G . Deletion of PUF3 decreases the proliferation of ρ0 cells . Serial dilutions ( tenfold dilution ) of the indicated strains were analyzed on SCD plates at 30 ℃ for 2 days .

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G. Deletion of PUF3 decreases the proliferation of ρ0 cells. Serial dilutions (tenfold dilution) of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.


H . Schematic illustration of the consensus Puf3 - binding motif Lapointe et al . , 2015 ( ) and the motif at Mia40 3 ' - UTR . Mutating the Puf3 - binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 cells . Mia40 was endogenously tagged with FLAG and its 3 ' - UTR was edited as indicated . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . RC : reverse complement . DE : deletion .

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H. Schematic illustration of the consensus Puf3-binding motif Lapointe et al., 2015() and the motif at Mia40 3'-UTR. Mutating the Puf3-binding motif of MIA40 mRNA abolishes Mia40 upregulation in ρ0 cells. Mia40 was endogenously tagged with FLAG and its 3'-UTR was edited as indicated. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. RC: reverse complement. DE: deletion.


I . Mutating the Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 cells . J . Mutating the Puf3 - binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS

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I. Mutating the Puf3-binding motif of MIA40 mRNA decreases ∆ψm of ρ0 cells. J. Mutating the Puf3-binding motif of MIA40 mRNA impairs the proliferation of ρ0 cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis


I . Mutating the Puf3 - binding motif of MIA40 mRNA decreases ∆ ψm of ρ0 cells .

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I. Mutating the Puf3-binding motif of MIA40 mRNA decreases ∆ψm of ρ0 cells.


K . Overexpression of MIA40 rescues ∆ ψm of ρ0 puf3 ∆ cells . Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains . L . Overexpression of MIA40 rescues the proliferation of ρ0 puf3 ∆ cells . Strains were prepared as in ( K ) . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Cells were cultured to mid - log phase , and stained with 125 nM TMRM for FACS

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K. Overexpression of MIA40 rescues ∆ψm of ρ0 puf3∆ cells. Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains. L. Overexpression of MIA40 rescues the proliferation of ρ0 puf3∆ cells. Strains were prepared as in (K). Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Cells were cultured to mid-log phase, and stained with 125 nM TMRM for FACS analysis


K . Overexpression of MIA40 rescues ∆ ψm of ρ0 puf3 ∆ cells . Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains .

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K. Overexpression of MIA40 rescues ∆ψm of ρ0 puf3∆ cells. Single copy plasmid expressing Mia40 driven by the MIA40 promoter was transformed into the indicated strains.


Puf3 is phosphorylated in WT cells and is hyper - phosphorylated in ρ0 cells . Puf3 - FLAG was immunoprecipitated from the indicated strains and treated with / without λ - phosphatase , and analyzed by regular and Phos - tag SDS - PAGE .

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Puf3 is phosphorylated in WT cells and is hyper-phosphorylated in ρ0 cells. Puf3-FLAG was immunoprecipitated from the indicated strains and treated with/without λ-phosphatase, and analyzed by regular and Phos-tag SDS-PAGE.


Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants . WT cells were treated with antimycin ( 10 μM ) , KCN ( 10 μM ) or oligomycin ( 10 μM ) for 3 hours , or shifted from SCD to SCEG medium for 3 hours . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Treatments and strains with Puf3 hyper - phosphorylation was highlighted in red . Glucose depletion was used as a positive control . Asterisk indicates a non - specific band .

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Puf3 phosphorylation in response to OXPHOS inhibitors and in OXPHOS mutants. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Treatments and strains with Puf3 hyper-phosphorylation was highlighted in red. Glucose depletion was used as a positive control. Asterisk indicates a non-specific band.


Puf3 - 15A mutation inhibits Puf3 hyper - phosphorylation in ρ0 cells . A cassette expressing WT or mutant Puf3 - FLAG ( 11A or 15A ) under the control of PUF3 promoter was inserted into the HO site in puf3 ∆ cells . The 15A mutant that blocks Puf3 hyper - phosphorylation is highlighted in red . Puf3 - FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos - tag SDS - PAGE .

