Figure 3C . AURKA interacts with the centriolar satellite proteins PCM1 , CEP131 , CEP72 , CSPP1 , KIAA0586 / Talpid3 and CP110 . HEK293T cells were transiently transfected with FLAG - BirA * or FLAG - BirA * - AURKA . Following 18 h biotin incubation , cells were lysed , and biotinylated proteins were precipitated by streptavidin beads . The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG , CEP72 , CEP131 , PCM1 , CEP63 , CSPP1 , KIAA0586 / Talpid3 , CP110 , BBS4 and SSX2IP . E . Endogenous interaction of PCM1 and AURKA . Lysates from synchronous and serum - starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control . The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA . F . AURKA co - localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain . U2OS cells were co - transfected with GFP - PCM1 - FKBP and HA - Kif5b ( 1 - 269 a . a . ) - FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours . Cells that were not treated with rapamycin were processed in parallel as a control . Fixed cells were stained with antibodies against GFP , AURKA and gamma - tubulin . DNA was stained with DAPI . Cell edges are outlined . Scale bar , 10 μm . G . Quantification of pericentrosomal levels of AURKA and gamma tubulin for F . n > 25 cells per experiment . Data represent mean value from two experiments per condition ± SEM ( * * * * p < 0 . 0001 ; * * p < 0 . 01 ; Unpaired Student ' s t - 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3C. AURKA interacts with the centriolar satellite proteins PCM1, CEP131, CEP72, CSPP1, KIAA0586/Talpid3 and CP110. HEK293T cells were transiently transfected with FLAG-BirA* or FLAG-BirA*-AURKA. Following 18 h biotin incubation, cells were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample and immunoprecipitated biotinylated proteins were run on a gel and immunoblotted with fluorescent streptavidin and antibodies against FLAG, CEP72, CEP131, PCM1, CEP63, CSPP1, KIAA0586/Talpid3, CP110, BBS4 and SSX2IP.E. Endogenous interaction of PCM1 and AURKA. Lysates from synchronous and serum-starved HEK293T were immunoprecipitated with AURKA antibody and rabbit IgG control. The initial sample and immunoprecipitated proteins were run on a gel and immunoblotted with antibodies against PCM1 and AURKA.F. AURKA co-localize with PCM1 at the peripheral satellite clusters upon chemical dimerization of satellites with Kif5b motor domain. U2OS cells were co-transfected with GFP-PCM1-FKBP and HA-Kif5b(1-269 a.a.)-FRB and treated with 500 nM rapamycin for 1 hour followed by fixation at 6 hours. Cells that were not treated with rapamycin were processed in parallel as a control. Fixed cells were stained with antibodies against GFP, AURKA and gamma-tubulin. DNA was stained with DAPI. Cell edges are outlined. Scale bar, 10 μm. G. Quantification of pericentrosomal levels of AURKA and gamma tubulin for F. n>25 cells per experiment. Data represent mean value from two experiments per condition ± SEM (****p < 0.0001; **p < 0.01; Unpaired Student's t-test.).


Figure 4A , B . Representative images and quantification of basal body levels of ( A ) AURKA and ( B ) phospho - AURKA ( p - AURKA ) in control and PCM1 - depleted cells . RPE1 cells were transfected with control or PCM1 siRNA # 1 or siRNA # 2 for 48 h . Following 24 h serum starvation , cells were fixed and stained with the indicated antibodies . Centrosomal AURKA and p - AURKA fluorescence intensities were measured from maximum projections , and average means of the levels in control cells were normalized to 1 . n > 500 cells per experiment . Data for ( A ) AURKA and ( B ) p - AURKA represent mean value from three experiments per condition ± SEM ( * * * * p < 0 . 0001 , Unpaired Student ' s t - test . ) Scale bar , 10 μmD . Cycloheximide chase experiment for quantification of AURKA half - life . Cells were transfected with control or PCM1 siRNA # 1 for 48 h , then treated with 200 nM cycloheximide along with serum starvation for indicated time points . AURKA intensities were quantified by immunoblotting for AURKA and vinculin ( loading control ) and AURKA levels were normalized to vinculin levels . Data represent mean value from three experiments per condition ± SEM ( * * p < 0 . 01 , Unpaired Student ' s t - 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 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

