A The induction of MtNPF6 . 7 and repression of MtNPF6 . 5 by NO3 - requires MtNLP1 . Plants were grown on FP plates and then treated with 5 mM KNO3 . expression shown is relative to UBQ ; * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) .

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

A The induction of MtNPF6.7 and repression of MtNPF6.5 by NO3- requires MtNLP1. Plants were grown on FP plates and then treated with 5 mM KNO3. expression shown is relative to UBQ; *p<0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate).


B The switch from Cl - to NO3 - as the major anion upon NO3 - addition is lost in mtnlp1 . Plants were grown on FP plates and then treated with 5 mM KNO3 treatment . * p < 0 . 05 , Student ' s t - test ; biological replicates : n = 4 ( 3 pooled root organs in each replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The switch from Cl- to NO3- as the major anion upon NO3- addition is lost in mtnlp1. Plants were grown on FP plates and then treated with 5 mM KNO3 treatment. *p< 0.05, Student's t-test; biological replicates: n=4 (3 pooled root organs in each replicate).C


( C ) The Hippo WW domain - containing proteins show binding specificity to the known Hippo PY motif - containing proteins . TAP - MS analysis of a series of WW domain - containing proteins were performed and their binding with the indicated Hippo PY motif - containing proteins was summarized in a heatmap .

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

(C) The Hippo WW domain-containing proteins show binding specificity to the known Hippo PY motif-containing proteins. TAP-MS analysis of a series of WW domain-containing proteins were performed and their binding with the indicated Hippo PY motif-containing proteins was summarized in a heatmap.


( E ) Validation of the binding specificity for the Hippo WW domain - containing proteins . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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

(E) Validation of the binding specificity for the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( F ) Validation of the binding specificity for the derived WW domains from the Hippo WW domain - containing proteins . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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

(F) Validation of the binding specificity for the derived WW domains from the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( C - G ) Validation of the identified 9 - amino acid sequence in determining the Hippo WW domain binding specificity . The requirement of the identified 9 - amino acid sequence for AMOT association was respectively examined for TAZ ( C ) , TAZ - WW domain ( D ) , KIBRA ( E ) , YAP ( F ) and SAV1 ( G ) . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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

(C-G) Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was respectively examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( B ) STXBP4 forms a complex with several Hippo PY motif - containing proteins . Immunoprecipitation was performed with STXBP4 antibody .

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

(B) STXBP4 forms a complex with several Hippo PY motif-containing proteins. Immunoprecipitation was performed with STXBP4 antibody.


( C ) The identified 9 - amino acid sequence is required for the association between STXBP4 and AMOT . HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay .

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

(C) The identified 9-amino acid sequence is required for the association between STXBP4 and AMOT. HEK293T cells were transfected with the indicated STXBP4 mutants and subjected to the pulldown assay.


( E ) The YAP - WW1 / SMAD7 - PY and STXBP4 - WW / SMAD7 - PY complexes show similar cumulative average trend and average binding free energy ( ∆ G ) within standard deviation ( the shaded region ) of one another .

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

(E) The YAP-WW1/SMAD7-PY and STXBP4-WW/SMAD7-PY complexes show similar cumulative average trend and average binding free energy (∆G) within standard deviation (the shaded region) of one another.


( F ) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation . Western blotting was performed with the indicated antibodies .

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

(F) Loss of STXBP4 inhibits YAP phosphorylation and LATS activation. Western blotting was performed with the indicated antibodies.


Loss of STXBP4 activates YAP . STXBP4 deficiency promotes YAP nuclear translocation ( G )

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

Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP nuclear translocation (G)


Loss of STXBP4 activates YAP . STXBP4 deficiency promotes YAP downstream gene transcription ( mean ± s . d . , n = 3 biological replicates ) ( H ) . Scale bar , 20 μm . * * * p < 0 . 001 ( Student ' s t - test ) .

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

Loss of STXBP4 activates YAP. STXBP4 deficiency promotes YAP downstream gene transcription (mean ± s.d., n=3 biological replicates) (H). Scale bar, 20 μm.*** p < 0.001 (Student's t-test).


( I ) WW domain is required for the STXBP4 - mediated YAP cytoplasmic translocation . STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed . HA - positive cells ( arrows ) from ~ 30 different views ( ~ 200 cells in total ) were randomly selected and quantified for YAP localization . Percentage of HA - positive cells with nuclear YAP enrichment is shown . Scale bar , 20 μm .

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

(I) WW domain is required for the STXBP4-mediated YAP cytoplasmic translocation. STXBP4 KO cells were transfected with the indicated STXBP4 constructs and immunofluorescent staining was performed. HA-positive cells (arrows) from ~30 different views (~200 cells in total) were randomly selected and quantified for YAP localization. Percentage of HA-positive cells with nuclear YAP enrichment is shown. Scale bar, 20 μm.


( J ) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition . The indicated cells were subjected to serum starvation ( treatment with no - serum medium for 12 hours ) , glucose starvation ( treatment with no - glucose medium for 6 hours ) or actin inhibition ( treatment with 0 . 5 μg / mL latrunculin B or 5 μM blebbistatin for 30 min ) . YAP phosphorylation was detected using phospho - tag gel , where the YAP phosphorylation level was indicated .

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

(J) Loss of STXBP4 attenuates YAP phosphorylation as induced by actin cytoskeleton inhibition. The indicated cells were subjected to serum starvation (treatment with no-serum medium for 12 hours), glucose starvation (treatment with no-glucose medium for 6 hours) or actin inhibition (treatment with 0.5 μg/mL latrunculin B or 5 μM blebbistatin for 30 min). YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.


( B ) STXBP4 forms a protein complex with α - catenin and a group of Hippo pathway proteins .

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

(B) STXBP4 forms a protein complex with α-catenin and a group of Hippo pathway proteins.


( C ) STXBP4 promotes the association of α - catenin with AMOT and LATS1 . HEK293T cells were transfected with the indicated SFB - tagged constructs and subjected to the pulldown assay .

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

(C) STXBP4 promotes the association of α-catenin with AMOT and LATS1. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pulldown assay.


( D ) Loss of STXBP4 diminishes the association of α - catenin with AMOT , LATS1 and YAP . HEK293A and STXBP4 KO cells were transfected with the SFB - tagged α - catenin construct and subjected to the pulldown assay .

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

(D) Loss of STXBP4 diminishes the association of α-catenin with AMOT, LATS1 and YAP. HEK293A and STXBP4 KO cells were transfected with the SFB-tagged α-catenin construct and subjected to the pulldown assay.


( E ) STXBP4 induces the co - localization between α - catenin and AMOT as well as LATS1 and YAP . HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed . Scale bar , 20 μm .

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

(E) STXBP4 induces the co-localization between α-catenin and AMOT as well as LATS1 and YAP. HEK293A cells were transfected with the indicated constructs and immunofluorescence was performed. Scale bar, 20 μm.


Identification of several STXBP4 missense mutations that disrupt its interaction with α - catenin and AMOT . The identified missense mutations respectively disrupted the STXBP4 - α - catenin ( G ) and STXBP4 - AMOT ( H ) complex formation .

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

Identification of several STXBP4 missense mutations that disrupt its interaction with α-catenin and AMOT. The identified missense mutations respectively disrupted the STXBP4- α-catenin (G) and STXBP4-AMOT (H) complex formation.


( I ) Inhibition of actin cytoskeleton promotes the STXBP4 - associated protein complex formation . HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre - immune serum and anti - STXBP4 serum under the indicated treatments .

