B Expression of MtNPFs in roots after transfer to media containing different of NO3 - for 30 minutes . C = KCl control , N = KNO3 ( Biological replicates : n = 3 ( 3 pooled root organs in each replicate ) , asterisk denotes significance p < 0 . 05 , Student ' s t - ) . means of samples were compared to that of their - matched KCl controls . Expression is shown relative to Ubiquitin .

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B Expression of MtNPFs in roots after transfer to media containing different levels of NO3- for 30 minutes. C=KCl control, N=KNO3 (Biological replicates: n = 3 (3 pooled root organs in each replicate), asterisk denotes significance p<0.05, Student's t-test). The means of all samples were compared to that of their concentration-matched KCl controls. Expression is shown relative to Ubiquitin.


A , B I - V responses in oocytes ( subtracted of water injected controls ) exposed to 40 mM Cl at pH5 . 8 ( A ) pH7 . 4 ( B ) . currents were measured while oocyte plasma was clamped at - 60 mv to between 20 - 140 mv for 120 ms with - 20 mv increments for each Cl - . Cl - - elicited current values in D were obtained by subtracting currents in water injected oocytes from MtNPF6 . 5 injected oocytes at each Cl - Data information : Value = mean ± , biological replicates : n =

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A, B I-V responses in oocytes (subtracted of the water injected controls) exposed to 40 mM Cl at pH5.8 (A) and pH7.4 (B). The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration Data information: Value = mean ± SEM, biological replicates: n = 4


C , D I - V responses in ( C ) water ( D ) MtNPF6 . 5 ( subtracted of water injected controls ) injected oocytes exposed to different concentrations of Cl - at pH5 . 8 . currents were measured while oocyte plasma was clamped at - 60 mv to between 20 - 140 mv for 120 ms with - 20 mv increments for each Cl - . Cl - - elicited current values in D were obtained by subtracting currents in water injected oocytes from MtNPF6 . 5 injected oocytes at each Cl - . Data information : Value = mean ± , biological replicates : n =

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C, D I-V responses in the (C) water and (D) MtNPF6.5 (subtracted of the water injected controls) injected oocytes exposed to different concentrations of Cl- at pH5.8. The currents were measured while the oocyte plasma membrane was clamped at -60 mv to between 20 and -140 mv for 120 ms with -20 mv increments for each Cl- concentration. The Cl- -elicited current values in D were obtained by subtracting the currents in water injected oocytes from the MtNPF6.5 injected oocytes at each Cl- concentration. Data information: Value = mean ± SEM, biological replicates: n = 6


C Oocytes were exposed to 1 . 17 mM 36Cl - for 2h with 1 10 mM NO3 - . Biological replicates : n = 6 ( 4 oocytes in each sample ) . D same data as in C is shown without AtNPF6 . 3 to allow for better comparison between MtNPFs . Biological replicates : n = 6 ( 4 oocytes in each sample ) . Data information : Asterisks denote significant difference : * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) . In boxplots , horizontal black bars represent median , black cross represent mean . Boxes represent middle 50 % of distribution , whiskers represent entire spread of data . Means were compared using Student ' s t - , α = 0 . 05 .

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C Oocytes were exposed to 1.17 mM 36Cl- for 2h with 1 and 10 mM NO3-. Biological replicates: n=6 (4 oocytes in each sample). D The same data as in C is shown without AtNPF6.3 to allow for better comparison between MtNPFs. Biological replicates: n=6 (4 oocytes in each sample). Data information: Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test). In the boxplots, horizontal black bars represent the median, and black cross represent the mean. Boxes represent the middle 50% of the distribution, and whiskers represent the entire spread of the data. Means were compared using Student's t-test, α= 0.05.