Figure 4A, B. Representative images and quantification of basal body levels of (A) AURKA and (B) phospho-AURKA (p-AURKA) in control and PCM1-depleted cells. RPE1 cells were transfected with control or PCM1 siRNA #1 or siRNA #2 for 48 h. Following 24 h serum starvation, cells were fixed and stained with the indicated antibodies. Centrosomal AURKA and p-AURKA fluorescence intensities were measured from maximum projections, and average means of the levels in control cells were normalized to 1. n>500 cells per experiment. Data for (A) AURKA and (B) p-AURKA represent mean value from three experiments per condition ± SEM (****p < 0.0001, Unpaired Student's t-test.) Scale bar, 10 μmD. Cycloheximide chase experiment for quantification of AURKA half-life. Cells were transfected with control or PCM1 siRNA#1 for 48 h, then treated with 200 nM cycloheximide along with serum starvation for indicated time points. AURKA intensities were quantified by immunoblotting for AURKA and vinculin (loading control) and AURKA levels were normalized to vinculin levels. Data represent mean value from three experiments per condition ± SEM (**p < 0.01, Unpaired Student's t-test)


Figure 5A . Representative immunofluorescence images of cilium assembly experiments in control and PCM1 - depleted RPE1 cells treated with DMSO or MLN8237 . Cells were transfected with control or PCM1 siRNA # 1 for 48 h and treated with DMSO ( vehicle control ) or 0 . 5 μM MLN8237 in serum starvation medium for 24 h . Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody ( Acet - tub ) and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 10 μm . B . Quantification of ciliogenesis efficiency for A . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * p < 0 . 01 ; ns , non - significant , Unpaired Student ' s t - test . ) . C . Quantification of cilium length for A . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * * p < 0 . 0001 ; ns , non - significant , Unpaired Student ' s t - test . ) . D . Representative immunofluorescence images of cilium assembly experiments in control and PCM1 - depleted RPE1 cells treated with DMSO or tubacin . Cells were transfected with control or PCM1 siRNA # 1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h . Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody ( Acet - tub ) and the centrosome with gamma - tubulin antibody . DNA was stained with DAPI . Scale bar , 10 μm E . Quantification of ciliogenesis efficiency for D . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * p < 0 . 05 ; ns , non - significant , Unpaired Student ' s t - test ) . F . Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium . RPE1 cells were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h and treated with DMSO or 0 . 5 μM MLN8237 . Cells were fixed and immunostained with antibodies against IFT88 , acetylated tubulin and gamma tubulin . DNA was stained with DAPI . Scale bar , 10 μm . G . Quantification of IFT88 centrosomal intensity and ciliary concentration for F . IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections , and average means of the levels in control cells were normalized to 1 . Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length . n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - test ) H , I . Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation . RPE1 cells were transfected with control or PCM1 siRNA # 1 for 48 h , serum starved for 24 h and treated with DMSO or 0 . 5 μM MLN8237 in serum stimulation medium for 2 h and 24 h . Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin , and percentage of ciliated cells were quantified . x - axis indicates the hours after serum stimulation . ( H ) n > 100 cells per experiment . Data represent mean value from three experiments per condition ± SEM . ( I ) n > 95 cells per experiment . Data represent mean value from three experiments per condition ± SEM ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , Unpaired Student ' s t - 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 O O O O O O O O O O O O O O O O O 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 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 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

Figure 5A. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or MLN8237. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm. B. Quantification of ciliogenesis efficiency for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; **p < 0.01; ns, non-significant, Unpaired Student's t-test.). C. Quantification of cilium length for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (****p < 0.0001; ns, non-significant, Unpaired Student's t-test.). D. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or tubacin. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm E. Quantification of ciliogenesis efficiency for D. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (*p < 0.05; ns, non-significant, Unpaired Student's t-test). F. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against IFT88, acetylated tubulin and gamma tubulin. DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of IFT88 centrosomal intensity and ciliary concentration for F. IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test) H, I. Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237 in serum stimulation medium for 2 h and 24 h. Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin, and percentage of ciliated cells were quantified. x-axis indicates the hours after serum stimulation. (H) n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM. (I) n>95 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test).


Figure 1 ( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d . Lung sections stained with Haematoxylin and eosin ( H & E ) ( A ) and quantification of lung lesion burden from H & E - stained sections ( B ) . Data information : Data are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results and each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc test were used for statistical analysis , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm . ( D - G ) H & E staining of lung sections from mice 4 weeks after intranasal infection with M . bovis BCG ( D and E ) or Mtb H37Rv ( F and G ) in the absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Quantification of lung inflamed regions shown in ( E and G ) . Data information : Data in are means + SD of indicated numbers of mice from 1 of n = 3 independent experiments with similar results and each symbol represents 1 mouse . Data are representative of n = 3 independent experiments . One - way ANOVA followed by Dunnett ' s post hoc test and Student ' s t test were used for statistical analysis , respectively . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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Figure 1(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d. Lung sections stained with Haematoxylin and eosin(H&E) (A) and quantification of lung lesion burden from H&E-stained sections (B). Data information: Data are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.(D-G) H&E staining of lung sections from mice 4 weeks after intranasal infection with M. bovis BCG (D and E) or Mtb H37Rv (F and G) in the absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Quantification of lung inflamed regions shown in (E and G). Data information: Data in are means + SD of indicated numbers of mice from 1 of n=3 independent experiments with similar results and each symbol represents 1 mouse. Data are representative of n=3 independent experiments. One-way ANOVA followed by Dunnett's post hoc test and Student's t test were used for statistical analysis, respectively. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Scale bar, 200 μm.


