Figure 5BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( A ) Representative traces of CFTR - dependent Cl - secretion measured by forskolin ( Fsk ) - induced increase of chloride current ( Isc ( μA / cm2 ) ) in small intestines mounted in Ussing chambers ; quantification of the peak CFTR inhibitor 172 ( CFTRinh172 ) - sensitive Isc ( ∆ Isc ) in tissue samples ( n = 5 ) . Mean ± SD of samples assayed . * * * p < 0 . 001 gliadin vs gliadin + VX - 770 ( Student ' s t - test ) . BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( B ) Immunoblot with anti - CFTR antibody and β - actin loading control . Densitometric analysis are mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 vehicle vs gliadin ; ° p < 0 . 05 , gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post test ) . Data information : The blots are representative of one experiment for group of treatment . BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( C ) Plasma markers of intestinal permeability in mice . Plasma concentration of FITC - dextran 4000 ( FITC - D4000 ) measured 1 h after gavage of a single dose of 600 mg FITCD4000 per kg body weight . Quantification of plasma concentration from n = 10 mice per group of treatment expressed as mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 vehicle vs gliadin ; ° ° ° p < 0 . 01 , gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post test ) . BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( D ) NLRP3 expression by immunoblotting with specific antibodies in small intestines from 3 mice . Densitometric analysis are mean ± SD of triplicates of independent pooled samples . * * p < 0 . 01 vehicle vs gliadin ; ° p < 0 . 05 , gliadin vs VX - 770 + gliadin . Data information : The blots are representative of one experiment for group of treatment . BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . ( E - F ) mRNA ( E ) or protein ( by specific ELISA ) ( F ) levels of IL - 15 , IL - 21 , IL - 17A and IFN - γ . Mean ± SD of triplicates of independent pooled samples . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 vehicle vs gliadin ; ° p < 0 . 05 , ° ° p < 0 . 01 , ° ° ° p < 0 . 001 gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post hoc test ) . BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . Transcript level of Rorc and Tbet ( G ) from small intestine homogenates . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 or * * * * p < 0 . 0001 gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post hoc test ) . BALB / c mice fed with a gluten - free diet for at least 3 generations , orally challenged with vehicle or gliadin for 4 weeks ( 5 mg / daily for one week and then 5 mg / daily thrice a week for 3 weeks ) in the presence or absence of intraperitoneal VX - 770 administered 15 minutes prior gliadin challenge ( n = 10 mice per group of treatment ) . protein levels ( by specific ELISA ) of IL - 10 and TGF - β ( H ) from small intestine homogenates . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 or * * * * p < 0 . 0001 gliadin vs VX - 770 + gliadin ( ANOVA , Bonferroni post hoc test ) . IFN - γ release ( ELISA ) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co - culture model upon 24 h challenge of confluent CaCo - 2 cells in the upper compartment with PT - gliadin ( I ) in presence or absence of VX - 770 . Mean ± SD of triplicates of independent pooled samples . * * * p < 0 . 001 , PT - gliadin vs medium ; ° ° ° p < 0 . 001 , PT - gliadin vs PT - gliadin + VX - 770 ( n = 4 ) ( ANOVA , Bonferroni post hoc test ) . IFN - γ release ( ELISA ) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co - culture model upon 24 h challenge of confluent CaCo - 2 cells in the upper compartment with combination of P31 - 43 and P57 - 68 ( J ) in presence or absence of VX - 770 . Mean ± SD of triplicates of independent pooled samples . ; * * * p < 0 . 001 , P57 - 68 or P31 - 43 vs P31 - 43 / P57 - 68 combination ( n = 6 ) ; ° ° ° p < 0 . 001 , P57 - 68 / P31 - 43 combination vs VX - 770 + P57 - 68 / P31 - 43 ( n = 3 ) , ( ANOVA , Bonferroni post hoc test ) . ( K ) IL - 10 release ( ELISA ) in culture supernatants by PBMC from 4 celiac patients cultured as in ( J ) . Mean ± SD of triplicates of independent pooled samples . * * * * p < 0 . 0001 , P57 - 68 / P31 - 43 combination vs P57 - 68 / P31 - 43 + VX - 770 treatment ( n = 3 ) ( ANOVA , Bonferroni post hoc test ) . ( L ) IFN - γ release ( ELISA ) into culture supernatants by PBMC from 3 celiac patients cultured in the lower compartment of a bidimensional co - culture model upon a 24 h challenge of confluent Caco - 2CFTR - KO cells in the upper compartment with a combination of P31 - 43 and P57 - 68 in the presence or absence of VX - 770 , as in J and K . * p < 0 . 05 , medium vs P57 - 68 / P31 - 43 combination ( n = 3 ) ( ANOVA , Bonferroni post hoc test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=5). Mean ± SD of samples assayed. ***p<0.001 gliadin vs gliadin+VX-770 (Student's t-test).BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (B) Immunoblot with anti-CFTR antibody and β-actin loading control. Densitometric analysis are mean ± SD of triplicates of independent pooled samples. **p<0.01 vehicle vs gliadin; °p<0.05, gliadin vs VX-770+gliadin (ANOVA, Bonferroni post test). Data information: The blots are representative of one experiment for group of treatment.BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (C) Plasma markers of intestinal permeability in mice. Plasma concentration of FITC-dextran 4000 (FITC-D4000) measured 1 h after gavage of a single dose of 600 mg FITCD4000 per kg body weight. Quantification of plasma concentration from n=10 mice per group of treatment expressed as mean ± SD of triplicates of independent pooled samples. ***p<0.001 vehicle vs gliadin; °°°p<0.01, gliadin vs VX-770+gliadin (ANOVA, Bonferroni post test).BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (D) NLRP3 expression by immunoblotting with specific antibodies in small intestines from 3 mice. Densitometric analysis are mean ± SD of triplicates of independent pooled samples. **p<0.01 vehicle vs gliadin; °p<0.05, gliadin vs VX-770+gliadin. Data information: The blots are representative of one experiment for group of treatment.BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). (E-F) mRNA (E) or protein (by specific ELISA) (F) levels of IL-15, IL-21, IL-17A and IFN-γ. Mean±SD of triplicates of independent pooled samples. *p<0.05, **p<0.01, ***p<0.001 vehicle vs gliadin; °p<0.05, °°p<0.01, °°°p<0.001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). Transcript level of Rorc and Tbet (G) from small intestine homogenates. Mean ± SD of triplicates of independent pooled samples. ***p<0.001 or ****p<0.0001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).BALB/c mice fed with a gluten-free diet for at least 3 generations, orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks) in the presence or absence of intraperitoneal VX-770 administered 15 minutes prior gliadin challenge (n=10 mice per group of treatment). protein levels (by specific ELISA) of IL-10 and TGF-β (H) from small intestine homogenates. Mean ± SD of triplicates of independent pooled samples. ***p<0.001 or ****p<0.0001 gliadin vs VX-770+gliadin (ANOVA, Bonferroni post hoc test).IFN-γ release (ELISA) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co-culture model upon 24 h challenge of confluent CaCo-2 cells in the upper compartment with PT-gliadin (I) in presence or absence of VX-770. Mean ± SD of triplicates of independent pooled samples. ***p<0.001, PT-gliadin vs medium; °°°p<0.001, PT-gliadin vs PT-gliadin + VX-770 (n=4) (ANOVA, Bonferroni post hoc test).IFN-γ release (ELISA) in culture supernatants by PBMC from 6 celiac patients or 4 controls cultured in the lower compartment of a bidimensional co-culture model upon 24 h challenge of confluent CaCo-2 cells in the upper compartment with combination of P31-43 and P57-68 (J) in presence or absence of VX-770. Mean ± SD of triplicates of independent pooled samples. ; ***p<0.001, P57-68 or P31-43 vs P31-43/P57-68 combination (n=6); °°°p<0.001, P57-68/P31-43 combination vs VX-770 + P57-68/P31-43 (n=3), (ANOVA, Bonferroni post hoc test).(K) IL-10 release (ELISA) in culture supernatants by PBMC from 4 celiac patients cultured as in (J). Mean ± SD of triplicates of independent pooled samples. ****p<0.0001, P57-68/P31-43 combination vs P57-68/P31-43+VX-770 treatment (n=3) (ANOVA, Bonferroni post hoc test).(L) IFN-γ release (ELISA) into culture supernatants by PBMC from 3 celiac patients cultured in the lower compartment of a bidimensional co-culture model upon a 24 h challenge of confluent Caco-2CFTR-KO cells in the upper compartment with a combination of P31-43 and P57-68 in the presence or absence of VX-770, as in J and K. *p<0.05, medium vs P57-68/P31-43 combination (n=3) (ANOVA, Bonferroni post hoc test).