A , B Relative expression of NPFs in M . truncatula roots grown in FP exposed to different NaCl treatments . Data information : * p < 0 . 05 , Student ' s t - ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ±

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A, B Relative expression of NPFs in M. truncatula roots grown in FP and exposed to different NaCl treatments. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM


D Relative expression of NPFs in M . truncatula roots grown in FP exposed to 100mM KCl treatments for different times . Data information : * p < 0 . 05 , Student ' s t - ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ±

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D Relative expression of NPFs in M. truncatula roots grown in FP and exposed to 100mM KCl treatments for different times. Data information: *p<0.05, Student's t-test; biological replicates: n=3 (3 pooled root organs in each replicate) ±SEM


A Seedling Cl - content determined by ion chromatography 2 h after treatment with 50mM NaCl . * p < 0 . 05 , Student ' s t - ; biological replicates : n = 4 . B Cl - content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3 . * p < 0 . 05 , Student ' s t - ; biological replicates : n = 4 .

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A Seedling Cl- content determined by ion chromatography 2 h after treatment with 50mM NaCl. *p<0.05, Student's t-test; biological replicates: n=4. B Cl- content determined by ion chromatography of seedlings supplemented with 5mM KNO3 or NH4NO3. *p<0.05, Student's t-test; biological replicates: n=4.


C On left , relative Cl - content determined by XRF of NO3 - starved seedlings after KNO3 supplementation . * p < 0 . 05 , Student ' s t - ; biological replicates : n = 3 - 5 . On right , a representative XRF image of Cl - signal across WT mutant roots at 0h timepoint . Five roots per genotype are shown . outlines indicate measurement areas . color legend indicates relative Cl - .

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C On the left, relative Cl- content determined by XRF of NO3- starved seedlings after KNO3 supplementation. *p<0.05, Student's t-test; biological replicates: n=3-5. On the right, a representative XRF image of Cl- signal across WT and mutant roots at the 0h timepoint. Five roots per genotype are shown. The outlines indicate the measurement areas. The color legend indicates relative Cl- concentration.


A Biomass ( fresh ) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl . * p < 0 . 05 , Student ' s t - ; biological replicates : n = 3 ( 2 - 5 pooled organs in each replicate ) .

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A Biomass (fresh weight) of seedlings grown on FP or FP supplemented with 50 or 100 mM NaCl. *p<0.05, Student's t-test; biological replicates: n=3 (2-5 pooled organs in each replicate).


B Image of seedlings grown on FP or 50 mM NaCl . Data information : roots were re - arranged to better display their , they did not exhibit curling / agravitropic growth .

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B Image of seedlings grown on FP or 50 mM NaCl. Data information: roots were re-arranged to better display their length, they did not exhibit curling/agravitropic growth.


B Changes in NO3 - Cl - content ( mol / g dry ) of NO3 - starved after NO3 - provision Data information : , * p < 0 . 05 , Student ' s t - ; biological replicates : n = 4 ± .

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B Changes in NO3- and Cl- content (mol/g dry weight) of NO3- starved plants after NO3- provision Data information: , *p< 0.05, Student's t-test; biological replicates: n=4 ±SEM.


E Relative MtNPF expression following treatment with 5mM KNO3 + 100mM NaCl Data information : expression shown is relative to Ubiquitin ; * p < 0 . 05 , Student ' s t - ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± .

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E Relative MtNPF expression following treatment with 5mM KNO3 +100mM NaCl Data information: expression shown is relative to Ubiquitin; *p< 0.05, Student's t-test; biological replicates: n= 3 (3 pooled root organs in each replicate) ±SEM.


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

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

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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 ) Hippo WW domain - containing show binding specificity to known Hippo PY motif - containing . TAP - MS of a series of WW domain - containing were performed their binding with indicated Hippo PY motif - containing was summarized in a .

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(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 binding specificity for Hippo WW domain - containing . HEK293T were transfected with indicated SFB - tagged constructs subjected to pulldown .

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(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 binding specificity for derived WW domains from Hippo WW domain - containing . HEK293T were transfected with indicated SFB - tagged constructs subjected to pulldown .

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

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(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 with several Hippo PY motif - containing . Immunoprecipitation was performed with STXBP4 antibody .

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


( C ) identified 9 - acid is required for association between STXBP4 AMOT . HEK293T were transfected with indicated STXBP4 mutants subjected to pulldown .