Figure 2 ( B ) Quantitative polymerase chain reaction ( qPCR ) analysis of Mmps including Mmp2 , Mmp9 , Mmp10 , Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG ( 1 μg / ml ) for indicated times . ( C ) Immunoblots of cell supernatants to analyze secreted MMP2 , MMP9 , MMP10 , MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG ( 1 μg / mL ) for indicated times ; GADPH of cell lysates served as a loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . ( H ) qPCR analysis of Mmps including Mmp9 , Mmp10 , Mmp12 , and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h ( MOI = 5 ) in absence or presence of AG aptamers ( 1 μg / ml ) . ( I ) Immunoblots of cell supernatants to analyze secreted MMP9 , MMP10 , MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in the absence or presence of AG aptamers ( 0 . 5 μg / ml ) ; GADPH of cell lysates served as the loading control . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Tukey ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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 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

Figure 2(B) Quantitative polymerase chain reaction (qPCR) analysis of Mmps including Mmp2, Mmp9, Mmp10, Mmp12 and Mmp13 mRNA from mouse peritoneal macrophages stimulated with AG (1 μg/ml) for indicated times. (C) Immunoblots of cell supernatants to analyze secreted MMP2, MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages stimulated with AG (1 μg/mL) for indicated times; GADPH of cell lysates served as a loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.(H) qPCR analysis of Mmps including Mmp9, Mmp10, Mmp12, and Mmp13 from mouse peritoneal macrophages infected with H37Rv for 24h (MOI=5) in absence or presence of AG aptamers (1 μg/ml). (I) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12 and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in the absence or presence of AG aptamers (0.5 μg/ml); GADPH of cell lysates served as the loading control. Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


Figure 3 ( G ) Immunoblots of cell supernatants to analyze secreted MMP9 , MMP10 , MMP12 , and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times ( MOI = 5 ) in absence or presence of inhibitor targeting ERK ( PD98059 ) at the concentration of 10 μM ; GADPH of cell lysates served as the loading control .

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Figure 3(G) Immunoblots of cell supernatants to analyze secreted MMP9, MMP10, MMP12, and MMP13 by mouse peritoneal macrophages infected with H37Rv for indicated times (MOI=5) in absence or presence of inhibitor targeting ERK (PD98059) at the concentration of 10 μM; GADPH of cell lysates served as the loading control.


Figure 4 ( A , B ) C57BL / 6 mice were left untreated ( NT ) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E ( A ) and quantification of lung lesion burden from H & E stained sections ( B ) . Data information : Data in ( A ) are representative of n = 3 independent experiments . Data in ( B ) are means + SD of indicated numbers of mice from one of n = 3 independent experiments and each symbol represents data from 1 mouse . One - way ANOVA followed by Dunnett ' s post hoc test ( B ) was used for statistical analysis . ns , not significant ; * * * * , p < 0 . 0001 . Scale bar , 200 μm .

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 4(A, B) C57BL/6 mice were left untreated (NT) or were intraperitoneally treated with indicated amounts of AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E (A) and quantification of lung lesion burden from H&E stained sections (B). Data information: Data in (A) are representative of n=3 independent experiments. Data in (B) are means + SD of indicated numbers of mice from one of n=3 independent experiments and each symbol represents data from 1 mouse. One-way ANOVA followed by Dunnett's post hoc test (B) was used for statistical analysis. ns, not significant; ****, p<0.0001. Scale bar, 200 μm.


Figure 5 ( A ) Surface plasmon resonance ( SPR ) assay of the direct interaction of AG with galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines . ( C ) SPR assay of the interaction of AG with CRD2 of galectin - 9 . Curve fittings to a 1 : 1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines .

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Figure 5(A) Surface plasmon resonance (SPR) assay of the direct interaction of AG with galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.(C) SPR assay of the interaction of AG with CRD2 of galectin-9. Curve fittings to a 1:1 Langmuir binding model calculated with TraceDrawer are shown as smooth black lines.