Figure 1 ( C ) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 ( ACT2 ) as a loading control . ( D ) qPCR quantification of shGAG and flGAG - POL normalized to ACT2 and to GLYCERALDEHYDE - 3 - PHOSPHATE DEHYDROGENASE C SUBUNIT ( GAPC ) levels . qPCR was performed on n = 3 biological replicates ; bars : standard error . ( * * ) = p - value < 0 . 01 ( two - sided t - test between indicated values ) . ( E ) Western analysis of GAG and RDR6 with Coomassie ( coom . ) staining as a loading control . Arrow indicates cognate RDR6 protein band . ( F - G ) sRNA - seq profiles from EVD de - repressed in the ddm1 ( F ) or ddm1 dcl1 ( G ) backgrounds . Different siRNA size categories are stacked . Nomenclature as in ( B ) .

O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control.(D) qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n=3 biological replicates; bars: standard error. (**) = p-value < 0.01 (two-sided t-test between indicated values).(E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band.(F-G) sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B).


Figure 2 ( B ) Expression levels of shGFP ( spliced ) and GFP - EVDint / ter - GUS ( unspliced ) transcripts , relative to ACT2 and AT4G26410 ( RHIP1 ) , in the WT or rdr6 background . qPCR was performed on three biological replicates and error bars represent the standard error on . ( * ) = p - value < 0 . 05 ( two - sided t - test against corresponding controls ) . ( C ) sRNA - seq profile mapped on the genomic 35S : GFP - EVDint / ter - GUS locus . ( RPM ) Reads per million . Positions indicated in nucleotides ( nt ) from the start of the 35S sequence . Dashed vertical lines : shGFP and GFP - GUS 3 ' ends .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. (*) = p-value < 0.05 (two-sided t-test against corresponding controls).(C) sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends.


Figure 3 ( D ) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2 . qPCR was performed on three biological replicates and error bars represent the standard error . ( ns . ) = non - significant , ( * ) = p - value < 0 . 05 , ( * * ) = p - value < 0 . 01 , ( two - sided t - test between indicated samples / targets ) . ( E ) High and low molecular - weight RNA analysis of EVD GAG ( GAG - ex1 ) and EVD intron ( GAG - in ) in two independent T1 bulks from each indicated line . The filled arrows on the right hand - side or with an asterisk on the blots correspond to the transcripts depicted in ( A - C ) . ACT2 : loading control for mRNAs ; tasiR255 , miR173 and U6 : loading controls for sRNAs . Hybridizations for GAG - ex2 and POL probes are found in Supp . Fig . 5A .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2. qPCR was performed on three biological replicates and error bars represent the standard error. (ns.) = non-significant, (*) = p-value < 0.05, (**) = p-value < 0.01, (two-sided t-test between indicated samples/targets).(E) High and low molecular-weight RNA analysis of EVD GAG (GAG-ex1) and EVD intron (GAG-in) in two independent T1 bulks from each indicated line. The filled arrows on the right hand-side or with an asterisk on the blots correspond to the transcripts depicted in (A-C). ACT2: loading control for mRNAs; tasiR255, miR173 and U6: loading controls for sRNAs. Hybridizations for GAG-ex2 and POL probes are found in Supp. Fig.5A.


Figure 4 ( B ) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in ( A ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 4(B) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in (A).


Figure 5 ( A ) Comparison of RNA isoforms and sRNA patterns generated by 35S : GFP - GUS and 35S : GFP - EVDint / ter - GUS . High and low molecular - weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background . mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig . 2A . EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs , respectively . ( B ) . Western blot analysis of the translation products from GFP and GFP - GUS transcripts . Coomassie ( coom . ) staining as a loading control . Black arrow : GFP - GUS fusion protein ; white arrow : GFP protein . ( C ) Nucleo - cytosolic distribution of 35S : EVD and 35S : GFP - EVDint / ter - GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR . RNA extracted from Total , nuclear ( Nucl ) and cytoplasmic ( Cyto ) fractions was reverse transcribed with random hexamers and oligo ( dT ) . snoRNA U5 is shown as a nuclear - only RNA control . ( D ) Same as in ( C ) but using exclusively oligo ( dT ) to reverse transcribe poly ( A ) + RNAs .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively.(B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein.(C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control. (D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.


Figure 6 ( B ) RIBO - seq coverage profiles from 35S : EVD in WT or rdr6 . RPM : Reads per million . ( C ) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P - sites to calculate codon coverage . The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) RIBO-seq coverage profiles from 35S:EVD in WT or rdr6. RPM: Reads per million. (C) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P-sites to calculate codon coverage. The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence.


Figure 1 ( B ) Schematic of marker - less GFP - tagging at the endogenous locus and representative live cell images of Mad1 - , Mad2 - , and Mad3 - GFP strains ( average intensity projections ) . ( C ) Representative images of single molecule mRNA FISH ( smFISH ) staining of S . pombe using probes against GFP ( red ) . DNA was stained with DAPI ( blue ) . The gamma - value was adjusted to make the cytoplasm visible ; cell shapes are outlined in blue . ( D ) Frequency distribution of mRNA numbers per cell determined by smFISH ; n = number of cells . Curves show fit to a Poisson distribution . ( E ) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP - tagged gene or the endogenous , untagged gene . Curves show fit to a Poisson distribution . The difference for mad1 + is statistically significant , that for mad2 + is not A lower mRNA number for untagged mad1 + was also observed in an independent strain . ( F ) Co - staining by smFISH using probes against mad1 + and GFP either in a strain expressing mad1 + - GFP as a positive control or in a strain expressing wild - type mad1 + and mad2 + - GFP . Cytoplasmic mad1 + ( green ) or GFP mRNA spots ( magenta ) were quantified as co - localizing or not with the respective other . For the mad1 + - GFP strain , 544 cells and a total of 1 , 641 mad1 spots and 1 , 839 GFP spots were analyzed ; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm . For the mad1 + mad2 + - GFP strain , 571 cells and a total of 1 , 107 mad1 spots and 1 , 537 GFP spots were analyzed ; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm .

O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Schematic of marker-less GFP-tagging at the endogenous locus and representative live cell images of Mad1-, Mad2-, and Mad3-GFP strains (average intensity projections).(C) Representative images of single molecule mRNA FISH (smFISH) staining of S. pombe using probes against GFP (red). DNA was stained with DAPI (blue). The gamma-value was adjusted to make the cytoplasm visible; cell shapes are outlined in blue. (D) Frequency distribution of mRNA numbers per cell determined by smFISH; n = number of cells. Curves show fit to a Poisson distribution.(E) Frequency distribution of mRNA numbers per cell using FISH probes against the endogenous genes and using either strains expressing the GFP-tagged gene or the endogenous, untagged gene. Curves show fit to a Poisson distribution. The difference for mad1+ is statistically significant, that for mad2+ is not A lower mRNA number for untagged mad1+ was also observed in an independent strain.(F) Co-staining by smFISH using probes against mad1+ and GFP either in a strain expressing mad1+-GFP as a positive control or in a strain expressing wild-type mad1+ and mad2+-GFP. Cytoplasmic mad1+ (green) or GFP mRNA spots (magenta) were quantified as co-localizing or not with the respective other. For the mad1+-GFP strain, 544 cells and a total of 1,641 mad1 spots and 1,839 GFP spots were analyzed; 48 cells were not considered since they did not contain at least one spot of each type in the cytoplasm. For the mad1+ mad2+-GFP strain, 571 cells and a total of 1,107 mad1 spots and 1,537 GFP spots were analyzed; 158 cells were not considered since they did not contain at least one spot of each type in the cytoplasm.