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Puf3-15A mutation inhibits Puf3 hyper-phosphorylation in ρ0 cells. A cassette expressing WT or mutant Puf3-FLAG (11A or 15A) under the control of PUF3 promoter was inserted into the HO site in puf3∆ cells. The 15A mutant that blocks Puf3 hyper-phosphorylation is highlighted in red. Puf3-FLAG was immunoprecipitated from the indicated strains and analyzed by regular and Phos-tag SDS-PAGE.


Puf3 - 15A mutation inhibits Puf3 hyper - phosphorylation upon oligomycin treatment and in ∆ V and ρ0 cells . WT cells were treated with antimycin ( 10 μM ) , KCN ( 10 μM ) or oligomycin ( 10 μM ) for 3 hours , or shifted from SCD to SCEG medium for 3 hours . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot . Asterisk indicates a non - specific band .

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Puf3-15A mutation inhibits Puf3 hyper-phosphorylation upon oligomycin treatment and in ∆V and ρ0 cells. WT cells were treated with antimycin (10 μM), KCN (10 μM) or oligomycin (10 μM) for 3 hours, or shifted from SCD to SCEG medium for 3 hours. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot. Asterisk indicates a non-specific band.


Puf3 - 15A mutation downregulates Puf3 - associated mitochondrial transcripts in ρ0 cells . ρ0 and ρ0 puf3 - 15A cells were cultured to log phase . RNAseq was performed with three biological replicates per genotype ( Dataset EV7 ) . All the transcripts and the Puf3 - associated transcripts encoding mitochondrial proteins ( gene lists in Table EV1D ) are shown . The significantly changed transcripts ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed transcripts are indicated .

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Puf3-15A mutation downregulates Puf3-associated mitochondrial transcripts in ρ0 cells. ρ0 and ρ0 puf3-15A cells were cultured to log phase. RNAseq was performed with three biological replicates per genotype (Dataset EV7). All the transcripts and the Puf3-associated transcripts encoding mitochondrial proteins (gene lists in Table EV1D) are shown. The significantly changed transcripts (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed transcripts are indicated.


Puf3 - 15A mutation inhibits the upregulation of Mia40 in ∆ V and ρ0 cells . Mia40 was endogenously tagged with FLAG . The selection markers were removed by the Cre - loxP system . Whole cell lysates were extracted and equal amount of proteins was loaded for western blot .

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Puf3-15A mutation inhibits the upregulation of Mia40 in ∆V and ρ0 cells. Mia40 was endogenously tagged with FLAG. The selection markers were removed by the Cre-loxP system. Whole cell lysates were extracted and equal amount of proteins was loaded for western blot.


Puf3 - 15A mutation decreases ∆ ψm in ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis . The red ] highlight two cell lines for comparison .

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Puf3-15A mutation decreases ∆ψm in ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.


Puf3 - 15A mutation has no effect on the proliferation of WT cells . Puf3 - 15A mutation inhibits the proliferation of ρ0 cells . Mia40 overexpression rescues the proliferation defect of ρ0 puf3 - 15A cells . Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains .

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Puf3-15A mutation has no effect on the proliferation of WT cells.   Puf3-15A mutation inhibits the proliferation of ρ0 cells.   Mia40 overexpression rescues the proliferation defect of ρ0 puf3-15A cells. Single copy plasmid expressing Mia40 driven by MIA40 promoter was transformed into the indicated strains.


Puf3 - 15A mutation has no effect on the proliferation of WT cells .

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Puf3-15A mutation has no effect on the proliferation of WT cells.


Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells , and represses ribosome and Ribi gene expression in ρ0 cells . ρ0 snf1 ∆ cells were prepared by acute EtBr ( 25 µg / ml ) treatment of snf1 ∆ cells in SCD for 2 days . RNAseq was performed with three biological replicates per genotype ( Dataset EV8 ) . All the transcripts and two categories of transcripts ( gene lists in Table EV1B ) are shown . Significantly changed genes ( P value < 0 . 05 ; fold change > 1 . 5 or < 0 . 67 ) are highlighted with dashed lines ; numbers of significantly changed genes are indicated .