Figure 6 ( I ) Immunoblot of lysates of shCtrl and shGalectin - 9 THP - 1 cells stimulated with AG ( 1 μg / ml ) for indicated times . Data are representative of n = 3 independent experiments . ( K ) qRT - PCR detection of MMP transcripts including Mmp9 , Mmp10 , and Mmp12 in shCtrl and shGalectin - 9 THP - 1 cells stimulated with AG ( 1 μg / ml ) for 24 h in absence or presence of ERK inhibitor PD98059 ( 10 μM ) . Data information : Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Dunnett ' s post hoc test were used for statistical analysis . ns , not significant ; * , p < 0 . 05 ; * * * , p < 0 . 001 * * * * , p < 0 . 0001 .

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 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

Figure 6(I) Immunoblot of lysates of shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for indicated times. Data are representative of n=3 independent experiments.(K) qRT-PCR detection of MMP transcripts including Mmp9, Mmp10, and Mmp12 in shCtrl and shGalectin-9 THP-1 cells stimulated with AG (1 μg/ml) for 24 h in absence or presence of ERK inhibitor PD98059 (10 μM). Data information: Data are means + SD averaged from 3 independent experiments performed with technical triplicates and each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Dunnett's post hoc test were used for statistical analysis. ns, not significant; *, p<0.05; ***, p<0.001 ****, p<0.0001.


Figure 7 ( D ) Confocal microscopy of mouse peritoneal macrophages left untreated ( NC ) ( upper row ) or stimulated with AG ( 1 μg / mL ) for 2 h ( middle row ) or infected with H37Rv for 3 h ( MOI = 5 ) ( bottom row ) , staining with anti - Galectin - 9 and anti - TAK1 antibody . DAPI , nuclei , blue . Scale bar , 5 μm . Data in the right graph show mean + SD of n = 12 fields from three independent experiments . The symbols indicate the colocalization ratio of at least 10 cells in each field . ( F ) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin - 9 KO mice stimulated with AG ( 1 μg / ml ) in absence or presence of TAK1 inhibitor 5Z - 7 - OZ ( 1 μM ) for indicated times . Data are representative of n = 3 independent experiments .

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 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

Figure 7(D) Confocal microscopy of mouse peritoneal macrophages left untreated (NC) (upper row) or stimulated with AG (1 μg/mL) for 2 h (middle row) or infected with H37Rv for 3 h (MOI=5) (bottom row), staining with anti-Galectin-9 and anti-TAK1 antibody. DAPI, nuclei, blue. Scale bar, 5 μm. Data in the right graph show mean + SD of n=12 fields from three independent experiments. The symbols indicate the colocalization ratio of at least 10 cells in each field.(F) Immunoblots of cell lysates of peritoneal macrophages isolated from WT or Galectin-9 KO mice stimulated with AG (1 μg/ml) in absence or presence of TAK1 inhibitor 5Z-7-OZ (1 μM) for indicated times. Data are representative of n=3 independent experiments.


Figure 8 ( B , WT or Galectin - 9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat ( 10 mg / kg ) given intraperitoneally prior to AG stimulation . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments . ( D , WT or Galectin - 9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers ( 1 μg ) once at a 1 week - interval . Lung sections stained with H & E Data information : Data are representative of n = 3 independent experiments .

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 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

Figure 8(B, WT or Galectin-9 KO mice were intraperitoneally treated with AG for 3 d in the absence or presence of the MMP inhibitor marimastat (10 mg/kg) given intraperitoneally prior to AG stimulation. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.(D, WT or Galectin-9 KO mice were intranasally infected with H37Rv for 4 weeks in absence or presence of intranasally administrated AG aptamers (1 μg) once at a 1 week-interval. Lung sections stained with H&E Data information: Data are representative of n=3 independent experiments.


Figure 1 . Recovery of infectious recombinant MHV - GFP . Cell culture supernatants containing viruses produced after virus rescue from two MHV - GFP YAC clones ( Clone1 , Clone 2 ) were used to infect 17Cl - 1 cells . At 48 hours post - infection , infected cells were visualised for GFP expression ( left panels ) and by bright field microscopy ( right panels ) . Mock represents 17Cl - 1 cells inoculated with supernatant from BHK - MHV - N cells electroporated without viral RNAs . Replication kinetics of parental MHV - GFP and recombinant MHV - GFP Clone 1 and Clone 2 . L929 cells were infected at an MOI of 0 . 1 . Cell culture supernatants were harvested at indicated time points post - infection and titrated by plaque assay . Data are representative of three independent experiments with two replicates per virus in each experiment . Error bars show standard deviation ( SD ) from the mean . pfu / ml , plaque forming units per millilitre .