Figure 2 ( A ) Cellular protein noise ( coefficient of variation , CV = std / mean ) in live - cell microscopy images of S . pombe ; n = 7 images ( Nmt1 - GFP ) , 11 ( Mad1 - GFP ) , 19 ( Mad2 - GFP ) , 10 ( Mad3 - GFP ) ; single images had 16 - 79 GFP - positive and 6 - 94 GFP - negative ( control ) cells . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Mad1 , Mad2 , and Mad3 all showed significantly lower noise than Nmt1 ( Wilcoxon rank sum test ; all p < 0 . 001 ) . ( B ) Simulations of stochastic gene expression noise from selected mRNA / protein half - life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell , and a mean protein number of 6000 per cell . The x - axis of each graph shows time , the y - axis shows mRNA number per cell ( blue ) or protein number per cell ( black ) . ( C ) Theoretical prediction for the coefficient of variation ( CV = std / mean ) of the protein number per cell , assuming different mRNA and protein half - lives , using the same underlying model as in B . Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3 . 5 , and a mean protein number per cell of 6000 ( approx . 100 nM ) . ( D ) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Lines are regression curves from generalized linear mixed model fits , excluding the measurements at t = 0 in order to accommodate for non - instantaneous labeling by 4tU . Act1 + and ecm33 + were used as long and short half - life controls , respectively ; qPCR was performed for the endogenous mRNAs . Half - lives ( 95 % confidence interval ) : mad1 + 5 . 6 min ( 4 . 3 - 8 . 4 ) , mad2 + 7 . 7 min ( 6 . 2 - 10 . 4 ) , mad3 + 5 . 2 min ( 4 . 3 - 6 . 9 ) , act1 + 61 . 8 min ( 37 . 2 - 172 . 3 ) , ecm33 + 5 . 0 min ( 4 . 5 - 5 . 7 ) . ( E ) Protein abundances after translation shut - off with cycloheximide ( CHX ) ; n = 3 experiments , error bars = std . Lines indicate fit to a one - phase exponential decay . Cdc2 and Cdc13 were used as long and short half - life controls , respectively .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Cellular protein noise (coefficient of variation, CV = std / mean) in live-cell microscopy images of S. pombe; n = 7 images (Nmt1-GFP), 11 (Mad1-GFP), 19 (Mad2-GFP), 10 (Mad3-GFP); single images had 16-79 GFP-positive and 6-94 GFP-negative (control) cells. Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Mad1, Mad2, and Mad3 all showed significantly lower noise than Nmt1 (Wilcoxon rank sum test; all p < 0.001).(B) Simulations of stochastic gene expression noise from selected mRNA/protein half-life combinations assuming a constantly active promoter Synthesis rates were set to obtain a mean mRNA number of 4 per cell, and a mean protein number of 6000 per cell. The x-axis of each graph shows time, the y-axis shows mRNA number per cell (blue) or protein number per cell (black). (C) Theoretical prediction for the coefficient of variation (CV = std / mean) of the protein number per cell, assuming different mRNA and protein half-lives, using the same underlying model as in B. Synthesis rates were adjusted to maintain a mean mRNA number per cell of 3.5, and a mean protein number per cell of 6000 (approx. 100 nM).(D) mRNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Lines are regression curves from generalized linear mixed model fits, excluding the measurements at t = 0 in order to accommodate for non-instantaneous labeling by 4tU. Act1+ and ecm33+ were used as long and short half-life controls, respectively; qPCR was performed for the endogenous mRNAs. Half-lives (95 % confidence interval): mad1+ 5.6 min (4.3 - 8.4), mad2+ 7.7 min (6.2 - 10.4), mad3+ 5.2 min (4.3 - 6.9), act1+ 61.8 min (37.2 - 172.3), ecm33+ 5.0 min (4.5 - 5.7).(E) Protein abundances after translation shut-off with cycloheximide (CHX); n = 3 experiments, error bars = std. Lines indicate fit to a one-phase exponential decay. Cdc2 and Cdc13 were used as long and short half-life controls, respectively.


Figure 3 ( A ) The mean CSC value for each S . pombe gene ( CSCg ) relative to protein number per cell by mass spectrometry CSC was determined using the mRNA half - life data Colored dots highlight proteins of interest . For Mad2 and Bub1 , no protein abundance data was available . ( B ) Cumulative frequency distribution of the CSCg values for protein - coding S . pombe genes . The position of spindle assembly checkpoint genes is highlighted . ( D ) Scatter plots of whole cell RNA counts versus cell length . Solid lines are regression curves from generalized linear mixed model fits ( gray : wild type , black : codon - optimized or ste13 ∆ ) . Dashed lines : 95 % bootstrap confidence bands for the regression curves . Model estimates of the ratio relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to five replicates per genotype . ( E ) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( dark = ste13 + , light = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded area : 95 % bootstrap confidence band for ste13 + ; dashed lines : 95 % bootstrap confidence band for ste13 ∆ . Half - life estimates are included with 95 % bootstrap confidence intervals in brackets .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) The mean CSC value for each S. pombe gene (CSCg) relative to protein number per cell by mass spectrometry CSC was determined using the mRNA half-life data Colored dots highlight proteins of interest. For Mad2 and Bub1, no protein abundance data was available. (B) Cumulative frequency distribution of the CSCg values for protein-coding S. pombe genes. The position of spindle assembly checkpoint genes is highlighted.(D) Scatter plots of whole cell RNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to five replicates per genotype.(E) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.


Figure 4 ( A ) Immunoblot of S . pombe protein extracts from cells expressing wild - type ( WT ) or codon - optimized ( co ) Mad2 - GFP or Mad3 - GFP probed with antibodies against GFP and Cdc2 ( loading control ) . Lanes 3 - 5 are a 1 : 1 dilution series of the extract from cells expressing the codon - optimized version . ( B ) Immunoblot of protein extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad2 , Mad3 , and tubulin ( loading control ) . A 1 : 1 dilution series was loaded for quantification . ( C ) Estimates of the protein concentration relative to wild - type conditions from experiments such as in ( A ) and ( B ) . Bars are experimental replicates , dots are technical replicates . Two - sided t - tests : p = 0 . 03 ( Mad2 - co ) , 0 . 004 ( Mad3 - co ) , 0 . 82 ( Mad2 ste13 ∆ ) , 0 . 15 ( Mad3 ste13 ∆ ) . ( D ) Whole - cell GFP concentration from individual live cell fluorescence microscopy experiments ( a . u . = arbitrary units ) . Boxes show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Codon - optimized concentration significantly higher than wild type for both genes ( GLMM ) . Mad2 - GFP : n = 468 and 413 ; Mad2 - co - GFP : n = 206 and 366 ; Mad3 - GFP : n = 224 and 127 ; Mad3 - co - GFP : n = 160 , 450 and 212 cells . ( E ) Representative images from one of the experiments in ( D ) . A single Z - slice is shown . Cells are outlined in gray .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad2-GFP or Mad3-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a 1:1 dilution series of the extract from cells expressing the codon-optimized version. (B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad2, Mad3, and tubulin (loading control). A 1:1 dilution series was loaded for quantification. (C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Two-sided t-tests: p = 0.03 (Mad2-co), 0.004 (Mad3-co), 0.82 (Mad2 ste13∆), 0.15 (Mad3 ste13∆).(D) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxes show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly higher than wild type for both genes (GLMM). Mad2-GFP: n = 468 and 413; Mad2-co-GFP: n = 206 and 366; Mad3-GFP: n = 224 and 127; Mad3-co-GFP: n = 160, 450 and 212 cells. (E) Representative images from one of the experiments in (D). A single Z-slice is shown. Cells are outlined in gray.