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

(I) Inhibition of actin cytoskeleton promotes the STXBP4-associated protein complex formation. HEK293A and the STXBP4 KO cells were subjected to immunoprecipitation using pre-immune serum and anti-STXBP4 serum under the indicated treatments.


( J ) The missense mutations of STXBP4 ( F ) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension . YAP phosphorylation was detected using phospho - tag gel , where the YAP phosphorylation level was indicated .

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

(J) The missense mutations of STXBP4 (F) diminished the ability of STXBP4 to rescue YAP phosphorylation in the STXBP4 KO cells with low actin cytoskeleton tension. YAP phosphorylation was detected using phospho-tag gel, where the YAP phosphorylation level was indicated.


STXBP4 is downregulated in human kidney cancer . The mRNA level of STXBP4 is analyzed in the Firebrowse web database ( A ) , where 14 , 729 tumor sample data generated by TCGA were included . The first quartile , median and third quartile values were indicated as the boxplots . Outliers were plotted as individual points . Error bars indicated the standard deviation above and below the mean of the data .

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

STXBP4 is downregulated in human kidney cancer. The mRNA level of STXBP4 is analyzed in the Firebrowse web database (A), where 14,729 tumor sample data generated by TCGA were included. The first quartile, median and third quartile values were indicated as the boxplots. Outliers were plotted as individual points. Error bars indicated the standard deviation above and below the mean of the data.


STXBP4 is downregulated in human kidney cancer . The expression of STXBP4 was also examined using kidney tissue microarray , where percentage of the indicated tissue samples with downregulated STXBP4 was shown ( B ) . The p value was calculated by using the paired Student ' s t - test .

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

STXBP4 is downregulated in human kidney cancer. The expression of STXBP4 was also examined using kidney tissue microarray, where percentage of the indicated tissue samples with downregulated STXBP4 was shown (B). The p value was calculated by using the paired Student's t-test.


( C ) Kaplan - Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level . Clinical data of STXBP4 were analyzed in TCGA - KIRC project containing total 611 patient samples . The p value was calculated by using the Log - rank ( Mantel - Cox ) test .

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

(C) Kaplan-Meier curves of overall survival of patients with ccRCC is stratified by STXBP4 expression level. Clinical data of STXBP4 were analyzed in TCGA-KIRC project containing total 611 patient samples. The p value was calculated by using the Log-rank (Mantel-Cox) test.


( D ) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray , where the indicated regions in the box were shown three times enlarged . Brown staining indicates positive immunoreactivity . Scale bar , 100 μm .

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

(D) Immunohistochemical staining of STXBP4 and YAP were performed in a kidney cancer tissue microarray, where the indicated regions in the box were shown three times enlarged. Brown staining indicates positive immunoreactivity. Scale bar, 100 μm.


( E ) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables . Statistical significance was determined by chi - square test . R , correlation coefficient . N , nuclear localization . C , cytoplasmic localization .

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

(E) Correlation analyses between STXBP4 and YAP in human normal kidney and clear cell carcinoma samples are shown as tables. Statistical significance was determined by chi-square test. R, correlation coefficient. N, nuclear localization. C, cytoplasmic localization.


( F ) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting .

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

(F) STXBP4 expression is examined in a panel of ccRCC cell lines by Western Blotting.


( G and H ) Both the association with α - catenin and the functional WW domain are required for the STXBP4 ' s tumor suppressive function in 786 - O cells . Overexpression of STXBP4 , but not the indicated STXBP4 missense mutants , significantly suppressed the 786 - O cell xenograft tumor formation . Xenograft tumors are shown in ( G ) , and the tumor weight is quantified in ( H ) ( n = 5 mice , mean ± s . d . ) . * * p < 0 . 01 ( Student ' s t - test ) . Scale bar , 1 cm .

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

(G and H) Both the association with α-catenin and the functional WW domain are required for the STXBP4's tumor suppressive function in 786-O cells. Overexpression of STXBP4, but not the indicated STXBP4 missense mutants, significantly suppressed the 786-O cell xenograft tumor formation. Xenograft tumors are shown in (G), and the tumor weight is quantified in (H) (n = 5 mice, mean ± s.d.). ** p < 0.01 (Student's t-test). Scale bar, 1 cm.


( B ) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . unparasitized hosts . Day - 4 of last instar larvae were used for this assay . Test larvae were parasitized by the wasp on day 0 of last instar .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. unparasitized hosts. Day-4 of last instar larvae were used for this assay. Test larvae were parasitized by the wasp on day 0 of last instar.


( C ) Survival of armyworm larvae injected with PBS , unparasitized , and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . * * P < 0 . 01 vs . PBS . Test larvae were exposed to the heat stress 4 h after injection of each sample .

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

(C) Survival of armyworm larvae injected with PBS, unparasitized, and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. PBS. Test larvae were exposed to the heat stress 4 h after injection of each sample.


( D ) Time - dependent survival of armyworm larvae injected with 0 . 8 µmol / larva N - acetyl - L - tyrosine ( NAT ) : post NAT - injection 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . * * P < 0 . 01 vs . zero time .

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

(D) Time-dependent survival of armyworm larvae injected with 0.8 µmol/larva N-acetyl-L-tyrosine (NAT): post NAT-injection 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. zero time.


( E ) Dose - dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . Injection of more than 10 nmol NAT / larva caused a significant difference compared with zero ( log rank test using R version 3 . 2 . 2 ) .

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

(E) Dose-dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min (data are means ± SEM; n=12). Injection of more than 10 nmol NAT/larva caused a significant difference compared with zero (log rank test using R version 3.2.2).


( F ) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . PBS . Each chemical ( 0 . 5 µmol / larva ) was dissolved in PBS ; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine .

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

(F) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. PBS. Each chemical (0.5 µmol/larva) was dissolved in PBS; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine.


( A ) HPLC chromatogram and L - MS spectrogram of N - acetyltyrosine purified from pooled human serum ( # 12181201 , Cosmo Bio Co . , Japan ) . The peak fraction purified by HPLC was confirmed to be N - acetyltyrosine by LC - MS ( inserted spectrogram ) .

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

(A) HPLC chromatogram and L-MS spectrogram of N-acetyltyrosine purified from pooled human serum (#12181201, Cosmo Bio Co., Japan). The peak fraction purified by HPLC was confirmed to be N-acetyltyrosine by LC-MS (inserted spectrogram).


( B ) NAT concentrations in the serum of mice ( Slc : ddY , 18 wks old , male ) exposed to heat ( 40oC ) and restraint stress ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NAT concentrations in the serum of mice (Slc:ddY, 18 wks old, male) exposed to heat (40oC ) and restraint stress (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. zero time.


( C ) Effects of NAT on corticosterone concentrations in the serum of mice ( Slc : ddY , 9 - 10 wks old , male ) exposed to restraint stress for 60 min ( data are means ± SEM ; n = 6 ) . * * P < 0 . 01 vs . without stress . Test mice drank 1 . 5 mg / ml NAT solution for 24 h before exposure to stress .

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

(C) Effects of NAT on corticosterone concentrations in the serum of mice (Slc:ddY, 9-10 wks old, male) exposed to restraint stress for 60 min (data are means ± SEM; n=6). ** P<0.01 vs. without stress. Test mice drank 1.5 mg/ml NAT solution for 24 h before exposure to stress.


( D ) Effect of NAT on stress - induced peroxidized lipid in the serum of mice ( Slc : ddY , 9 wks old , male ) forced to swim for 60 min ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . PBS .

