducers , followed by p53 immunoblot or immunofluorescence microscopy to visualize GFP - LC3 redistribution .

O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD 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 HDM2 siRNA on autophagy of WT p53 − / − HCT116 .

O O O O O B-SMALL_MOLECULE B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_LINE 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 absence of p53 . Nutlin - 3 RITA were added to p53 − / − HCT116 , followed by of GFP - LC3 .

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

O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 transfected with siRNA targeting Atg5 or Beclin 1 , p53 were determined by immunoblot after 6 h of treatment with rapamycin , tunicamycin or thapsigargin .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 RBEKI allele ( red ) or RBEKO allele ( bright red ) normalized for total number of mice ( absolute numbers per genotype / absolute number of mice per respective breeding strategy ) expected Mendelian is indicated by dashed . Statistical using 2 - revealed no statistical difference between wild - type mutant mice ( RBEKI allele p = 0 . 33 ; RBEKO allele p = 0 . 11 ) .

O O B-GENEPROD O O O O B-ORGANISM B-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-GENEPROD 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 RBEKO / KO mice probed with antibodies to CtBP2 ( to detect B - domain of RIBEYE CtBP2 ) to EGFP ( to detect GCamP3 in RIBEYE A - domain GCamP3 fusion referred to as RIB - G3 ) .

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

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 RBEKI / KI mice for EGFP ( green , to detect GCamP3 in RIB - G3 ) SV2 ( red , to label synapses ) demonstrates expression of A - domain / GCamP3 fusion in knockin mice ( OPL , outer plexiform layer ; INL , inner layer ; IPL , inner plexiform layer ; GCL , granule layer ) . Scale bar = 5 µm .

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

O O O B-GENEPROD O O O B-TISSUE O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 ) mGluR6 ( red , to label photoreceptorsynapses ) in cryostat sections from littermate RBEWT / WT RBEKO / KOmouseretinas demonstrates that KO abolishes RIBEYE expression ( OPL , outer plexiform layer ) . Scale bars : 5 µm .

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

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

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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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

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


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

O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 of CPAP - interacting in interphase centrosomes compared to vehicle treatment . At least 150 centrosomes ( for vehicle ) ; 130 centrosomes ( for CCB02 ) were analyzed in each condition . Error bars , mean ± . ( N ) = 3 . Ordinary Two - way ANOVA * * * P < 0 .

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

(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