Figure 5 ( B ) Scatter plots of whole cell mRNA counts versus cell length . Solid lines are regression curves from generalized linear mixed model fits ( gray : wild type , black : codon - optimized or ste13 ∆ ) . Dashed lines : 95 % bootstrap confidence bands for the regression curves . Model estimates of the ratio relative to wild - type mRNA are included with bootstrap 95 % confidence interval in brackets . Two to three replicates per genotype . ( C ) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool ( two independent experiments ) . Solid lines : regression curves from generalized linear mixed model fits ( dark = ste13 + , light = ste13 ∆ ) , excluding t = 0 to accommodate for non - instantaneous labeling by 4tU . Shaded area : 95 % bootstrap confidence band for ste13 + ; dashed lines : 95 % bootstrap confidence band for ste13 ∆ . Half - life estimates are included with 95 % bootstrap confidence intervals in brackets . ( D ) Comparison between mean CSC values for selected genes ( CSCg ) and mRNA half - life measured with or without deletion of ste13 + .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Scatter plots of whole cell mRNA counts versus cell length. Solid lines are regression curves from generalized linear mixed model fits (gray: wild type, black: codon-optimized or ste13∆). Dashed lines: 95 % bootstrap confidence bands for the regression curves. Model estimates of the ratio relative to wild-type mRNA are included with bootstrap 95 % confidence interval in brackets. Two to three replicates per genotype.(C) Time course of RNA abundances by qPCR following metabolic labeling and removal of the labeled pool (two independent experiments). Solid lines: regression curves from generalized linear mixed model fits (dark = ste13+, light = ste13∆), excluding t = 0 to accommodate for non-instantaneous labeling by 4tU. Shaded area: 95 % bootstrap confidence band for ste13+; dashed lines: 95 % bootstrap confidence band for ste13∆. Half-life estimates are included with 95 % bootstrap confidence intervals in brackets.(D) Comparison between mean CSC values for selected genes (CSCg) and mRNA half-life measured with or without deletion of ste13+.


Figure 6 ( A ) Immunoblot of S . pombe protein extracts from cells expressing wild - type ( WT ) or codon - optimized ( co ) Mad1 - GFP probed with antibodies against GFP and Cdc2 ( loading control ) . Lanes 3 - 5 are a dilution series of the extract from wild - type cells . ( B ) Immunoblot of protein extracts from wild - type ( WT ) or ste13 ∆ strains probed with antibodies against Mad1 and tubulin ( loading control ) . A 1 : 1 dilution series was loaded for quantification . ( C ) Estimates of the protein concentration relative to wild - type conditions from experiments such as in ( A ) and ( B ) . Bars are experimental replicates , dots are technical replicates . Blue lines indicate the mean of all experiments . Two - sided t - tests : p = 0 . 005 ( Mad1 - co , n = 4 experimental replicates ) ; p = 0 . 16 ( Mad1 ste13 ∆ , n = 2 ) . ( D ) Whole - cell GFP concentration from individual live cell fluorescence microscopy experiments ( a . u . = arbitrary units ) . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Codon - optimized concentration significantly lower than wild type ( generalized linear mixed model ) . Mad1 - GFP : n = 197 and 224 ; Mad1 - co - GFP : n = 80 and 377 cells . ( E ) Representative images from one of the experiments in ( D ) . An average projection of three Z - slices is shown ; cells are outlined in gray . ( F ) Live - cell imaging for time spent in mitosis . The alp7 + gene was deleted to increase the likelihood of spindle assembly checkpoint activation . Localization of Plo1 - tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis Exp1 : n = 73 ( WT ) and 94 cells ( co ) ; Exp2 : n = 126 ( WT ) and 152 cells ( co ) . Boxplots show median and interquartile range ( IQR ) ; whiskers extend to values no further than 1 . 5 times the IQR from the first and third quartile , respectively . Measurements for individual cells are shown in addition ( gray circles if measurement was exact , red triangles if end of mitosis was not captured because imaging ended ) . Difference between WT and co : p = 0 . 14 ( Exp1 ) and 0 . 15 ( Exp2 ) by Kolmogorov - Smirnov test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Immunoblot of S. pombe protein extracts from cells expressing wild-type (WT) or codon-optimized (co) Mad1-GFP probed with antibodies against GFP and Cdc2 (loading control). Lanes 3-5 are a dilution series of the extract from wild-type cells. (B) Immunoblot of protein extracts from wild-type (WT) or ste13∆ strains probed with antibodies against Mad1 and tubulin (loading control). A 1:1 dilution series was loaded for quantification. (C) Estimates of the protein concentration relative to wild-type conditions from experiments such as in (A) and (B). Bars are experimental replicates, dots are technical replicates. Blue lines indicate the mean of all experiments. Two-sided t-tests: p = 0.005 (Mad1-co, n = 4 experimental replicates); p = 0.16 (Mad1 ste13∆, n = 2).(D) Whole-cell GFP concentration from individual live cell fluorescence microscopy experiments (a.u. = arbitrary units). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Codon-optimized concentration significantly lower than wild type (generalized linear mixed model). Mad1-GFP: n = 197 and 224; Mad1-co-GFP: n = 80 and 377 cells. (E) Representative images from one of the experiments in (D). An average projection of three Z-slices is shown; cells are outlined in gray.(F) Live-cell imaging for time spent in mitosis. The alp7+ gene was deleted to increase the likelihood of spindle assembly checkpoint activation. Localization of Plo1-tdTomato to spindle pole bodies was used to judge entry into and exit from mitosis Exp1: n = 73 (WT) and 94 cells (co); Exp2: n = 126 (WT) and 152 cells (co). Boxplots show median and interquartile range (IQR); whiskers extend to values no further than 1.5 times the IQR from the first and third quartile, respectively. Measurements for individual cells are shown in addition (gray circles if measurement was exact, red triangles if end of mitosis was not captured because imaging ended). Difference between WT and co: p = 0.14 (Exp1) and 0.15 (Exp2) by Kolmogorov-Smirnov test.