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

(D) Effect of NAT on stress-induced peroxidized lipid in the serum of mice (Slc:ddY, 9 wks old, male) forced to swim for 60 min (data are means ± SEM; n=10). *P<0.05 vs. PBS.


( A ) Survival of Drosophila melanogaster ( W1118 ) larvae fed with 2 µmol NAT / g - diet 2 d after heat stress at 38 . 5oC for 60 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . zero time .

(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.


( B ) Effects of NAT feeding on expression of antioxidant enzymes , catalase , SOD1 , and SOD2 ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . zero time .

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

(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.


( C ) Representative images of nuclei ( left ) and FoxO ( middle ) in the fat body of Drosophila larvae before and after NAT feeding for 12 h . White signals in the merged image ( right ) show the overlap of both signals . Scale bar : 50 µm . ( D ) Relative whiteness intensities before and after NAT feeding ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . zero time .

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

(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm. (D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time.


( E ) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae ( data are means ± SEM ; n = 8 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time .

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

(E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.


( F ) Effects of N - acetyltransferase ( Dat ) knockdown in gut enteroendocrine cells ( TK - Gut - Gal4 > UAS - dsDat ) on survival of Drosophila larvae ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . without NAT .

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

(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.


( A ) Survival of Drosophila S2 cells treated with preheating , 100 µM NAT , and 100 µM NAT with 10 mM 2 - aminobicyclo - ( 2 , 2 , 1 ) - heptane - 2 - carboxylic acid ( BCH ) after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , those were removed by changing the medium . * P < 0 . 05 , * * P < 0 . 01 vs . PBS .

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

(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS.


( B ) Survival of Drosophila S2 cells pretreated with 100 µM NAT , 100 µM NAT with 30 µg / ml antimycin A , and preheating with or without 30 µg / ml antimycin A 48 h after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , they were removed by changing the medium . * P < 0 . 05 vs . PBS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS.


( C ) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells . Upper : representative images of time - dependent changes in MitoRed localization in mitochondria after adding NAT . Scale bar : 20 µm . Lower : relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader . Different letters above bars represent significant differences [ P < 0 . 05 ( data are means ± SEM , n = 12 ) ] .

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

(C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)].


( D ) ROS concentrations in S2 cells after adding 100 µM NAT ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 vs . zero time .

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

(D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time.


( F ) Effects of dsRNA targeting of FoxO on NAT - dependent expression of Keap1 ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies .

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

(F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies.


( G ) Effect of NAT on growth of HCT116 colon cancer cells in mice ( BALB / cSlc - nu / nu , 6 wks old , female ) ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Test mice began to freely drink 5 mg / ml NAT solution for 7 d before transplantation of the tumor cells on day 0 : the average amount of NAT consumed by each mouse was about 8 . 8 mg / day ( 0 . 4 - 0 . 5 g / kg weight / day ) . HCT116 cells ( 5 . 0 x 106 cells ) were transplanted into each test mouse using a 1 ml syringe with a 27G needle .

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

(G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.


a , Percentage of ciliated cells in control ( Ctrl ) and IFT20 − MEFs ( * P = 0 . 030 , n = 3 ) and wild - type ( WT ) and Ift88 − / − KECs ( * * P = 0 . 003 , 25 cells each , n = 3 ) after 24 h of serum removal . Arrows , cilia .

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

a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.030, n = 3) and wild-type (WT) and Ift88−/− KECs (**P = 0.003, 25 cells each, n = 3) after 24 h of serum removal. Arrows, cilia.


b , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ctrl and IFT20 − MEFs ( top , * * P = 0 . 0003 , n = 7 ) , and in WT and Ift88 − / − KECs ( bottom , * P = 0 . 035 , n = 9 ) . PI , protease inhibitors ; S , serum .

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

b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. c


c , Autophagic flux in WT and Ift88 − / − KECs by mCherry - GFP - LC3 puncta quantification . Puncta : yellow , autophagosomes ; red only , autophagolysosomes ; total , both together ( * * P = 0 . 008 , * P = 0 . 025 ) .

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

c, Autophagic flux in WT and Ift88−/−KECs by mCherry-GFP-LC3 puncta quantification. Puncta: yellow, autophagosomes; red only, autophagolysosomes; total, both together (**P = 0.008, *P = 0.025).


. d , LC3 immunoblot ( left ) for quantification of autophagosome formation ( right ) . ( * P = 0 . 048 , n = 4 ) .

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

. d, LC3 immunoblot (left) for quantification of autophagosome formation (right). (*P = 0.048, n = 4).


e , Electron microscopy images ( left ) and morphometric quantification of autophagic vacuoles ( AV , right ) in Ctrl and IFT20 − MEFs ( top , * * P = 0 . 001 , 10 fields in 2 experiments ) and KECs WT and Ift88 − / − ( bottom , * P = 0 . 037 , 5 fields ) . Arrows , autophagic vacuoles ( AV , black ) and endosomal compartments ( yellow ) .

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

e, Electron microscopy images (left) and morphometric quantification of autophagic vacuoles (AV, right) in Ctrl and IFT20− MEFs (top, **P = 0.001, 10 fields in 2 experiments) and KECs WT and Ift88−/− (bottom, *P = 0.037, 5 fields). Arrows, autophagic vacuoles (AV, black) and endosomal compartments (yellow).


f , Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml − 1 platelet - derived growth factor ( PDGF ) treatment . ( * * P = 0 . 001 , * * * P = 0 . 0009 , 121 , 230 and 162 cells , n = 5 ) .

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

f, Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml−1 platelet-derived growth factor (PDGF) treatment. (**P = 0.001, ***P = 0.0009, 121, 230 and 162 cells, n = 5).


g , LC3immunoblot ( top ) for autophagic flux quantification ( bottom ) upon PDGF treatment ( * P = 0 . 005 , n = 3 ) . Scale bars in a , 10 µm . n . s . , statistically non - significant . Mean ± s . d . in b , d and mean ± s . e . m . in other panels .

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

g, LC3immunoblot (top) for autophagic flux quantification (bottom) upon PDGF treatment (*P = 0.005, n = 3). Scale bars in a, 10 µm. n.s., statistically non-significant. Mean ± s.d. in b, d and mean ± s.e.m. in other panels.


a , GFP - LC3 puncta pattern ( left ) and quantification ( right ) in MEFs treated with purmorphamine ( Purmo ; top , * P = 0 . 028 , n = 4 ) , in Ptc − / − MEFs ( middle , * P = 0 . 015 , n = 4 ) and after myc - GLI1 overexpression ( bottom , * P = 0 . 012 , n = 3 ) .

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

a, GFP-LC3 puncta pattern (left) and quantification (right) in MEFs treated with purmorphamine (Purmo; top, *P = 0.028, n = 4), in Ptc−/− MEFs (middle, *P = 0.015, n = 4) and after myc-GLI1 overexpression (bottom, *P = 0.012, n = 3).


b , Autophagic flux by mCherry - GFP - LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum . Yellow , autophagosomes ; red only , autophagolysosomes ; total , both compartments . ( * P = 0 . 0323 , 40 fields ) .

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

b, Autophagic flux by mCherry-GFP-LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum. Yellow, autophagosomes; red only, autophagolysosomes; total, both compartments. (*P = 0.0323, 40 fields).


c , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ctrl ( * * P = 0 . 001 ) and MEFs knocked down for SMO ( SMO − ) ( * * P = 0 . 0001 ) upon purmorphamine treatment ( * P = 0 . 009 , n = 4 ) .