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 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

Figure 1.Recovery of infectious recombinant MHV-GFP. Cell culture supernatants containing viruses produced after virus rescue from two MHV-GFP YAC clones (Clone1, Clone 2) were used to infect 17Cl-1 cells. At 48 hours post-infection, infected cells were visualised for GFP expression (left panels) and by bright field microscopy (right panels). Mock represents 17Cl-1 cells inoculated with supernatant from BHK-MHV-N cells electroporated without viral RNAs.Replication kinetics of parental MHV-GFP and recombinant MHV-GFP Clone 1 and Clone 2. L929 cells were infected at an MOI of 0.1. Cell culture supernatants were harvested at indicated time points post-infection and titrated by plaque assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. pfu/ml, plaque forming units per millilitre.


Figure 2 . Rescue of recombinant MERS - CoV and MERS - CoV - GFP . Following delivery of viral RNAs into BHK - 21 cells via electroporation the cells were co - cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells . Infected cells were visualised by bright field microscopy for recombinant MERS - CoV ( upper panel ; 5 days post infection ) , and for GFP expression for recombinant MERS - CoV - GFP ( lower panel , 3 days post infection ) . Mock represents VeroB4 cells inoculated with supernatant from BHK - 21 cells electroporated without viral RNAs . Replication kinetics of the MERS - CoV - EMC ( laboratory adapted isolate ) , rMERS - CoV and rMERS - CoV - GFP . VeroB4 cells were infected at an MOI of 0 . 01 . Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay . Data are representative of three independent experiments with two replicates per virus in each experiment . Error bars show standard deviation ( SD ) from the mean . TCID50 / ml , 50 % tissue culture infectious dose per millilitre .

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

Figure 2.Rescue of recombinant MERS-CoV and MERS-CoV-GFP. Following delivery of viral RNAs into BHK-21 cells via electroporation the cells were co-cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells. Infected cells were visualised by bright field microscopy for recombinant MERS-CoV (upper panel; 5 days post infection), and for GFP expression for recombinant MERS-CoV-GFP (lower panel, 3 days post infection). Mock represents VeroB4 cells inoculated with supernatant from BHK-21 cells electroporated without viral RNAs.Replication kinetics of the MERS-CoV-EMC (laboratory adapted isolate), rMERS-CoV and rMERS-CoV-GFP. VeroB4 cells were infected at an MOI of 0.01. Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. TCID50/ml, 50% tissue culture infectious dose per millilitre.


Figure 3 . Rescue of recombinant rSARS - CoV - 2 and rSARS - CoV - 2 - GFP . The experimental setting is illustrated in the upper panel . Two clones of each construct 1 - 6 were used to prepare YAC DNAs and in - vitro transcribed viral genome RNAs were electroporated into BHK - 21 cells and / or BHK - SARS - N together with an mRNA encoding the SARS - CoV - 2 N protein . Electroporated cells were co - cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12 - well plates to perform plaque assay for rSARS - CoV - 2 constructs 1 - 3 and fluorescence microscopy for rSARS - CoV - 2 - GFP constructs 4 - 6 . Plaque assays are shown in the lower panel for SARS - CoV - 2 clones 1 . 1 , 2 . 2 , and 3 . 1 . VeroE6 cells were infected with 10 − 1 , 10 − 2 , and 10 − 3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non - infected cells ( Mock ) . The expression of GFP in SARS - CoV - 2 - GFP infected VeroE6 cells is shown for clones 4 . 1 , 5 . 2 , and 6 . 1 . Mock represents non - infected VeroE6 cells .

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

Figure 3.Rescue of recombinant rSARS-CoV-2 and rSARS-CoV-2-GFP. The experimental setting is illustrated in the upper panel. Two clones of each construct 1-6 were used to prepare YAC DNAs and in-vitro transcribed viral genome RNAs were electroporated into BHK-21 cells and/or BHK-SARS-N together with an mRNA encoding the SARS-CoV-2 N protein. Electroporated cells were co-cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12-well plates to perform plaque assay for rSARS-CoV-2 constructs 1-3 and fluorescence microscopy for rSARS-CoV-2-GFP constructs 4-6. Plaque assays are shown in the lower panel for SARS-CoV-2 clones 1.1, 2.2, and 3.1. VeroE6 cells were infected with 10−1, 10−2, and 10−3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non-infected cells (Mock). The expression of GFP in SARS-CoV-2-GFP infected VeroE6 cells is shown for clones 4.1, 5.2, and 6.1. Mock represents non-infected VeroE6 cells.


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 ) .

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 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 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 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

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).