Figure 7 ( A ) Top : Immunoprecipitation ( IP ) with anti - GFP or anti - Mad1 from extracts of haploid S . pombe cells expressing both untagged and GFP - tagged Mad1 , probed with antibodies against Mad1 and tubulin ; in = input ( 2 . 5 % of extract for IP ) , sup = supernatant after IP . Bottom : Comparison between the observed ( obs . ) and the expected ( expect . ) ratio between Mad1 - GFP and untagged Mad1 in the IP given their ratio in the input ; two and one experiment ( s ) , respectively . One more GFP - IP from the same strain was unquantifiable , because no second band was visible in the IP . ( C ) Top : Anti - GFP immunoprecipitation ( IP ) and Strep pull - down ( PD ) from extracts of diploid cells expressing Mad1 - GFP and Mad1 - Strep from the two endogenous loci ; membrane probed with anti - Mad1 ; in = input ( 7 % of extract for IP / PD ) , sup = supernatant after IP / PD . Bottom : as in ( A ) , 2 experiments each . The experiment shown at the top and two more GFP - IPs from the same strain were unquantifiable , because no second band was visible in the IP . ( D ) In vitro translation ( IVT ) of Mad1 - flag - His and untagged Mad1 in the presence of 35S - labelled Methionine and Cysteine , followed by Mad1 immunoprecipitation ( IP ) or His pull - down ( PD ) ; in = input ( 9 . 5 % of extract for IP / PD ) , sup = supernatant after IP / PD . An IVT with only untagged Mad1 was used to check for specificity of the His PD ( right side ) . Shown is the autoradiograph after SDS - PAGE with quantification of the Mad1 - flag - His to untagged Mad1 ratio in select lanes . ( E ) Test for mRNA dimerization by single molecule mRNA FISH ; probes against mad1 + and GFP . Top : Schematic of genotypes . Bottom left : Intensity of cytoplasmic mad1 + mRNA spots in the different strains . For the 2 copy strain , a mad1 + spot was classified as mad1 + - GFP if it was colocalizing with a GFP spot , and as mad1 + otherwise . Colors as indicated in the schematic . Vertical solid line : peak of each density plot ; dashed line : theoretical position of a double - intensity peak . Number of spots analyzed : mad1 + ( 1 copy strain ) = 921 , mad1 + ( 2 copy strain ) = 637 , mad1 + - GFP ( 2 copy strain ) = 982 , mad1 + - GFP ( 1 copy strain ) = 1699 . Bottom right : Counts of cytoplasmic mad1 + or mad1 + - GFP mRNA from the same experiment with generalized linear mixed model fits as lines . Number of cells : 1 copy strain mad1 + = 478 , 2 copy strain = 327 , 1 copy strain mad1 + - GFP = 466 . ( F ) Experiment similar to ( E ) , except that cells expressing both mad1 + - GFP and mad3 + - GFP from the respective endogenous locus were probed with FISH probes against mad1 + and GFP mRNA . A GFP spot was classified as mad1 + - GFP if it was colocalizing with a mad1 + spot ( arrowheads ) , and as mad3 + - GFP otherwise . The intensity of GFP spots was quantified . Vertical solid line : peak of each density plot ; dashed line : theoretical position of a double - intensity peak . Number of spots analyzed : mad1 + - GFP = 987 , mad3 + - GFP = 1299 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Top: Immunoprecipitation (IP) with anti-GFP or anti-Mad1 from extracts of haploid S. pombe cells expressing both untagged and GFP-tagged Mad1, probed with antibodies against Mad1 and tubulin; in = input (2.5 % of extract for IP), sup = supernatant after IP. Bottom: Comparison between the observed (obs.) and the expected (expect.) ratio between Mad1-GFP and untagged Mad1 in the IP given their ratio in the input ; two and one experiment(s), respectively. One more GFP-IP from the same strain was unquantifiable, because no second band was visible in the IP.(C) Top: Anti-GFP immunoprecipitation (IP) and Strep pull-down (PD) from extracts of diploid cells expressing Mad1-GFP and Mad1-Strep from the two endogenous loci; membrane probed with anti-Mad1; in = input (7 % of extract for IP/PD), sup = supernatant after IP/PD. Bottom: as in (A), 2 experiments each. The experiment shown at the top and two more GFP-IPs from the same strain were unquantifiable, because no second band was visible in the IP.(D) In vitro translation (IVT) of Mad1-flag-His and untagged Mad1 in the presence of 35S-labelled Methionine and Cysteine, followed by Mad1 immunoprecipitation (IP) or His pull-down (PD); in = input (9.5 % of extract for IP/PD), sup = supernatant after IP/PD. An IVT with only untagged Mad1 was used to check for specificity of the His PD (right side). Shown is the autoradiograph after SDS-PAGE with quantification of the Mad1-flag-His to untagged Mad1 ratio in select lanes.(E) Test for mRNA dimerization by single molecule mRNA FISH; probes against mad1+ and GFP. Top: Schematic of genotypes. Bottom left: Intensity of cytoplasmic mad1+ mRNA spots in the different strains. For the 2 copy strain, a mad1+ spot was classified as mad1+-GFP if it was colocalizing with a GFP spot, and as mad1+ otherwise. Colors as indicated in the schematic. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+ (1 copy strain) = 921, mad1+ (2 copy strain) = 637, mad1+-GFP (2 copy strain) = 982, mad1+-GFP (1 copy strain) = 1699. Bottom right: Counts of cytoplasmic mad1+ or mad1+-GFP mRNA from the same experiment with generalized linear mixed model fits as lines. Number of cells: 1 copy strain mad1+ = 478, 2 copy strain = 327, 1 copy strain mad1+-GFP = 466.(F) Experiment similar to (E), except that cells expressing both mad1+-GFP and mad3+-GFP from the respective endogenous locus were probed with FISH probes against mad1+ and GFP mRNA. A GFP spot was classified as mad1+-GFP if it was colocalizing with a mad1+ spot (arrowheads), and as mad3+-GFP otherwise. The intensity of GFP spots was quantified. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+-GFP = 987, mad3+-GFP = 1299.


Figure 1A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM , 10 µM , or 20 µM CDDP for 3 days . The explants were labeled with Myosin 7A ( red ) to identify hair cells and NF200 ( green ) to highlight auditory nervefibers . Scale bar = 20 µm . nf : nerve fibers , OHCs : outer hair cells , IHCs : inner hair cells . B Dose - response curves of CDDP - induced loss of OHCs ( blue line ) and IHCs ( red line ) in basal cochlear regions . Data are expressed as mean ± SEM ( n = 5 cochleae per condition and per time point ) . C Effect over time on OHCs and IHCs treated with either culture medium alone ( light blue and red lines for OHCs and IHCs , respectively ) , or 10 µM CDDP ( blue and red lines for OHCs and IHCs , respectively ) . Data are expressed as mean ± SEM ( n = 5 cochleae per condition and per time point ) . D Representative Western blot analysis using antibodies against β - actin and cleaved caspase 3 in whole cochlear extracts . E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group ( n = 6 cochleae per condition and per time point ) . Actin served as a loading control . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( * P ≤ 0 . 028 , * * P = 0 . 0007 , cochleae at the different times after CDDP exposure vs . control 12h ) . F , G Confocal images showing the basal region of cochlear explants treated with either culture medium alone or containing 10 µM CDDP for 1 ( F ) or 2 ( G ) days . Hair cells were identified using Myosin 7A ( red ) , phosphatidylserine sites on the cell membrane surface were detected using fluorochrome - labeled Annexin V ( green in F ) , and apoptotic DNA fragmentation was identified using a TUNEL apoptosis kit ( green in G ) . The white arrow heads indicate cell surface Annexin V - positive labeling ( lower left ) and TUNEL - positive nuclei ( lower right ) . Scale bar = 15 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Confocal images showing the basal regions of cochlear explants treated with either culture medium alone or medium containing 5 µM, 10 µM, or 20 µM CDDP for 3 days. The explants were labeled with Myosin 7A (red) to identify hair cells and NF200 (green) to highlight auditory nervefibers. Scale bar=20 µm. nf: nerve fibers, OHCs: outer hair cells, IHCs: inner hair cells.B Dose-response curves of CDDP-induced loss of OHCs (blue line) and IHCs (red line) in basal cochlear regions. Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).C Effect over time on OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively). Data are expressed as mean ± SEM (n=5 cochleae per condition and per time point).D Representative Western blot analysis using antibodies against β-actin and cleaved caspase 3 in whole cochlear extracts.E Histogram representing the change in cleaved caspase 3 expression levels over time in control and CDDP group (n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P ≤ 0.028, **P=0.0007, cochleae at the different times after CDDP exposure vs. control 12h).F, G Confocal images showing the basal region of cochlear explants treated with either culture medium alone or containing 10 µM CDDP for 1 (F) or 2 (G) days. Hair cells were identified using Myosin 7A (red), phosphatidylserine sites on the cell membrane surface were detected using fluorochrome-labeled Annexin V (green in F), and apoptotic DNA fragmentation was identified using a TUNEL apoptosis kit (green in G). The white arrow heads indicate cell surface Annexin V-positive labeling (lower left) and TUNEL-positive nuclei (lower right). Scale bar=15 µm.