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

c, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl (**P = 0.001) and MEFs knocked down for SMO (SMO−) (**P = 0.0001) upon purmorphamine treatment (*P = 0.009, n = 4).


d , Autophagic flux by mCherry - GFP - LC3 puncta quantification in WT and Ift88 − / − KECs upon purmorphamine treatment ( * P = 0 . 046 , * * P = 0 . 008 , 40 fields ) .

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

d, Autophagic flux by mCherry-GFP-LC3 puncta quantification in WT and Ift88−/− KECs upon purmorphamine treatment (*P = 0.046, **P = 0.008, 40 fields).


. e , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ift88 − / − KECs overexpressing myc - GLI1 ( * * P = 0 . 00006 , n = 3 ) .

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

. e, LC3 immunoblot (left) for autophagic flux quantification (right) in Ift88−/− KECs overexpressing myc-GLI1 (**P = 0.00006, n = 3).


f , Autophagic flux by mCherry - GFP - LC3puncta quantification in MEFs treated with cyclopamine upon serum removal ( * P = 0 . 011 , * * P = 0 . 013 , 25 fields ) . Scale bars , 10 µm . Mean ± s . d . in c , f and mean ± s . e . m . in other panels .

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

f, Autophagic flux by mCherry-GFP-LC3puncta quantification in MEFs treated with cyclopamine upon serum removal (*P = 0.011, **P = 0.013, 25 fields). Scale bars, 10 µm. Mean ± s.d. in c, f and mean ± s.e.m. in other panels.


a , Co - immunostaining ( top ) and 3D reconstruction ( middle and bottom ) for the indicated autophagy - related proteins ( ATGs , green ) and acetylated tubulin ( red , ciliary axoneme marker ) in KECs maintained in the absence of serum for 24 h . Yellow arrows , colocalization .

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

a, Co-immunostaining (top) and 3D reconstruction (middle and bottom) for the indicated autophagy-related proteins (ATGs, green) and acetylated tubulin (red, ciliary axoneme marker) in KECs maintained in the absence of serum for 24 h. Yellow arrows, colocalization.


b , Schematic representation of the ATGs associated with the axoneme ( top left ) . Co - immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off . Individual channels and merged images are shown .

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

b, Schematic representation of the ATGs associated with the axoneme (top left). Co-immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off. Individual channels and merged images are shown.


c , Co - immunostaining ( bottom ) and 3D reconstruction ( top ) for ATGs ( green ) and gamma - tubulin ( red , basal body marker ) in the same cells and conditions . Arrows , colocalization ( yellow ) , no colocalization ( white ) .

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

c, Co-immunostaining (bottom) and 3D reconstruction (top) for ATGs (green) and gamma-tubulin (red, basal body marker) in the same cells and conditions. Arrows, colocalization (yellow), no colocalization (white).


d - f , Percentage of cells with colocalizing ATGs in the basal body ( BB ) in WT and Ift88 − / − KECs in the presence or absence of serum . d , Serum - and IFT - dependent association of ATGs with the BB ( VPS34 , † P = 0 . 04 , * * P = 0 . 002 , n = 4 ; ATG16L , † † P = 0 . 0003 , * * P = 0 . 0009 , 15 cells each per experiment , n = 7 ) . e , IFT - dependent but serum - independent association of ATGs with the BB ( * * P = 0 . 001 , * * * P = 9 . 33768 × 10 − 6 , n = 4 ; * P = 0 . 018 , 15 cells each per experiment , n = 5 ) . f , BB association of ATGs independent on serum or IFT ( ATG5 , n = 5 ; LC3 , n = 8 ; 15 cells each per experiment ) . Scale bars , 10 & amp ; amp ; mgr ; m . Mean ± s . e . m .

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

d-f, Percentage of cells with colocalizing ATGs in the basal body (BB) in WT and Ift88−/− KECs in the presence or absence of serum. d, Serum- and IFT-dependent association of ATGs with the BB (VPS34, †P = 0.04, **P = 0.002, n = 4; ATG16L, ††P = 0.0003, **P = 0.0009, 15 cells each per experiment, n = 7). e, IFT-dependent but serum-independent association of ATGs with the BB (**P = 0.001, ***P = 9.33768 × 10−6, n = 4; *P = 0.018, 15 cells each per experiment, n = 5). f, BB association of ATGs independent on serum or IFT (ATG5, n = 5; LC3, n = 8; 15 cells each per experiment). Scale bars, 10 &amp;mgr;m. Mean ± s.e.m.


a , b , Immunoblot for the indicated proteins of WT and Ift88 − / − KECs subjected to co - immunoprecipitation of IFT20 ( a ) and ATG16L ( b ) . Inp , 1 / 10 input ; IP , immunoprecipitate ; FT , 1 / 10 flow - through .

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

a, b, Immunoblot for the indicated proteins of WT and Ift88−/− KECs subjected to co-immunoprecipitation of IFT20 (a) and ATG16L (b). Inp, 1/10 input; IP, immunoprecipitate; FT, 1/10 flow-through.


c , Immunoblot for ATG16L and clathrin in MEF homogenate ( Hom ) , and the pellets from 1 h centrifugation at 100 , 000g ( organelles ( Org ) ) , 300 , 000g ( 300k ) and 500 , 000g ( vesicles , 500k ) .

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

c, Immunoblot for ATG16L and clathrin in MEF homogenate (Hom), and the pellets from 1 h centrifugation at 100,000g (organelles (Org)), 300,000g (300k) and 500,000g (vesicles, 500k).


d , Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl , IFT20 − and IFT88 − MEFs in the presence ( top ) and absence ( bottom ) of serum .

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

d, Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl, IFT20− and IFT88− MEFs in the presence (top) and absence (bottom) of serum.


e , Immunogold electron microscopy for ATG16L ( 15 - nm particles ) and IFT20 ( 10 - nm particles ) in vesicles from MEFs isolated by the same method as in c . Right , quantification of IFT20 and ATG16L presence in the vesicles ( 11 fields ) . Mean ± s . e . m .

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

e, Immunogold electron microscopy for ATG16L (15-nm particles) and IFT20 (10-nm particles) in vesicles from MEFs isolated by the same method as in c. Right, quantification of IFT20 and ATG16L presence in the vesicles (11 fields). Mean ± s.e.m.


a , b , Ciliated Atg5 − / − MEFs in serum + ( * P = 0 . 038 , 25 cells each per experiment , n = 3 ) ( a ) or serum − ( * P = 0 . 006 , nonlinear fit regression , 25 cells each per experiment , n = 3 ) ( b ) .

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

a, b, Ciliated Atg5−/− MEFs in serum+ (*P = 0.038, 25 cells each per experiment, n = 3) (a) or serum− (*P = 0.006, nonlinear fit regression, 25 cells each per experiment, n = 3) (b).


c , Scanning electron microscopy ( SEM ) of primary cilia . Arrows , cilia - associated vesicles ; arrowheads , ciliary pocket .

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

c, Scanning electron microscopy (SEM) of primary cilia. Arrows, cilia-associated vesicles; arrowheads, ciliary pocket.


. d , Cilia length quantification from SEM ( * P = 0 . 027 n = 28 ; * * P = 0 . 001 , † † P = 0 . 0001 , n = 23 ) .

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

. d, Cilia length quantification from SEM (*P = 0.027 n = 28; **P = 0.001, ††P = 0.0001, n = 23).