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

Snf1 maintains glycolysis and glucose transporter gene expression in both WT and ρ0 cells, and represses ribosome and Ribi gene expression in ρ0 cells. ρ0 snf1∆ cells were prepared by acute EtBr (25 µg/ml) treatment of snf1∆ cells in SCD for 2 days. RNAseq was performed with three biological replicates per genotype (Dataset EV8). All the transcripts and two categories of transcripts (gene lists in Table EV1B) are shown. Significantly changed genes (P value < 0.05; fold change > 1.5 or < 0.67) are highlighted with dashed lines; numbers of significantly changed genes are indicated.


Snf1 represses ribosome biogenesis in WT and ρ0 cells . Polysomes of the indicated strains were fractionated by sucrose gradients ( 10 % - 50 % ) , and the gradients were separated and measured at A254 .

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Snf1 represses ribosome biogenesis in WT and ρ0 cells. Polysomes of the indicated strains were fractionated by sucrose gradients (10%-50%), and the gradients were separated and measured at A254.


Deletion of SNF1 decreases ∆ ψm in both ∆ V and ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis

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Deletion of SNF1 decreases ∆ψm in both ∆V and ρ0 cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis


Deletion of SNF1 decreases ∆ ψm in both ∆ V and ρ0 cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS

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Deletion of SNF1 decreases the proliferation of both ∆ V and ρ0 cells . Serial dilutions of the indicated strains were analyzed on SCD plates at 30 ℃ for 2 days .

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Deletion of SNF1 decreases the proliferation of both ∆V and ρ0 cells. Serial dilutions of the indicated strains were analyzed on SCD plates at 30℃ for 2 days.


Deletion of MIG1 in ρ0 snf1 ∆ cells rescues ∆ ψm . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis

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Deletion of MIG1 in ρ0 snf1∆ cells rescues ∆ψm. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis


Deletion of MIG1 in ρ0 snf1 ∆ cells rescues ∆ ψm . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS

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Deletion of MIG1 in ρ0 snf1 ∆ cells partially rescues cell proliferation . A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains . Serial dilutions ( tenfold dilution ) were analyzed on SCD plates at 30 ℃ for 2 days . V : empty vector .

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Deletion of MIG1 in ρ0 snf1∆ cells partially rescues cell proliferation. A plasmid overexpressing MIG1 under the control of MIG1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.


Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1 ∆ cells fully rescues cell proliferation . A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains . Serial dilutions ( tenfold dilution ) were analyzed on SCD plates at 30 ℃ for 2 days . V : empty vector .

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Combining MIG1 deletion and NOG2 overexpression in ρ0 snf1∆ cells fully rescues cell proliferation. A plasmid overexpressing NOG2 under the control of TEF1 promoter was transformed into the indicated strains. Serial dilutions (tenfold dilution) were analyzed on SCD plates at 30℃ for 2 days. V: empty vector.


Heat map plots of the mRNA level changes of chaperones , ubiquitin - proteasome pathway , and autophagy genes ( gene list in Table EV1F ) in the indicated strains ( data from Dataset EV1 and Dataset EV10 ) . Selected genes for growth analysis in B are highlighted in red .

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Heat map plots of the mRNA level changes of chaperones, ubiquitin-proteasome pathway, and autophagy genes (gene list in Table EV1F) in the indicated strains (data from Dataset EV1 and Dataset EV10). Selected genes for growth analysis in B are highlighted in red.


Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3 ∆ cells . Cells were cultured from 0 . 05 OD in SCD , and OD600 was measured every 2 hours . Data are mean ± SD from three biological replicates .

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Deletion of HSC82 selectively impairs the proliferation of ρ0 puf3∆ cells. Cells were cultured from 0.05 OD in SCD, and OD600 was measured every 2 hours. Data are mean ± SD from three biological replicates.


Deletion of HSC82 selectively decreases ∆ ψm in ρ0 puf3 ∆ cells . Cells were cultured to mid - log phase and stained with 125 nM TMRM for FACS analysis . The red ] highlight two cell lines for comparison .