Figure 2A , F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A ( red ) and γH2AX ( green in A ) , or Myosin 7A ( red ) and 53BP1 ( green in F ) . The white arrow heads indicate CDDP - induced increase of γH2AX foci in both OHCs and IHCs . Scale bars : A and F = 24 µm . B , G Higher magnification images of representative OHC and IHCnuclei from all conditions tested . Scale bar = 5 µm . C , H Histograms displaying green fluorescent signal intensity of x - projections and 3D reconstruction images from OHC and IHCnuclei presented in B and G , respectively . F0 corresponds to background noise , gray dashed lines represent the threshold used to detect specific foci labeling . 3D images were reconstructed according to the threshold defined in the histograms of all conditions tested . Scale bar = 5 µm . D , E , I Quantification analysis of γH2AX foci number ( D ) ; total volume of foci per nucleus ( E ) ; 53BP1 foci number ( I ) from OHCs and IHCs treated with either culture medium alone ( light blue and red lines for OHCs and IHCs , respectively ) , or 10 µM CDDP ( blue and red lines for OHCs and IHCs , respectively , n = 50 nuclei per condition and per time point ) . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( D : * * P = 0 . 007 ; E and I : * * P ≤ 0 . 009 . CDDP - exposed OHCsvs . CDDP - exposed IHCs ) . J Higher magnification images showing representative OHCnuclei from the basal region of the organ of Corti treated with 10 µM CDDP for 1 day and immunolabeled for 53BP1 ( red ) , γH2AX ( green ) and counterstained with Hoechst ( blue ) . The 3D image shows co - localization of smaller sized 53BP1 foci within the γH2AX foci . Scale bar = 5 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and γH2AX (green in A), or Myosin 7A (red) and 53BP1 (green in F). The white arrow heads indicate CDDP-induced increase of γH2AX foci in both OHCs and IHCs. Scale bars: A and F=24 µm.B, G Higher magnification images of representative OHC and IHCnuclei from all conditions tested. Scale bar=5 µm.C, H Histograms displaying green fluorescent signal intensity of x-projections and 3D reconstruction images from OHC and IHCnuclei presented in B and G, respectively. F0 corresponds to background noise, gray dashed lines represent the threshold used to detect specific foci labeling. 3D images were reconstructed according to the threshold defined in the histograms of all conditions tested. Scale bar=5 µm.D, E, I Quantification analysis of γH2AX foci number (D); total volume of foci per nucleus (E); 53BP1 foci number (I) from OHCs and IHCs treated with either culture medium alone (light blue and red lines for OHCs and IHCs, respectively), or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (D: **P=0.007; E and I: **P ≤ 0.009. CDDP-exposed OHCsvs. CDDP-exposed IHCs).J Higher magnification images showing representative OHCnuclei from the basal region of the organ of Corti treated with 10 µM CDDP for 1 day and immunolabeled for 53BP1 (red), γH2AX (green) and counterstained with Hoechst (blue). The 3D image shows co-localization of smaller sized 53BP1 foci within the γH2AX foci. Scale bar=5 µm.


Figure 3A Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A ( red ) and p - ATM ( green ) . Scale bar = 15 µm . B Higher magnification images showing representative OHC and IHCnuclei from control and CDDP - exposed cells . Scale bar = 5 µm . C Histograms displaying green fluorescent signal intensity of x - projections of OHC and IHCnuclei from control and CDDP - exposed cells . D Quantification analysis of p - ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone ( light blue and red lines for OHCs and IHCs , respectively ) or 10 µM CDDP ( blue and red lines for OHCs and IHCs , respectively , n = 50 nuclei per condition and per time point ) . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( * * P < 0 . 008 , * * * P = 0 . 00005 , CDDP - exposed OHCsvs . CDDP - exposed IHCs ) . E Higher magnification confocal and 3D images showing the co - localization of p - ATM foci ( green ) with 53BP1 foci ( red ) in an OHCnucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day . Scale bar = 5 µm . F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A ( red ) and p - Chk2 ( green ) . Scale bar = 15 µm . G Histograms of green fluorescent signal intensity from OHC and IHCnuclei of a CDDP - treated organ of Corti explant . H Quantification of Chk2 - positive nuclei in both IHCs ( red bars ) and OHCs ( blue bars ) from control and CDDP - treated organ of Corti cultures ( n = 50 nuclei per condition and per time point ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * P = 0 . 045 , * * * P = 0 . 0002 , CDDP - exposed IHCsvs . control IHCs ; CDDP - exposed OHCsvs . control OHCs ) . I Representative western blots using antibodies against p - p53 ( serine 15 ) , p53 and β - actin in control and 10 µM CDDP - exposed whole cochlear extracts . J - K Histograms representing the levels of total p53 protein ( J ) and p53 phosphorylation ( K ) in control ( gray bars ) and CDDP - exposed cochleae ( green bars , n = 6 cochleae per condition and per time point ) . Actin served as a loading control . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( J : * P ≤ 0 . 023 ; K : * P = 0 . 032 , * * P = 0 . 007 , * * * P ≤ 0 . 0008 , cochleae at the different times after CDDP exposure vs . control 12h ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-ATM (green). Scale bar=15 µm.B Higher magnification images showing representative OHC and IHCnuclei from control and CDDP-exposed cells. Scale bar=5 µm.C Histograms displaying green fluorescent signal intensity of x-projections of OHC and IHCnuclei from control and CDDP-exposed cells.D Quantification analysis of p-ATM foci numbers in both OHCs and IHCs exposed to either culture medium alone (light blue and red lines for OHCs and IHCs, respectively) or 10 µM CDDP (blue and red lines for OHCs and IHCs, respectively, n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (**P<0.008, ***P=0.00005, CDDP-exposed OHCsvs. CDDP-exposed IHCs).E Higher magnification confocal and 3D images showing the co-localization of p-ATM foci (green) with 53BP1 foci (red) in an OHCnucleus from the basal region of the organ of Corti culture treated with 10 µM CDDP for 1 day. Scale bar=5 µm.F Confocal images showing the basal region of organ of Corti cultures treated with either culture medium alone or medium containing 10 µM CDDP for 1 day and immunolabeled for Myosin 7A (red) and p-Chk2 (green). Scale bar=15 µm.G Histograms of green fluorescent signal intensity from OHC and IHCnuclei of a CDDP-treated organ of Corti explant.H Quantification of Chk2-positive nuclei in both IHCs (red bars) and OHCs (blue bars) from control and CDDP-treated organ of Corti cultures (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (*P=0.045, ***P=0.0002, CDDP-exposed IHCsvs. control IHCs; CDDP-exposed OHCsvs. control OHCs).I Representative western blots using antibodies against p-p53 (serine 15), p53 and β-actin in control and 10 µM CDDP-exposed whole cochlear extracts.J-K Histograms representing the levels of total p53 protein (J) and p53 phosphorylation (K) in control (gray bars) and CDDP-exposed cochleae (green bars, n=6 cochleae per condition and per time point). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (J: *P ≤ 0.023; K: *P=0.032, **P=0.007, ***P ≤ 0.0008, cochleae at the different times after CDDP exposure vs. control 12h).


Figure 4A 3D images of OHCnuclei taken from the basal regions of organ of Corti cultures treated with medium alone , 10 µM KU55933 , 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day and immunolabeled for 53BP1 ( green ) and Hoechst 33342 ( blue ) . Scale bar = 5 µm . B Quantification analysis of 53BP1 foci number per nucleus in both IHCs ( red bars ) and OHCs ( blue bars ) for all conditions ( n = 50 nuclei per condition and per time point ) . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P ≤ 0 . 0004 , CDDPvs . control , or CDDPvs . CDDP + KU55933 ) . C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone , 10 µM KU55933 , 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A ( red ) . Scale bar = 24 µm . D Histograms representing the numbers of surviving IHCs ( red bars ) and OHCs ( blue bars ) for all treatment conditions after 5 days ( n = 5 cochleae per condition and per time point ) . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( * P = 0 . 03 , * * P = 0 . 004 , * * * P ≤ 0 . 0008 , CDDPvs . control , or CDDPvs . CDDP + KU55933 ) . E 3D images of OHCnuclei from the basal regions of organ of Corti cultures treated with culture medium alone , 100 µM PFT - α , 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT - α for 1 day and immunolabeled for 53BP1 ( green ) and Hoechst 33342 ( blue ) . Scale bar = 5 µm . F Quantification of 53BP1 foci number per nucleus in both IHCs ( red bars ) and OHCs ( blue bars ) for all conditions ( n = 50 nuclei per condition and per time point ) . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P ≤ 0 . 0007 , CDDPvs . control ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 3D images of OHCnuclei taken from the basal regions of organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.B Quantification analysis of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0004, CDDPvs. control, or CDDPvs. CDDP+KU55933).C Confocal images showing the basal region of the organ of Corti cultures treated with medium alone, 10 µM KU55933, 10 µM CDDP or 10 µM CDDP in combination with 10 µM KU55933 for 5 days and immunolabeled for Myosin 7A (red). Scale bar=24 µm.D Histograms representing the numbers of surviving IHCs (red bars) and OHCs (blue bars) for all treatment conditions after 5 days (n=5 cochleae per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (*P=0.03, **P =0.004, ***P ≤ 0.0008, CDDPvs. control, or CDDPvs. CDDP+KU55933).E 3D images of OHCnuclei from the basal regions of organ of Corti cultures treated with culture medium alone, 100 µM PFT-α, 10 µM CDDP or 10 µM CDDP in combination with 100 µM PFT-α for 1 day and immunolabeled for 53BP1 (green) and Hoechst 33342 (blue). Scale bar=5 µm.F Quantification of 53BP1 foci number per nucleus in both IHCs (red bars) and OHCs (blue bars) for all conditions (n=50 nuclei per condition and per time point). Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0007, CDDPvs. control).