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

e, f, GLI1 (*P = 0.04, n = 3) (e) and GLI2 (***P = 0.0005, **P = 0.0046, †P = 0.0132, n = 3) (f) messenger RNA expression in Atg5−/− MEFs.


g , h , IFT20 immunoblot in Atg5 − / − ( g ) and WT MEFs with lysosomal inhibitors ( NL ) ( h ) .

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

g, h, IFT20 immunoblot in Atg5−/− (g) and WT MEFs with lysosomal inhibitors (NL) (h).


i , IFT20 immunoblot in cytosol ( Cyt ) , homogenate ( Hom ) and autophagosomes ( APG ) . j , IFT20 protein levels in Atg5 − / − MEFs ( * P = 0 . 038 , n = 6 ) . k , IFT20 immunoblot in Atg5 − / − MEFs . n . s . , statistically non - significant . Mean ± s . d . in d and j and mean ± s . e . m . in other panels .

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

i, IFT20 immunoblot in cytosol (Cyt), homogenate (Hom) and autophagosomes (APG). j, IFT20 protein levels in Atg5−/− MEFs (*P = 0.038, n = 6). k, IFT20 immunoblot in Atg5−/− MEFs. n.s., statistically non-significant. Mean ± s.d. in d and j and mean ± s.e.m. in other panels.


( A ) Breast cancer cell lines were sorted using CD44 for luminal MCF - 7 cells ( left panel ) or EpCAM for MDA - MB - 231 cells ( right panel ) . Serial mammosphere assays were performed using 4000 cells for each cell fraction , counting spheres after 3 weeks . Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate .

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

(A) Breast cancer cell lines were sorted using CD44 for luminal MCF-7 cells (left panel) or EpCAM for MDA-MB-231 cells (right panel). Serial mammosphere assays were performed using 4000 cells for each cell fraction, counting spheres after 3 weeks. Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate.


( B ) Extreme limiting dilution assay ( ELDA ) of MCF - 7 cells . Sorted cells were serially diluted ( 1250 , 625 , 312 , 156 and 78 cells ) and then seeded in 96 - well plates . After one week , the cells were fixed , stained and counted . The lower limit for well repopulation in CD44 + cells was 312 cells , compared with 615 cells for both unsorted and CD44 - populations . The data obtained were evaluated using the ELDA software . p values that test the single - hit hypothesis using the likelihood ratio test are shown . The curves are depicted in Fig

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Extreme limiting dilution assay (ELDA) of MCF-7 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in CD44+ cells was 312 cells, compared with 615 cells for both unsorted and CD44- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1


( C ) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL , Vimentin and TWIST in MCF - 7 / CD44 + cells . The fold change was calculated using GAPDH as an internal control using the 2 - ΔΔCT method comparing stem or non - stem cells versus the total cell line . The box and whiskers graph shows the range , median and quartile interval values from 3 independent experiments .

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

(C) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL, Vimentin and TWIST in MCF-7/CD44+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cell line. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.


( D ) miR - 10b overexpression affecting stemness and EMT factors . Relative quantification of miR - 10b ( relative to RNU6B ) , HOXD10 and RHOC ( relative to GAPDH ) was performed in MCF - 7 / CD44 + fractions compared to non - enriched cells . Box plots show the range , median and quartile interval values from 3 independent experiments . A two - tailed Student ' s t test was used to compare the two groups ( p < 0 . 05 ) . SEM , standard error of the mean ; TLDA , TaqMan Low Density Arrays ; and EMT , epithelial - mesenchymal transition .

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

(D) miR-10b overexpression affecting stemness and EMT factors. Relative quantification of miR-10b (relative to RNU6B), HOXD10 and RHOC (relative to GAPDH) was performed in MCF-7/CD44+ fractions compared to non-enriched cells. Box plots show the range, median and quartile interval values from 3 independent experiments. A two-tailed Student's t test was used to compare the two groups (p < 0.05). SEM, standard error of the mean; TLDA, TaqMan Low Density Arrays; and EMT, epithelial-mesenchymal transition.


( E ) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA - MB - 231 / EpCAM + cells . The fold change was calculated using GAPDH as an internal control using the 2 - ΔΔCT method comparing stem or non - stem cells versus the total cells . The box and whiskers graph shows the range , median and quartile interval values from 3 independent experiments .

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

(E) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA-MB-231/EpCAM+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cells. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.


( F ) Extreme Limiting Dilution Assay ( ELDA ) of MDA - MB - 231 cells . Sorted cells were serially diluted ( 1250 , 625 , 312 , 156 and 78 cells ) and then seeded in 96 - well plates . After one week , the cells were fixed , stained and counted . The lower limit for well repopulation in EpCAM + cells was 156 cells compared with 625 cells for both unsorted and EpCAM - populations . The data obtained were evaluated using the ELDA software . p values that test the single - hit hypothesis using the likelihood ratio test are shown . The curves are depicted in Fig

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

(F) Extreme Limiting Dilution Assay (ELDA) of MDA-MB-231 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in EpCAM+ cells was 156 cells compared with 625 cells for both unsorted and EpCAM- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1


( G ) Nine miRNAs are commonly regulated in both CD44 + and EpCam + breast cancer cells . Quantitative RT - PCR of miRNAs was performed on sorted MCF - 7 and MDA - MB - 231 cell populations using TaqMan Low Density Arrays ( TLDA ) . Results were plotted as a heat map comparing CD44 + to CD44 - cells ( MCF - 7 ) and EpCam + to EpCam - cells . The scale is shown below the map .

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

(G) Nine miRNAs are commonly regulated in both CD44+ and EpCam+ breast cancer cells. Quantitative RT-PCR of miRNAs was performed on sorted MCF-7 and MDA-MB-231 cell populations using TaqMan Low Density Arrays (TLDA). Results were plotted as a heat map comparing CD44+ to CD44- cells (MCF-7) and EpCam+ to EpCam- cells. The scale is shown below the map.


( A ) Upper panel , left : MCF - 7 / CD44 + cells were transfected with anti - miR - 10b or a scrambled control , and qRT - PCR was performed . Upper panel , right : An aliquot of these cells was subjected to mammosphere - forming assays . The graph shows the mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments . Lower panel , left : Expression of miR - 10b relative to RNU6B in MCF - 7 cells stably transfected with a plasmid containing the miR - 10b gene versus an empty vector . Results are the mean ± SEM values from 3 independent experiments . Lower panel , right : miR - 10b - overexpressing MCF - 7 cells analyzed by serial mammosphere assays . The mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments was plotted on each bar .

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

(A) Upper panel, left: MCF-7/CD44+ cells were transfected with anti-miR-10b or a scrambled control, and qRT-PCR was performed. Upper panel, right: An aliquot of these cells was subjected to mammosphere-forming assays. The graph shows the mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments. Lower panel, left: Expression of miR-10b relative to RNU6B in MCF-7 cells stably transfected with a plasmid containing the miR-10b gene versus an empty vector. Results are the mean ± SEM values from 3 independent experiments. Lower panel, right: miR-10b-overexpressing MCF-7 cells analyzed by serial mammosphere assays. The mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments was plotted on each bar.


( B ) Upper panel : Mammosphere formation was performed by seeding 20 , 000 MDA - MB - 231 / EpCAM + cells , and after 3 weeks , the cells were disaggregated , counted , and reseeded . Stable miR - 10b - overexpressing cells from 3 independent clones were tested in 3 passages , and cell number was plotted as the mean ± SEM .