Figure 1A . Representative FACS plots of freshly isolated epidermal cells ( Epi d0 ) and day 14 ( d14 ) cultures of epidermal cells in different growth conditions and media . KGM 2D : 2D in basal KGM ; 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . B . CD34 + α6 + cells were quantified by flow cytometry from d14 as in ( A ) ( mean ± SD ; n = 3 - 5 ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 , Kruskal - Wallis / Dunn ' s post test ) . 3C 2D : 2D in KGM + Y27632 + VEGF - A + FGF - 2 ; Y : 3D - Matrigel in KGM + Y27632 ; YV : Y + VEGF - A ; YF : Y + FGF - 2 ; 3C : Y + VEGF - A + FGF - 2 . C . Absolute numbers of CD34 + α6 + cells from Epi d0 and d14 cultures as in ( A - B ) . Fold enrichment over Epi d0 is shown ( mean ± SEM , n = 3 - 5 ; * p ≤ 0 . 05 , Mann - Whitney U test ) . D . Epidermal cells grown for 14 days in the indicated conditions . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . 3C 2D : 2D in 3C medium . Scale bars 30µm . E . Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells . See also Appendix Appendix figure S1 .

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 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 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

Figure 1A. Representative FACS plots of freshly isolated epidermal cells (Epi d0) and day 14 (d14) cultures of epidermal cells in different growth conditions and media. KGM 2D: 2D in basal KGM; 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. B. CD34+α6+ cells were quantified by flow cytometry from d14 as in (A) (mean ±SD; n=3-5; *p ≤ 0.05; ***p ≤ 0.001, Kruskal-Wallis/Dunn's post test). 3C 2D: 2D in KGM + Y27632 + VEGF-A + FGF-2; Y: 3D-Matrigel in KGM + Y27632; YV: Y + VEGF-A; YF: Y + FGF-2; 3C: Y + VEGF-A + FGF-2. C. Absolute numbers of CD34+α6+ cells from Epi d0 and d14 cultures as in (A-B). Fold enrichment over Epi d0 is shown (mean ±SEM, n=3-5; *p ≤ 0.05, Mann-Whitney U test).D. Epidermal cells grown for 14 days in the indicated conditions. 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2. 3C 2D: 2D in 3C medium. Scale bars 30µm.E. Immunofluorescence analysis of 3C cultures showing the expression of the HFSC markers CD34 and SOX9 in a subset of cells. See also Appendix Appendix figure S1.


Figure 2A . Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells ( Epi d0 ; control ) ( mean ± SEM ; n = 5 ; * p ≤ 0 . 05 , Mann - Whitney U - test ) . 3C : 3D - Matrigel in KGM + Y27632 + VEGF - A + FGF - 2 . B . Full thickness skin reconstitution assay with freshly isolated epidermal cells ( Epi d0 ) or 3C cultures shows that cells cultured in 3C retain their self - renewal capacity and multipotency . A representative recipient of 4 mice / condition is shown . Right panel shows quantification of hair follicle density . Note that 3C cultures give rise to more hair compared to controls ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U test ) . C . Long - term culture of cells in 3C was performed as shown in the left panel . 3C cultures maintain a stable population of CD34 + α6 + long term ( data are shown using a Box - and - Whisker plot : box indicates 25th and 75th percentiles ; error bars represent 10th and 90th percentiles ; middle line is the median ; n = 3 - 5 independent experiments ) . p : passage . D . Full thickness skin reconstitution assay using 3C cells from passage 1 ( p1 ) and passage 15 ( p15 ) show that cells cultured in 3C maintain their multipotency and self - renewal capacity in long - term cultures . A representative recipient of 4 mice / condition is shown . See also Appendix figure S2 .

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

Figure 2A. Colony forming assays show increased proliferative potential of cells cultured in 3C compared to freshly isolated epidermal cells (Epi d0; control) (mean ±SEM; n=5; *p ≤ 0.05, Mann-Whitney U-test). 3C: 3D-Matrigel in KGM + Y27632 + VEGF-A + FGF-2.B. Full thickness skin reconstitution assay with freshly isolated epidermal cells (Epi d0) or 3C cultures shows that cells cultured in 3C retain their self-renewal capacity and multipotency. A representative recipient of 4 mice/condition is shown. Right panel shows quantification of hair follicle density. Note that 3C cultures give rise to more hair compared to controls (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).C. Long-term culture of cells in 3C was performed as shown in the left panel. 3C cultures maintain a stable population of CD34+α6+ long term (data are shown using a Box-and-Whisker plot: box indicates 25th and 75th percentiles; error bars represent 10th and 90th percentiles; middle line is the median; n=3-5 independent experiments). p: passage.D. Full thickness skin reconstitution assay using 3C cells from passage 1 (p1) and passage 15 (p15) show that cells cultured in 3C maintain their multipotency and self-renewal capacity in long-term cultures. A representative recipient of 4 mice/condition is shown. See also Appendix figure S2.