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

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(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 LATS activation . Western blotting was performed with indicated antibodies .

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(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 translocation ( G )

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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 - ) .

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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 STXBP4 - mediated YAP translocation . STXBP4 KO were transfected with indicated STXBP4 constructs immunofluorescent was performed . HA - positive ( arrows ) from ~ 30 different views ( ~ 200 in total ) were randomly selected quantified for YAP localization . of HA - positive with YAP enrichment is shown . Scale bar , 20 μm .

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(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 . indicated 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 YAP phosphorylation level was indicated .

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(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 with α - catenin a group of Hippo pathway .

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


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

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(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 association of α - catenin with AMOT , LATS1 YAP . HEK293A STXBP4 KO were transfected with SFB - tagged α - catenin construct subjected to pulldown .

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(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 co - localization between α - catenin AMOT as well as LATS1 YAP . HEK293A were transfected with indicated constructs immunofluorescence was performed . Scale bar , 20 μm .

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(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 AMOT . identified missense mutations respectively disrupted STXBP4 - α - catenin ( G ) STXBP4 - AMOT ( H ) formation .

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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 STXBP4 - associated formation . HEK293A STXBP4 KO were subjected to immunoprecipitation using pre - immune serum anti - STXBP4 serum under indicated treatments .

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

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

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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 . expression of STXBP4 was also examined using kidney tissue microarray , where of indicated tissue samples with downregulated STXBP4 was shown ( B ) . p value was calculated by using paired Student ' s t - .

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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 with ccRCC is stratified by STXBP4 expression level . Clinical data of STXBP4 were analyzed in TCGA - KIRC project containing total 611 samples . p value was calculated by using Log - rank ( Mantel - Cox ) .

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

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(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 YAP in human normal kidney clear carcinoma samples are shown as tables . Statistical significance was determined by chi - square . R , correlation coefficient . N , localization . C , localization .

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(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 lines by Western Blotting .

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


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

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

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

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(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 ± ; n = 12 ) . * * P < 0 . 01 vs . zero time .

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(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 ± ; n = 12 ) . Injection of more than 10 nmol NAT / larva caused a significant difference compared with zero ( log rank using R version 3 . 2 . 2 ) .

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(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 ± ; 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 .

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

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

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

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(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 serum of mice ( Slc : ddY , 9 wks old , ) forced to swim for 60 min ( data are means ± ; n = 10 ) . * P < 0 . 05 vs . PBS .

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(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 ± ; n = 10 ) . * * P < 0 . 01 vs . zero time .

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(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 , SOD2 ( data are means ± ; n = 10 ) . * P < 0 . 05 vs . zero time .

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

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(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 fat of Drosophila larvae ( data are means ± ; n = 8 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time .

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(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 ( TK - Gut - Gal4 > UAS - dsDat ) on survival of Drosophila larvae ( data are means ± ; n = 15 ) . * * P < 0 . 01 vs . without NAT .

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(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 treated with preheating , 100 µM NAT , 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 ± ; n = 10 ) . After pretreatment with chemicals for 12 h , those were removed by changing medium . * P < 0 . 05 , * * P < 0 . 01 vs . PBS .

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

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

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(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 after adding 100 µM NAT ( data are means ± ; n = 12 ) . * P < 0 . 05 vs . zero time .

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(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 ± ; n = 12 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies .

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

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

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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 ( right ) in Ctrl IFT20 − MEFs ( top , * * P = 0 . 0003 , n = 7 ) , in WT Ift88 − / − KECs ( bottom , * P = 0 . 035 , n = 9 ) . PI , protease inhibitors ; S , serum .

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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 Ift88 − / − KECs by mCherry - GFP - LC3 . : yellow , autophagosomes ; red only , autophagolysosomes ; total , both together ( * * P = 0 . 008 , * P = 0 . 025 ) .

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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 of autophagosome formation ( right ) . ( * P = 0 . 048 , n = 4 ) .