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

(B) Upper panel: Mammosphere formation was performed by seeding 20,000 MDA-MB-231/EpCAM+ cells, and after 3 weeks, the cells were disaggregated, counted, and reseeded. Stable miR-10b-overexpressing cells from 3 independent clones were tested in 3 passages, and cell number was plotted as the mean ± SEM.


( C ) In vivo limiting dilution assays . miR - 10 - OE or control cells were subcutaneously injected in the backs of mice at 3 doses ( 1 , 000 , 10 , 000 and 50 , 000 cells for MCF - 7 and 5000 , 10 000 and 20 000 for SKBR - 3 ) . Mice were sacrificed after eight weeks , and tumor burden was analyzed using the ELDA software . Plots are depicted in Fig EV2 . The tables show the number of mice with tumors versus the total number of mice injected . Below the tables , the estimated frequency ( freq ) of stem cells for each group and p is shown . Stem cell frequency for MCF - 7 cells was 1 in 34252 ( range 94888 - 12364 ) in miR - 10b - OE cells versus 1 in 238965 ( Range 1817801 - 31414 ) in miR - scr cells . Stem cell frequency for SKBR3 cells was 1 in 5013 ( Range 11135 - 2257 ) in miR - 10b - OE cells versus 1 in 18778 ( Range 45397 - 7767 ) in miR - scr cells .

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

(C) In vivo limiting dilution assays. miR-10-OE or control cells were subcutaneously injected in the backs of mice at 3 doses (1,000, 10,000 and 50,000 cells for MCF-7 and 5000, 10 000 and 20 000 for SKBR-3). Mice were sacrificed after eight weeks, and tumor burden was analyzed using the ELDA software. Plots are depicted in Fig EV2. The tables show the number of mice with tumors versus the total number of mice injected. Below the tables, the estimated frequency (freq) of stem cells for each group and p is shown. Stem cell frequency for MCF-7 cells was 1 in 34252 (range 94888-12364) in miR-10b-OE cells versus 1 in 238965 (Range 1817801-31414) in miR-scr cells. Stem cell frequency for SKBR3 cells was 1 in 5013 (Range 11135-2257) in miR-10b-OE cells versus 1 in 18778 (Range 45397-7767) in miR-scr cells.


( D ) Stemness and EMT marker mRNA levels following miR - 10b modulation . miR - 10b , OCT4 / 3 , SNAIL and Vimentin expression was quantified in breast cancer cell lines using transient transfection of miR - 10b versus a miR - Scr vector ( left ) or anti - miR - 10b versus anti - miR - ctl ( right ) . Log - scale plots show the mean and SEM for the relative expression of each gene from triplicate independent transient transfections , each performed three times . A Mann - Whitney U test was used to compare expression between groups . SOX2 , OCT4 and Vimentin were significantly modulated by up - or down - regulation of miR - 10b ( p = 0 . 0043 , p = 0 . 0054 and p = 0 . 000908 , respectively ) . SEM , standard error of the mean .

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

(D) Stemness and EMT marker mRNA levels following miR-10b modulation. miR-10b, OCT4/3, SNAIL and Vimentin expression was quantified in breast cancer cell lines using transient transfection of miR-10b versus a miR-Scr vector (left) or anti-miR-10b versus anti-miR-ctl (right). Log-scale plots show the mean and SEM for the relative expression of each gene from triplicate independent transient transfections, each performed three times. A Mann-Whitney U test was used to compare expression between groups. SOX2, OCT4 and Vimentin were significantly modulated by up- or down-regulation of miR-10b (p=0.0043, p=0.0054 and p=0.000908, respectively). SEM, standard error of the mean.


( B ) MCF - 7 , SKBR - 3 and T47 - D breast cancer cells were transfected with gain - or loss - of - function constructs / shRNAs and their respective controls ( a miR - 10b - OE plasmid or anti - miR - 10b ) . MREs of the validated target HOXD10 and of the putative targets PTEN and PIK3CA were cloned into pMIR - Report , and dual luciferase reporter assays were performed . A mutated control miR - 10b ( MRE MUT PTEN ) was also included . Bars show the mean ± SEM from 6 independent transfections .

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

(B) MCF-7, SKBR-3 and T47-D breast cancer cells were transfected with gain- or loss-of-function constructs/shRNAs and their respective controls (a miR-10b-OE plasmid or anti-miR-10b). MREs of the validated target HOXD10 and of the putative targets PTEN and PIK3CA were cloned into pMIR-Report, and dual luciferase reporter assays were performed. A mutated control miR-10b (MRE MUT PTEN) was also included. Bars show the mean ± SEM from 6 independent transfections.


( C ) PTEN and PIK3CA expression after miR - 10b modulation . Left panel shows miR - 10b , PTEN and PIK3CA expression by qRT - PCR in MCF - 7 cells with ectopic miR - 10b expression . Right panel shows miR - 10b , PTEN and PIK3CA expression by qRT - PCR in MDA - MB - 231 cells after antagomiR - 10b transfection . Data are from three independent transient transfections performed by triplicate .

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

(C) PTEN and PIK3CA expression after miR-10b modulation. Left panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MCF-7 cells with ectopic miR-10b expression. Right panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MDA-MB-231 cells after antagomiR-10b transfection. Data are from three independent transient transfections performed by triplicate.


( D ) To further confirm PTEN targeting , we performed gain - or loss - of - function experiments in breast cancer cell lines ( MCF - 7 , MCF - 7 / CD44 + , SKBR - 3 and T47D ) by overexpressing miR - 10b or inhibiting it with a miR - 10b - OE plasmid or anti - miR - 10b shRNAs . PTEN and miR - 10b levels were measured by qRT - PCR . The log2 - fold change of PTEN ( left panel ) and PIK3CA ( right panel ) mRNA levels obtained through the 2 - ΔΔCT method were plotted against their respective miR - 10b expression levels . Correlation analysis was performed [ PTEN ( r = - 0 . 80 ) , PI3KCA ( r = 0 . 43 ) ] . A two - tailed Student ' s t test was used to compare the two groups ( * p < 0 . 05 ) . SEM , standard error of the mean .

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

(D) To further confirm PTEN targeting, we performed gain- or loss-of-function experiments in breast cancer cell lines (MCF-7, MCF-7/CD44+, SKBR-3 and T47D) by overexpressing miR-10b or inhibiting it with a miR-10b-OE plasmid or anti-miR-10b shRNAs. PTEN and miR-10b levels were measured by qRT-PCR. The log2-fold change of PTEN (left panel) and PIK3CA (right panel) mRNA levels obtained through the 2-ΔΔCT method were plotted against their respective miR-10b expression levels. Correlation analysis was performed [PTEN (r=-0.80), PI3KCA (r=0.43)]. A two-tailed Student's t test was used to compare the two groups (*p < 0.05). SEM, standard error of the mean.


( E ) PTEN protein modulation by miR - 10b . Upper panel : PTEN protein levels in SKBR - 3 , and MCF - 7 cells were analyzed by Western blot . GAPDH was used as a loading control with cells cultured as in ( B ) . Lower panel : Densitometric analysis of Western blot assays performed with three biological replicates .

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

(E) PTEN protein modulation by miR-10b. Upper panel: PTEN protein levels in SKBR-3, and MCF-7 cells were analyzed by Western blot. GAPDH was used as a loading control with cells cultured as in (B). Lower panel: Densitometric analysis of Western blot assays performed with three biological replicates.