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Deletion of HSC82 selectively decreases ∆ψm in ρ0 puf3∆ cells. Cells were cultured to mid-log phase and stained with 125 nM TMRM for FACS analysis. The red ] highlight two cell lines for comparison.


A , B cRNA or water as a control was injected into oocytes and uptake was assessed two days after injection for 14h in N free MBS . Data information : biological replicates : n = 5 with 2 oocytes in each sample . Asterisks denote significant difference : * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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A, B cRNA or water as a control was injected into oocytes and uptake was assessed two days after injection for 14h in N free MBS. Data information: biological replicates: n = 5 with 2 oocytes in each sample. Asterisks denote significant difference: **P<0.01, *P<0.05 (Student's t-test).


A , B The oocytes were exposed to different concentrations of NO3 - in N free MBS for 1 . 5 h at pH5 . 5 . Net NO3 - accumulation was fitted with both Michaelis - Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity . Values are the mean ± SE ( biological replicates : n = 3 ) with 3 - 4 oocytes in each sample .

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A, B The oocytes were exposed to different concentrations of NO3- in N free MBS for 1.5 h at pH5.5. Net NO3- accumulation was fitted with both Michaelis-Menten and Linear Regression in A for high affinity range and Linear Regression in B for low affinity. Values are the mean ± SE (biological replicates: n=3) with 3-4 oocytes in each sample.


A Histochemical stained roots from composite plants transformed with proMtNPF6 . 5 : GUS ( left column ) , proMtNPF6 . 7 : GUS ( middle column ) , proMtNPF6 . 6 : GUS ( right column ) .

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A Histochemical stained roots from composite plants transformed with proMtNPF6.5:GUS (left column), proMtNPF6.7:GUS (middle column), proMtNPF6.6:GUS (right column).


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 .

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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 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 =

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

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 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 =

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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 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 .

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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 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 ) ±

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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 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

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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 - 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 .

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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 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 .

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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 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 ) .

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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 length , 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 - 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 .

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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 - test ; biological replicates : n = 3 ( 3 pooled root organs in each replicate ) ± SEM .

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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 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 ) .

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 complex with several Hippo PY motif - containing proteins . 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 ) 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 .

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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 ) 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 .

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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 and LATS activation . Western blotting was performed with the 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 nuclear 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 - test ) .

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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 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 .

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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 . 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 .

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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 protein complex with α - catenin and a group of Hippo pathway proteins .

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(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 .

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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 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 .

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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 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 .

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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 and AMOT . The identified missense mutations respectively disrupted the STXBP4 - α - catenin ( G ) and STXBP4 - AMOT ( H ) complex 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 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 .

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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 ) 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 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 .

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

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(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 .

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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 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 .

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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 , 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 .

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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 ± SEM ; 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 ± 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 ) .

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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 ± 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 ± SEM ; 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 , and SOD2 ( data are means ± SEM ; 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 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 .

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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 the fat body of Drosophila larvae ( data are means ± SEM ; 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 cells ( TK - Gut - Gal4 > UAS - dsDat ) on survival of Drosophila larvae ( data are means ± SEM ; 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 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 .

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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 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 .

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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 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 ) ] .

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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 cells after adding 100 µM NAT ( data are means ± SEM ; 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 ± 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 .

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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 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 B-SMALL_MOLECULE O B-EXP_ASSAY O B-CELL_LINE B-DISEASE I-DISEASE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O B-ORGANISM 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 .

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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 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 .

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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 and Ift88 − / − KECs by mCherry - GFP - LC3 puncta quantification . Puncta : 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 quantification 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 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 ) .

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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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 puncta quantification in WT and 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 quantification ( 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 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 .

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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 ) 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 .

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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 .

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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 ) .

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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 .

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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 .

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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 ) .

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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 and ATG16L in the same fractions isolated from Ctrl , IFT20 − and IFT88 − MEFs in the presence ( top ) and 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 ) 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 .

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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 ) .

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

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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 ) .

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


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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 ) .

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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 .

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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 .

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(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 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 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 B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) 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 .

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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 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 .

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(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 .

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(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 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 O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

(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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-ORGANISM 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 B-ORGANISM O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-CELL_LINE 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

(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.