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR 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 , 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 , 230 162 , n = 5 ) .

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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 ( 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 mean ± s . e . m . in other panels .

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

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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 upon purmorphamine treatment in MEFs in presence of serum . Yellow , autophagosomes ; red only , autophagolysosomes ; total , both compartments . ( * P = 0 . 0323 , 40 fields ) .

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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 ( right ) in Ctrl ( * * P = 0 . 001 ) MEFs knocked down for SMO ( SMO − ) ( * * P = 0 . 0001 ) upon purmorphamine treatment ( * P = 0 . 009 , n = 4 ) .

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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 in WT Ift88 − / − KECs upon purmorphamine treatment ( * P = 0 . 046 , * * P = 0 . 008 , 40 fields ) .

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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 ( right ) in Ift88 − / − KECs overexpressing myc - GLI1 ( * * P = 0 . 00006 , n = 3 ) .

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. 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 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 mean ± s . e . m . in other panels .

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

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-CELL_TYPE O O O O B-TISSUE 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 ATGs associated with axoneme ( top left ) . Co - immunostaining for ATGs acetylated tubulin in cilia isolated by peeling off . channels merged are shown .

O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-SUBCELLULAR 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 ) 3D reconstruction ( top ) for ATGs ( green ) gamma - tubulin ( red , basal marker ) in same conditions . Arrows , colocalization ( yellow ) , no colocalization ( ) .

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

O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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

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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 ) IFT20 ( 10 - nm particles ) in vesicles from MEFs isolated by same method as in c . Right , of IFT20 ATG16L presence in vesicles ( 11 fields ) . Mean ± s . e . m .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR 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 each per experiment , n = 3 ) ( a ) or serum − ( * P = 0 . 006 , nonlinear fit regression , 25 each per experiment , n = 3 ) ( b ) .

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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 ( ) of cilia . Arrows , cilia - associated vesicles ; arrowheads , ciliary pocket .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O

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


O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE 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 ) WT MEFs with lysosomal inhibitors ( NL ) ( h ) .

O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O B-CELL_LINE 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 ) autophagosomes ( APG ) . j , IFT20 in Atg5 − / − MEFs ( * P = 0 . 038 , n = 6 ) . k , IFT20 immunoblot in Atg5 − / − MEFs . n . s . , statistically non - significant . Mean ± s . d . in d j mean ± s . e . m . in other panels .

O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O 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 lines were sorted using CD44 for luminal MCF - 7 ( left panel ) or EpCAM for MDA - MB - 231 ( right panel ) . Serial mammosphere assays were performed using 4000 for each fraction , counting spheres after 3 . Plots show mean ± of mammospheres formed with 4000 from 3 independent experiments performed in triplicate .

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

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

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

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

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

(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 OCT4 EMT markers SNAIL Vimentin in MDA - MB - 231 / EpCAM + . fold change was calculated using GAPDH as an internal control using 2 - ΔΔCT method comparing or non - versus total . box whiskers graph shows range , median quartile interval values from 3 independent experiments .

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

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

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

(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 + EpCam + breast cancer . Quantitative RT - PCR of miRNAs was performed on sorted MCF - 7 MDA - MB - 231 populations using TaqMan Low Density Arrays ( TLDA ) . Results were plotted as a heat map comparing CD44 + to CD44 - ( MCF - 7 ) EpCam + to EpCam - . scale is shown below map .

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

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O 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 + , after 3 , were disaggregated , counted , reseeded . Stable miR - 10b - overexpressing from 3 independent clones were tested in 3 passages , number was plotted as mean ± .

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

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(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 EMT marker mRNA following miR - 10b modulation . miR - 10b , OCT4 / 3 , SNAIL Vimentin expression was quantified in breast cancer 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 mean for relative expression of each gene from triplicate independent transient transfections , each performed three times . A Mann - Whitney U was used to compare expression between groups . SOX2 , OCT4 Vimentin were significantly modulated by up - or down - regulation of miR - 10b ( p = 0 . 0043 , p = 0 . 0054 p = 0 . 000908 , respectively ) . , standard error of mean .