( F ) PTEN immunohistochemistry analysis of MCF - 7 - miR - 10b - OE or control MCF - 7 - miR - Scr - OE tumorxenografts .

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

(F) PTEN immunohistochemistry analysis of MCF-7-miR-10b-OE or control MCF-7-miR-Scr-OE tumorxenografts.


( G ) miR - 10b overexpression does not increase mammosphere formation in PTEN - depleted cells . MCF - 7 - miR - Scr - OE cells ( left panels ) and MCF - 7 - miR - 10b - OE ( right panels ) were transfected with a Dicer - substrate RNA directed toward PTEN , and after 24 h , quantitative RT - PCR assays for PTEN , SOX2 , OCT4 , SNAIL and Vimentin were performed . Additionally , the same cells were analyzed for mammosphere formation . The graph shows the mean and standard error of the relative increase of each marker or number of mammospheres in PTEN - depleted cells ( black bars ) and in cells transfected with a scrambled control ( white bars ) in three independent biological experiments performed by triplicate .

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

(G) miR-10b overexpression does not increase mammosphere formation in PTEN-depleted cells. MCF-7-miR-Scr-OE cells (left panels) and MCF-7-miR-10b-OE (right panels) were transfected with a Dicer-substrate RNA directed toward PTEN, and after 24 h, quantitative RT-PCR assays for PTEN, SOX2, OCT4, SNAIL and Vimentin were performed. Additionally, the same cells were analyzed for mammosphere formation. The graph shows the mean and standard error of the relative increase of each marker or number of mammospheres in PTEN-depleted cells (black bars) and in cells transfected with a scrambled control (white bars) in three independent biological experiments performed by triplicate.


( H ) T47 - D cells were transfected with antagomiR - 10b , a dsi - RNA for PTEN or both and mammospheres produced as described in material and methods . Bars show mean and SEM from three independent experiments . * p = 0 . 003 by Student ´ s T test , after ANOVA showing statistical differences in groups .

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

(H) T47-D cells were transfected with antagomiR-10b, a dsi-RNA for PTEN or both and mammospheres produced as described in material and methods. Bars show mean and SEM from three independent experiments. * p=0.003 by Student´s T test, after ANOVA showing statistical differences in groups.


( I ) PTEN overexpression compensates for miR - 10b overexpression . Left panel : MDA - 468 cells were transfected with a plasmid overexpressing wild - type PTEN or a version containing a mutated MRE in its 3 ' - UTR , with or without a plasmid containing miR10b and PTEN mRNA expression was measured by end - point RT - PCR . Middle panel : Relative expression of stemness factor SNAIL . The plots show the relative expression from biological triplicate analyses ( mean and SE shown ) . Right panel : Relative expression of stemness factor SOX2 . The plots show the relative expression from biological triplicate analyses ( mean and SE shown ) .

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

(I) PTEN overexpression compensates for miR-10b overexpression. Left panel: MDA-468 cells were transfected with a plasmid overexpressing wild-type PTEN or a version containing a mutated MRE in its 3'-UTR, with or without a plasmid containing miR10b and PTEN mRNA expression was measured by end-point RT-PCR. Middle panel: Relative expression of stemness factor SNAIL. The plots show the relative expression from biological triplicate analyses (mean and SE shown). Right panel: Relative expression of stemness factor SOX2. The plots show the relative expression from biological triplicate analyses (mean and SE shown).


( A ) AKT phosphorylation in CSCs from MCF - 7 cells sorted by CD44 . GAPDH protein levels were used as a loading control .

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

(A) AKT phosphorylation in CSCs from MCF-7 cells sorted by CD44. GAPDH protein levels were used as a loading control.


( B ) Upper panel . Cultures were serum - starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002 . PTEN , PI3KCA , GSK3 and phospho - AKT Ser - 473 protein levels were analyzed by Western blot . GAPDH was used as a loading control . AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p - AKT - Ser - 473 antibody and using chimeric GSK - 3 as a substrate . A two - tailed Student ' s t test was used to compare the two groups ( p < 0 . 05 ) . SEM , standard error of the mean . Lower panel . SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN , miR - 10b or both and phospho - AKT , AKT or tubulin levels analyzed by Western Blot assays . Control is empty vector - transfected cells .

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

(B) Upper panel. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002. PTEN, PI3KCA, GSK3 and phospho-AKT Ser-473 protein levels were analyzed by Western blot. GAPDH was used as a loading control. AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p-AKT-Ser-473 antibody and using chimeric GSK-3 as a substrate. A two-tailed Student's t test was used to compare the two groups (p<0.05). SEM, standard error of the mean. Lower panel. SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN, miR-10b or both and phospho-AKT, AKT or tubulin levels analyzed by Western Blot assays. Control is empty vector-transfected cells.


( C ) Clonogenic ability of miR - 10b - depleted CD44 + MCF - 7 cells after PI3K pathway inhibition . Cultures were serum - starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002 . Cells transfected with anti - miR - 10b or a scrambled control ( anti - miR - ctl ) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors . After 3 weeks , colonies were quantified . Bars show the mean ± SEM for 3 independent experiments .

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

(C) Clonogenic ability of miR-10b-depleted CD44+ MCF-7 cells after PI3K pathway inhibition. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002. Cells transfected with anti-miR-10b or a scrambled control (anti-miR-ctl) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.


( D ) miR - 10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation . MCF - 7 stable miR - Scr - OE or miR - 10b - OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors . After 3 weeks , colonies were quantified . Bars show the mean ± SEM for 3 independent experiments .

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

(D) miR-10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation. MCF-7 stable miR-Scr-OE or miR-10b-OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.


Module - trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples . Gene modules ( y axis ) are denoted by an arbitrary color name . Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype / disease stage ( x axis ) and p values . A value of 1 ( red ) quantifies the strongest positive correlation ( genes are upregulated ) , - 1 ( blue ) the strongest negative correlation ( genes are downregulated ) , and 0 ( white ) no correlation .

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

Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation.


Microarray analysis was performed following doxycycline - regulated specific AR - V7 depletion ( using a tet - plKO backbone ) in 22Rv1 PC cells compared to doxycycline - treated shGFP controls . The genes that were significantly regulated by shAR - V7 ( in either direction , p value < 0 . 05 ) were distributed among the gene modules defined by WGCNA in panel A . Upregulated genes ( red ) are those in which expression decreased following AR - V7 depletion and conversely downregulated genes ( blue ) are those that increased following AR - V7 depletion .

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

Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.


Hornberg et al . , 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases , grouped by their relative levels of AR - VS , mainly AR - V7 . ( High - levels of AR - V7 ( top quartile ) or lower levels of AR - V7 ( quartiles 1 - 3 ) . Data are plotted as the mean ± s . e . m . . Non - parametric Mann - Whitney test was performed ( two - tailed ) . Note that BUB1B expression was not measured in these microarrays . * * Significant at a p value < 0 . 05 . N ( AR - V7 low ) = 20 ; N ( AR - V7 high ) = 10 .

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

Hornberg et al., 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases, grouped by their relative levels of AR-VS, mainly AR-V7. (High-levels of AR-V7 (top quartile) or lower levels of AR-V7 (quartiles 1-3). Data are plotted as the mean ± s.e.m.. Non-parametric Mann-Whitney test was performed (two-tailed). Note that BUB1B expression was not measured in these microarrays. ** Significant at a p value < 0.05. N (AR-V7 low) = 20; N (AR-V7 high) = 10.