Figure 5A Representative auditory brainstem response ( ABR ) waveforms evoked by 16 kHz tone - bursts in p53wt mice treated with saline ( black plot ) or CDDP ( green plot ) , and p53 - / - mice treated with saline ( light blue plot ) or CDDP ( dark blue plot ) for 5 days . B ABR thresholds recorded in p53wt mice before ( gray plot ) and after 5 days of saline ( black plot ) or CDDP treatments ( green plot ) , and ABR thresholds recorded in p53 - / - mice before ( light gray plot ) , and after 5 days of saline ( light blue plot ) or CDDP treatment ( dark blue plot ) . C Mean ABR threshold from 4 kHz to 32 kHz derived from B . Data are expressed as mean ± SEM ( saline treated - group : n = 7 , CDDP treated - group : n = 12 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * P ≤ 0 . 008 , p53wt + CDDP , d5 vs . p53wt , before or p53wt + CDDP , d5 vs . p53 - / - + CDDP , d5 ) . D Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP - treated p53wt and p53 - / - mice after 5 days . Scale bar = 15µm . E Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from the cochlear apex provided from saline - treated p53wt mice ( black bars ) , CDDP - treated p53wt mice ( green bars ) or p53 - / - mice ( blue bars ) , after 5 days ( n = 5 per group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , p53wt + CDDP , d5 vs . p53wt + saline , d5 or p53wt + CDDP , d5 vs . p53 - / - + CDDP , d5 ) . F ABR thresholds from p53wt mice recorded prior to ( gray plot ) or after 5 days systemic treatment with : DMSO ( yellow plot ) , CDDP + DMSO ( green plot ) , CDDP + PFT - α ( pink plot ) . G Mean ABR threshold from 4 kHz to 32 kHz derived from F . Data are expressed as mean ± SEM ( DMSO treated - group : n = 7 ; CDDP + DMSO treated - group : n = 12 ; CDDP + PFT - α - treated group : n = 12 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P ≤ 0 . 0005 , CDDP + DMSO , d5 vs . before or CDDP + DMSO , d5 vs . CDDP + PFT - α , d5 ) . H Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from the cochlear apical end taken from p53wt mice treated with : DMSO ( yellow bars ) , CDDP + DMSO ( green bars ) , CDDP + PFT - α ( pink bars ) , after 5 days ( n = 5 per group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , CDDP + DMSO , d5 vs . DMSO , d5 or CDDP + DMSO , d5 vs . CDDP + PFT - α , d5 ) . I ABR thresholds from p53wt mice recorded prior to ( gray plot and black plot for left and right ear , respectively ) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear ( green plot ) , and of PFT - α into the right ear ( pink plot ) . J Mean ABR threshold from 4 kHz to 32 kHz derived from I . Data are expressed as mean ± SEM ( n = 14 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P = 0 . 0003 , CDDP + left ear , DMSOvs . before , left ear or CDDP + left ear , DMSOvs . CDDP + right ear , PFT - α . K Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1 . 1 , 2 . 6 , 3 . 5 or 4 . 1 mm from the cochlear apical end taken from the DMSO - treated left ear ( green bars ) and PFT - α treated right ear ( pink bars ) of the same CDDP - treated p53wt mice after 5 days ( n = 5 per group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , CDDP + right ear , PFT - α vs . CDDP + left ear , DMSO ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Representative auditory brainstem response (ABR) waveforms evoked by 16 kHz tone-bursts in p53wt mice treated with saline (black plot) or CDDP (green plot), and p53-/- mice treated with saline (light blue plot) or CDDP (dark blue plot) for 5 days.B ABR thresholds recorded in p53wt mice before (gray plot) and after 5 days of saline (black plot) or CDDP treatments (green plot), and ABR thresholds recorded in p53-/- mice before (light gray plot), and after 5 days of saline (light blue plot) or CDDP treatment (dark blue plot).C Mean ABR threshold from 4 kHz to 32 kHz derived from B. Data are expressed as mean ± SEM (saline treated-group: n=7, CDDP treated-group: n=12). One-way ANOVA test followed by post hoc Tukey's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt, before or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).D Representative scanning electron microscopy micrographs showing the basal regions of cochleae from CDDP-treated p53wt and p53-/- mice after 5 days. Scale bar=15µm.E Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apex provided from saline-treated p53wt mice (black bars), CDDP-treated p53wt mice (green bars) or p53-/- mice (blue bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, p53wt+CDDP, d5 vs. p53wt+saline, d5 or p53wt+CDDP, d5 vs. p53-/- + CDDP, d5).F ABR thresholds from p53wt mice recorded prior to (gray plot) or after 5 days systemic treatment with: DMSO (yellow plot), CDDP+DMSO (green plot), CDDP+PFT-α (pink plot).G Mean ABR threshold from 4 kHz to 32 kHz derived from F. Data are expressed as mean ± SEM (DMSO treated-group: n=7; CDDP+DMSO treated-group: n=12; CDDP+ PFT-α-treated group: n=12). One-way ANOVA test followed by post hoc Tukey's test (***P ≤ 0.0005, CDDP+DMSO, d5 vs. before or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).H Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from p53wt mice treated with: DMSO (yellow bars), CDDP+DMSO (green bars), CDDP+PFT-α (pink bars), after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP+DMSO, d5 vs. DMSO, d5 or CDDP+DMSO, d5 vs. CDDP+PFT-α, d5).I ABR thresholds from p53wt mice recorded prior to (gray plot and black plot for left and right ear, respectively) or after 5 days of systemic treatment with CDDP plus intratympanic injection of DMSO into the left ear (green plot), and of PFT-α into the right ear (pink plot).J Mean ABR threshold from 4 kHz to 32 kHz derived from I. Data are expressed as mean ± SEM (n=14). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003 , CDDP+left ear, DMSOvs. before, left ear or CDDP + left ear, DMSOvs. CDDP + right ear, PFT-α.K Cytocochleograms representing the percentage of surviving hair cells in four cochlear regions located at 1.1, 2.6, 3.5 or 4.1 mm from the cochlear apical end taken from the DMSO-treated left ear (green bars) and PFT-α treated right ear (pink bars) of the same CDDP-treated p53wt mice after 5 days (n=5 per group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP + right ear, PFT-α vs. CDDP+left ear, DMSO).