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

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

O O O B-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD 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 lines ( MCF - 7 , MCF - 7 / CD44 + , SKBR - 3 T47D ) by overexpressing miR - 10b or inhibiting it with a miR - 10b - OE plasmid or anti - miR - 10b shRNAs . PTEN miR - 10b were measured by qRT - PCR . log2 - fold change of PTEN ( left panel ) PIK3CA ( right panel ) mRNA obtained through 2 - ΔΔCT method were plotted against their respective miR - 10b expression . Correlation was performed [ PTEN ( r = - 0 . 80 ) , PI3KCA ( r = 0 . 43 ) ] . A two - Student ' s t was used to compare two groups ( * p < 0 . 05 ) . , standard error of mean .

O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 modulation by miR - 10b . Upper panel : PTEN in SKBR - 3 , MCF - 7 were analyzed by Western blot . GAPDH was used as a loading control with cultured as in ( B ) . Lower panel : Densitometric of Western blot assays performed with three biological replicates .

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(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 of MCF - 7 - miR - 10b - OE or control MCF - 7 - miR - Scr - OE tumorxenografts .

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(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 . MCF - 7 - miR - Scr - OE ( left panels ) MCF - 7 - miR - 10b - OE ( right panels ) were transfected with a Dicer - substrate directed toward PTEN , after 24 h , quantitative RT - PCR assays for PTEN , SOX2 , OCT4 , SNAIL Vimentin were performed . Additionally , same were analyzed for mammosphere formation . graph shows mean standard error of relative increase of each marker or number of mammospheres in PTEN - depleted ( black bars ) in transfected with a scrambled control ( bars ) in three independent biological experiments performed by triplicate .

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

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

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(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 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 PTEN mRNA expression was measured by end - point RT - PCR . Middle panel : Relative expression of stemness factor SNAIL . plots show relative expression from biological triplicate analyses ( mean SE shown ) . Right panel : Relative expression of stemness factor SOX2 . plots show relative expression from biological triplicate analyses ( mean SE shown ) .

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(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 sorted by CD44 . GAPDH were used as a loading control .

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

O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY 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 after PI3K pathway inhibition . Cultures were serum - starved for 18 h then stimulated with insulin or exposed to PI3K pathway inhibitors wortmannin or LY294002 . transfected with anti - miR - 10b or a scrambled control ( anti - miR - ctl ) were seeded in soft agar at clonal density exposed to insulin or PIK3CA inhibitors . After 3 , colonies were quantified . Bars show mean ± for 3 independent experiments .

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(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 colony numbers in response to insulin or PI3K inactivation . MCF - 7 stable miR - Scr - OE or miR - 10b - OE were seeded in soft agar at clonal density exposed to insulin or PIK3CA inhibitors . After 3 , colonies were quantified . Bars show mean ± for 3 independent experiments .

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(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 Pearson correlation value between gene expression of each module within noted phenotype / disease stage ( x axis ) p values . A value of 1 ( red ) quantifies strongest positive correlation ( genes are upregulated ) , - 1 ( blue ) strongest negative correlation ( genes are downregulated ) , 0 ( ) no correlation .

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

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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 expression of seven genes in human CRPC bone metastases , grouped by their relative of AR - VS , mainly AR - V7 . ( High - of AR - V7 ( top quartile ) or lower of AR - V7 ( quartiles 1 - 3 ) . Data are plotted as mean ± s . e . m . . Non - parametric Mann - Whitney was performed ( two - ) . 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-ORGANISM B-DISEASE I-DISEASE I-DISEASE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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.


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

O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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.


proliferation was examined in CRPC 22Rv1 following depletion of mRNAs for seven genes or shGFP controls , using shRNA against coding region for each gene ( shRNA # 2 ) . number was measured using a non - perturbing restricted dye 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 ( p value < 0 . 0001 , two - ) Dunn ' s multiple comparisons were performed .

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

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


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

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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.


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

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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.