The Kaplan - Meier curves for Disease - Free Survival ( DFS ) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset ( 465 samples ) ( upper graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of the seven genes were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0009 ; for death : p - value = 0 . 026 . An independent dataset was analyzed ( Prostate Adenocarcinoma MSKCC , Cancer Cell 2010 , 123 samples ) ( lower graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0007 ; for death : p - value = 0 . 00546 .

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

The Kaplan-Meier curves for Disease-Free Survival (DFS) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset (465 samples) (upper graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of the seven genes were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0009; for death: p-value = 0.026. An independent dataset was analyzed (Prostate Adenocarcinoma MSKCC, Cancer Cell 2010, 123 samples) (lower graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0007; for death: p-value = 0.00546.


Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls , using shRNA against the coding region for each gene ( shRNA # 2 ) . Cell number was measured using a non - perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System . Data shown are mean ± s . e . m . of 8 to 12 replicates normalized to their shGFP control . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed .

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

Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls, using shRNA against the coding region for each gene (shRNA #2). Cell number was measured using a non-perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System. Data shown are mean ± s.e.m. of 8 to 12 replicates normalized to their shGFP control. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed.


Representative images of 22Rv1 stably depleted of BUB1B , or control ( shGFP ) are shown .

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

Representative images of 22Rv1 stably depleted of BUB1B, or control (shGFP) are shown.


22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription . The assay was conducted in 2 % CSS to measure AR ligand - independent transcriptional activity . Data represent two independent experiments performed in triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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

22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription. The assay was conducted in 2% CSS to measure AR ligand-independent transcriptional activity. Data represent two independent experiments performed in triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The expression of FKBP5 and UBE2C determined by RT - qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes . Cells were cultured in 2 % CSS . Data represent two independent experiments performed in duplicate or triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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

The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The CRPC cell line 22Rv1 was cultured in 2 % CSS media and was treated for 72 hours with vehicle ( DMSO ) , doxorubicin ( DOX ) , N9 - isopropylolomoucine ( N - 9 ) , or the combination of DOX and N - 9 at different concentrations . Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each . The data were analyzed using Compusyn software , and a normalized isobologram was built . The table shows the Combination Index ( CI ) for the different drug combinations . CI = 1 represents additivity , CI < 1 synergism , and CI > 1 antagonistic effects .

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

The CRPC cell line 22Rv1 was cultured in 2% CSS media and was treated for 72 hours with vehicle (DMSO), doxorubicin (DOX), N9-isopropylolomoucine (N-9), or the combination of DOX and N-9 at different concentrations. Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each. The data were analyzed using Compusyn software, and a normalized isobologram was built. The table shows the Combination Index (CI) for the different drug combinations. CI=1 represents additivity, CI<1 synergism, and CI>1 antagonistic effects.


The non - tumorigenic prostate epithelial cell line RWPE1 , the AR - null PC cell line PC3 , the androgen dependent cell line LNCaP , and the CRPC cell lines C4 - 2B and 22Rv1 were treated for 72 hours with vehicle ( DMSO ) , DOX ( 100 ng / mL [ 184 nM ] ) , N - 9 ( 200 ng / mL [ 613 nM ] ) , or the combination of DOX ( 100 ng / mL [ 184 nM ] ) and N - 9 ( 200 ng / mL [ 613 nM ] ) . C4 - 2B and 22Rv1 cells were kept in 10 % CSS media , and the other cell lines in 10 % FBS . Cell confluence was monitored using the Incucyte Zoom System . Data represent two independent experiments , with four to six replicates each , showing the mean ± s . e . m . , and normalized to vehicle controls ( Kruskal - Wallis test , P value < 0 . 0001 , two - tailed ) . * Significant at a p value < 0 . 05 , * * p value < 0 . 01 , * * * p value < 0 . 001 .

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

The non-tumorigenic prostate epithelial cell line RWPE1, the AR-null PC cell line PC3, the androgen dependent cell line LNCaP, and the CRPC cell lines C4-2B and 22Rv1 were treated for 72 hours with vehicle (DMSO), DOX (100 ng/mL [184 nM]), N-9 (200 ng/mL [613 nM]), or the combination of DOX (100 ng/mL [184 nM]) and N-9 (200 ng/mL [613 nM]). C4-2B and 22Rv1 cells were kept in 10% CSS media, and the other cell lines in 10% FBS. Cell confluence was monitored using the Incucyte Zoom System. Data represent two independent experiments, with four to six replicates each, showing the mean ± s.e.m., and normalized to vehicle controls (Kruskal-Wallis test, P value <0.0001, two-tailed). *Significant at a p value < 0.05, ** p value < 0.01, *** p value < 0.001.


The non - tumorigenic prostate cell line RWPE - 1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle ( DMSO ) or the combination of DOX and N - 9 at 100 ng / mL and N - 9 200 ng / mL , respectively . Cell confluence was monitored using the Incucyte Zoom System .

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

The non-tumorigenic prostate cell line RWPE-1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle (DMSO) or the combination of DOX and N-9 at 100 ng/mL and N-9 200 ng/mL, respectively. Cell confluence was monitored using the Incucyte Zoom System.


( A ) Distribution of antimycin A resistance scores for wild isolates , compared to standard laboratory strain ( red vertical line ) . Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg / L antimycin A . Fitness was estimated based on colony size on solid media , corrected for spatial and plate effects After quality control , we obtained quantitative fitness scores for 154 strains , with a signal - to - noise ratio of 29 . 8 and an unexplained variance of 0 . 12 .

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

(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.


( B ) Volcano plot of a genome - wide association using mixed - model linear regression of antimycin A resistance for 118 , 527 genetic variants . Variants with moderate or high impact are shown in blue . Red dot : the variant at locus I : 3845516 , which causes a T343A change in the Pyk1 amino - acid sequence . This variant was among the top scoring ( effect size = - 0 . 645 , rank 34 ; p = 0 . 0008 , rank 70 ) .

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

(B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).


( C ) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus . Strains carrying a C at this genomic locus ( orange box ) generally have higher antimycin resistance than strains carrying the reference allele T ( blue box ) . As is standard , this boxplot and all other boxplots in this manuscript show the median of the data as central line , the quartiles as box and the extend of the rest of the distribution as whiskers . Points which are 1 . 5 times the inter - quartile range beyond the high and low quartiles are considered outliers and shown individually .

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

(C) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.


( B ) Left boxplot : two glycolytic intermediates directly upstream of PYK were strongly depleted in the A - strain ( nT - strain = 9 , nA - strain = 8 ) . Right barplot : PYK activity was directly measured using a lactate dehydrogenase - coupled colorimetric enzyme activity assay , showing the mean and standard deviation of substrate conversion rate for 3 biological replicates , each measured in technical duplicates . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left boxplot: two glycolytic intermediates directly upstream of PYK were strongly depleted in the A-strain (nT-strain=9, nA-strain=8). Right barplot: PYK activity was directly measured using a lactate dehydrogenase-coupled colorimetric enzyme activity assay, showing the mean and standard deviation of substrate conversion rate for 3 biological replicates, each measured in technical duplicates. Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( C ) Concentrations of fructose - 1 , 6 - bisphosphate , which correlate with glycolytic flux , are significantly higher in A - strain ( ratio = 1 . 35 , nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

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

(C) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( D ) Boxplots for selected metabolomics data indicate differences in energy and redox status between T - and A - strains ( nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

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

(D) Boxplots for selected metabolomics data indicate differences in energy and redox status between T- and A-strains (nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)