Figure 6A ABR thresholds recorded prior to ( gray plot ) or at day 28 in HBCx - 90 ( TP53 wt ) bearing - mice received systemic treatment with : DMSO ( black plot ) , PFT - α ( yellow plot ) , CDDP ( green plot ) , CDDP + PFT - α ( pink plot ) . B Mean ABR threshold from 4 kHz to 32 kHz derived from A . Data are expressed as mean ± SEM ( before : n = 20 ; DMSO : n = 5 ; PFT - α : n = 5 ; CDDP : n = 5 ; CDDP + PFT - α : n = 5 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P = 0 . 0002 , CDDP vs . before ; CDDP vs . CDDP + PFT - α . E ABR thresholds recorded prior to ( gray plot ) or at day 35 in HBCx - 14 ( TP53 mutant ) bearing - mice received systemic treatment with : DMSO ( black plot ) , PFT - α ( yellow plot ) , CDDP ( green plot ) , CDDP + PFT - α ( pink plot ) . F Mean ABR threshold from 4 kHz to 32 kHz derived from E . Data are expressed as mean ± SEM ( before : n = 30 ; DMSO : n = 5 ; PFT - α : n = 5 ; CDDP : n = 10 ; CDDP + PFT - α : n = 10 ) . One - way ANOVA test followed by post hoc Tukey ' s test ( * * * P = 0 . 0003 , CDDPvs . before or CDDPvs . CDDP + PFT - α . C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1 . 1 , 2 . 6 , and 3 . 5 mm from the cochlear apical end . These cochleae ( n = 5 per group ) were collected at 28 days from the beginning of treatment with DMSO ( black bars ) , PFT - α ( yellow bars ) , CDDP ( green bars ) , or CDDP + PFT - α ( pink bars ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 007 , CDDP , d28 vs . DMSO , d28 or CDDP , d28 vs . CDDP + PFT - α , d28 ) . G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1 . 1 , 2 . 6 , and 3 . 5 mm from the cochlear apical end . These cochleae ( n = 5 per group ) were collected at 35 days from the beginning of treatment with DMSO ( black bars ) , PFT - α ( yellow bars ) , CDDP ( green bars ) , or CDDP + PFT - α ( pink bars ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * P ≤ 0 . 008 , CDDP , d35 vs . DMSO , d35 or CDDP , d35 vs . CDDP + PFT - α , d35 ) . D Tumor growth curves alongside images of dissected tumors collected on day 28 . Note the partial inhibition of growth in CDDP and CDDP + PFT - α - treated mice . H Tumor growth curves alongside images of dissected tumors collected on day 35 . Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP + PFT - α - treated mice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx-90 (TP53 wt) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).B Mean ABR threshold from 4 kHz to 32 kHz derived from A. Data are expressed as mean ± SEM (before: n=20; DMSO: n=5; PFT-α: n=5; CDDP: n=5; CDDP+PFT-α: n=5). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0002, CDDP vs. before; CDDP vs. CDDP+PFT-α.E ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx-14 (TP53 mutant) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).F Mean ABR threshold from 4 kHz to 32 kHz derived from E. Data are expressed as mean ± SEM (before: n=30; DMSO: n=5; PFT-α: n=5; CDDP: n=10; CDDP+PFT-α: n=10). One-way ANOVA test followed by post hoc Tukey's test (***P = 0.0003, CDDPvs. before or CDDPvs. CDDP+PFT-α.C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.007, CDDP, d28 vs. DMSO, d28 or CDDP, d28 vs. CDDP+PFT-α, d28).G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP, d35 vs. DMSO, d35 or CDDP, d35 vs. CDDP+PFT-α, d35).D Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP+PFT- α-treated mice.H Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP+PFT- α-treated mice.


Figure 7A Representative confocal images of microvessels in transversal tumor sections from HBCx - 14 ( TP53mutant ) tumors treated with either DMSO , CDDP or a combination of CDDP and PFT - α . The sections were immunolabeled for CD31 ( red ) and viewed with a 20x objective . The basal - like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8 . Upper right panel is a 2D projection from the white boxed area in upper left panel . The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area . The white line shows the vessel perimeter . Scale bars : left panel = 50 µm , right panels = 35 µm . The tumor samples were collected at day 21 . B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula : vessel / vascular area = area of CD31 - positive objects + lumen area per field area × 100 % . The both HBCx - 14 ( TP53mutant ) and HBCx - 90 ( TP53wt ) tumors from the different treated groups were collected at day 21 ( n = 4 sections / tumor and 3 - 4 tumors / group ) . Data are expressed as mean ± SEM . Kruskal - Wallis test followed by post hoc Dunn ' s test ( * * * P ≤ 0 . 0004 , CDDPvs . DMSO or CDDPvs . CDDP + PFT - α ) . C Representative confocal images of cleaved caspase 3 positive cells ( red ) in the transversal tumor sections and viewed with a 20x objective . The stromal compartments were immunolabeled in green with an antibody against vimentin . HBCx - 14 ( TP53mutant ) tumors from the different treated groups were collected at day 18 . Scale bar = 50 µm . c - cas - 3 : cleaved caspase 3 . D Representative western blot analysis using antibodies against β - actin , p53 and p21 in tumor extracts from HBCx - 14 ( TP53mutant ) and HBCx - 90 ( TP53 wt ) tumors treated with either DMSO or CDDP and collected at day 18 . Note the higher CDDP - induced increase of p53 and p21 expression in TP53wt HBCx - 90tumors . E , F Representative western blot analysis using antibodies against β - actin , p - Chk1 , Beclin 1 , LC3 - I / II and Rab7 in tumor extracts from HBCx - 14 ( TP53mutant ) and HBCx - 90 ( TP53 wt ) tumors treated with either DMSO , PFT - α , CDDP or a combination of CDDP and PFT - α . The tumor samples were collected at day 18 . G , H , I Histograms representing the levels of Beclin 1 , LC3 - II and Rab7 in HBCx - 14 and HBCx - 90tumors treated with the different regimens ( n = 3 - 4 tumors per group , all experiments were performed in triplicate ) . Actin served as a loading control . Data are expressed as mean ± SEM . One - way ANOVA test followed by post hoc Tukey ' s test ( G : * P = 0 . 03 , * * P = 0 . 006 , * * * P ≤ 0 . 0004 ; H : * P = 0 . 02 , * * * P = 0 . 0005 ; I : * P = 0 . 03 , * * * P ≤ 0 . 0006 , CDDPvs . DMSO or CDDPvs . CDDP + PFT - α ) .

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Figure 7A Representative confocal images of microvessels in transversal tumor sections from HBCx-14 (TP53mutant) tumors treated with either DMSO, CDDP or a combination of CDDP and PFT-α. The sections were immunolabeled for CD31 (red) and viewed with a 20x objective. The basal-like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars: left panel=50 µm, right panels=35 µm. The tumor samples were collected at day 21.B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31-positive objects + lumen area per field area × 100 %. The both HBCx-14 (TP53mutant) and HBCx-90 (TP53wt) tumors from the different treated groups were collected at day 21 (n=4 sections/tumor and 3-4 tumors/group). Data are expressed as mean ± SEM. Kruskal-Wallis test followed by post hoc Dunn's test (***P ≤0.0004, CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).C Representative confocal images of cleaved caspase 3 positive cells (red) in the transversal tumor sections and viewed with a 20x objective. The stromal compartments were immunolabeled in green with an antibody against vimentin. HBCx-14 (TP53mutant) tumors from the different treated groups were collected at day 18. Scale bar=50 µm. c-cas-3: cleaved caspase 3.D Representative western blot analysis using antibodies against β-actin, p53 and p21 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP-induced increase of p53 and p21 expression in TP53wt HBCx-90tumors.E, F Representative western blot analysis using antibodies against β-actin, p-Chk1, Beclin 1, LC3-I/II and Rab7 in tumor extracts from HBCx-14 (TP53mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO, PFT-α, CDDP or a combination of CDDP and PFT-α. The tumor samples were collected at day 18.G, H, I Histograms representing the levels of Beclin 1, LC3-II and Rab7 in HBCx-14 and HBCx-90tumors treated with the different regimens (n=3-4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey's test (G: *P = 0.03, **P =0.006 , ***P ≤ 0.0004 ; H: *P =0.02, ***P = 0.0005 ; I: *P =0.03, ***P ≤ 0.0006 , CDDPvs. DMSO or CDDPvs. CDDP+PFT-α).