Figure 3B . Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A . 3C cultures cluster with purified CD34 + α6 + HFSCs ( n = 3 biological replicates ) . E . Heatmap of selected genes differentially expressed in 3C - CD34 + α6 + and 3C - CD34 ¯ α6 + cells . Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers ( triangles ) in 3C - CD34 + α6 + cells , and upregulation of genes implicated in lineage progression in 3C - CD34 ¯ α6 + cells ( circles ) . G . RT - qPCR analyses of FACS - purified CD34 + α6 + and CD34 - α6 + cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C - CD34 + α6 + cells compared to freshly isolated CD34 - α6 + progenitors . 3C - CD34 - α6 + cells show intermediated expression ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . H . Both 3C - CD34 + α6 + and 3C - CD34 - α6 + cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S3 and Tables S1 - S3 .

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 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 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 3B. Heatmap and Euclidian distance dendrogram of quantified transcripts from RNAseq data generated as shown in panel A. 3C cultures cluster with purified CD34+α6+ HFSCs (n=3 biological replicates).E. Heatmap of selected genes differentially expressed in 3C-CD34+α6+ and 3C-CD34¯α6+ cells. Note upregulation of multiple key HFSC lineage identity transcription factors and bulge markers (triangles) in 3C-CD34+α6+ cells, and upregulation of genes implicated in lineage progression in 3C-CD34¯α6+ cells (circles).G. RT-qPCR analyses of FACS-purified CD34+α6+ and CD34-α6+ cells from either 3C cultures or from freshly isolated epidermis show upregulation of HFSC identity in both freshly isolated and 3C-CD34+α6+ cells compared to freshly isolated CD34-α6+ progenitors. 3C- CD34-α6+ cells show intermediated expression (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).H. Both 3C-CD34+α6+ and 3C-CD34-α6+ cells show upregulation of a panel of cell cycle genes and genes shown to be enriched in HFSC progeny (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S3 and Tables S1-S3.


Figure 4A . FACS analysis of CD34 + α6 + cells over time shows that 3C cultures reach a ~ 50 : 50 population equilibrium after 10 days of culture ( mean ± SEM ; n = 3 ) . B . Cells were EdU - labeled for 24 h ( day 10 - 11 ) prior to analysis by flow cytometry to quantify proliferating cells . Percentage of EdU + cells is indicated ( n = 4 ; mean ± SD ; * p ≤ 0 . 05 , Mann - Whitney U - test ) . D . Simulated population proportions of CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs derived from the mathematical model describing steady state cultures . Experimental data from ( A ) are shown as reference ( in grey ) . See also Appendix figure S4 and Dataset EV4 .

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 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 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 4A. FACS analysis of CD34+α6+ cells over time shows that 3C cultures reach a ~50:50 population equilibrium after 10 days of culture (mean ±SEM; n=3).B. Cells were EdU-labeled for 24 h (day 10-11) prior to analysis by flow cytometry to quantify proliferating cells. Percentage of EdU+ cells is indicated (n=4; mean ±SD; *p ≤ 0.05, Mann-Whitney U-test).D. Simulated population proportions of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs derived from the mathematical model describing steady state cultures. Experimental data from (A) are shown as reference (in grey). See also Appendix figure S4 and Dataset EV4.


Figure 5A . CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs were FACS - purified from total epidermis ( d0 ) to ≥ 98 % purity post sorting ( d0 post - sort ) , and grown 14 days in 3C cultures ( d14 3C ) . A significant population of CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs is generated from both purified populations after 14 days . A representative of 3 independent experiments is shown ( mean ± SD ) . B - C . Time course analyses of 3C cultures established from FACS - purified populations of CD34 + α6 + HFSCs ( B ) or CD34 ¯ α6 + non - HFSCs ( C ) from 3C cultures . Both cultures reach population equilibrium between day 6 and 10 ( n = 4 ; mean ± SD ) . D - E . Simulated population proportions of CD34 + α6 + HFSCs ( D ) and CD34 ¯ α6 + non - HFSCs ( E ) from the mathematical model describing behavior of pure cell populations . The model parameters were estimated from the experimental data in B - C ( shown in grey as reference ) . G . Lineage tracing of FACS - purified CD34 + α6 + HFSCs and CD34 ¯ α6 + non - HFSCs from EGFP ¯ and EGFP + 3C cultures , respectively . CD34 + α6 + and CD34 ¯ α6 + were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry . Presence of CD34 + α6 + EGFP + cells in cultures at d10 indicates non - HFSC to HFSC interconversion . H . Lineage tracing as in ( F ) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non - HFSCs . I . Quantification of the proportion of CD34 + α6 + cells that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled cells are indicated ) . J . Quantification of the proportion of CD34 ¯ α6 + cells that maintain their fate over time ( experiments with both EGFP + or EGFP ¯ labeled cells are indicated ) . Data in panels G - J show n = 4 ; mean ± SD . See also Appendix figure S5 .

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 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 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 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 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 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

Figure 5A. CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs were FACS-purified from total epidermis (d0) to ≥98% purity post sorting (d0 post-sort), and grown 14 days in 3C cultures (d14 3C). A significant population of CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs is generated from both purified populations after 14 days. A representative of 3 independent experiments is shown (mean ±SD).B-C. Time course analyses of 3C cultures established from FACS-purified populations of CD34+α6+ HFSCs (B) or CD34¯α6+ non-HFSCs (C) from 3C cultures. Both cultures reach population equilibrium between day 6 and 10 (n=4; mean ±SD).D-E. Simulated population proportions of CD34+α6+ HFSCs (D) and CD34¯α6+ non-HFSCs (E) from the mathematical model describing behavior of pure cell populations. The model parameters were estimated from the experimental data in B-C (shown in grey as reference).G. Lineage tracing of FACS-purified CD34+α6+ HFSCs and CD34¯α6+ non-HFSCs from EGFP¯ and EGFP+ 3C cultures, respectively. CD34+α6+ and CD34¯α6+ were mixed at the indicated ratios at d0 and EGFP expression was used to trace the cells of origin over time by flow cytometry. Presence of CD34+α6+ EGFP+ cells in cultures at d10 indicates non-HFSC to HFSC interconversion.H. Lineage tracing as in (F) shows that 3C cultures reach equilibrium between day 6 and 10 independent of the initial proportions of HFSCs and non-HFSCs.I. Quantification of the proportion of CD34+α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated).J. Quantification of the proportion of CD34¯α6+ cells that maintain their fate over time (experiments with both EGFP+ or EGFP¯ labeled cells are indicated). Data in panels G-J show n=4; mean ±SD. See also Appendix figure S5.


Figure 6A - B . Inhibitor treatment scheme ( A ) and FACS plots ( B ) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin ( mean ± SD ; n = 3 ) . C . Quantification of experiments as in ( B ) shows enrichment of HFSCs upon cyclopamine ( Cyclo ) treatment , whereas dorsomorphin ( Dorso ) treatment leads to depletion of HFSCs ( mean ± SD ; n = 3 ; * p ≤ 0 . 05 , Kruskal - Wallis ) . D . Heatmap of significantly ( padj value < 0 . 05 ) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h . HFSC identity genes ( triangles ) and genes implicated in HFSC lineage progression ( circles ) are indicated . E . RT - qPCR analysis of 3C cultures treated as in ( D ) confirms that Id 1 , 2 and 3 are significantly upregulated in Cyclo treated cells ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , Mann - Whitney U test ) . F . RT - qPCR analysis of 3C cultures treated as in ( A ) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment ( mean ± SEM ; n = 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . H . Quantification of experiments as in E ( mean ± SD ; n = 3 - 4 ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , Kruskal - Wallis ) . See also Appendix figure S6 and Dataset EV5 .

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 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 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 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 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

Figure 6A-B. Inhibitor treatment scheme (A) and FACS plots (B) of 3C cultures treated with the Shh inhibitor cyclopamine or the BMP inhibitor dorsomorphin (mean ±SD; n=3). C. Quantification of experiments as in (B) shows enrichment of HFSCs upon cyclopamine (Cyclo) treatment, whereas dorsomorphin (Dorso) treatment leads to depletion of HFSCs (mean ±SD; n=3; *p ≤ 0.05, Kruskal-Wallis).D. Heatmap of significantly (padj value<0.05) differentially expressed genes from RNAseq analysis of 3C cultures treated with Cyclo or Dorso for 48h. HFSC identity genes (triangles) and genes implicated in HFSC lineage progression (circles) are indicated.E. RT-qPCR analysis of 3C cultures treated as in (D) confirms that Id 1,2 and 3 are significantly upregulated in Cyclo treated cells (mean ±SEM; n=4; *p ≤ 0.05, Mann-Whitney U test).F. RT-qPCR analysis of 3C cultures treated as in (A) shows upregulation of HFSC identity genes upon cyclopamine treatment whereas regulators of HFSC quiescence are downregulated upon dorsomorphin treatment (mean ±SEM; n=4; *p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis).H. Quantification of experiments as in E (mean ±SD; n=3-4; * p ≤ 0.05, ** p ≤ 0.01, Kruskal-Wallis). See also Appendix figure S6 and Dataset EV5.