Figure 5D ) VPS34 CI components expressed with Ulk1 1 - 427 WT or KI as indicated . After starvation for 1 hr in the presence or absence of MRT68921 ( ULK inhibitor ) , cells were lysed and VPS34 CI coimmunoprecipitated via ATG14 - ZZ . VPS15 S861 phosphorylation was increased in the presence of active Ulk1 . VPS15 pS861 / total VPS15 was quantified , mean + / - SEM , n = 3 . E ) HEK293A were transfected with VPS34 CII components and Ulk1 1 - 427 WT or KI and treated as indicated before VPS34 CII co - immunopurification via UVRAG - ZZ . Samples were analysed by Western blot as in D , revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII . Quantifications show mean of 4 independent experiments + / - SEM , * * * p < 0 . 001 [ one - tailed ANOVA ] .

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Figure 5D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3.E) HEK293A were transfected with VPS34 CII components and Ulk1 1-427 WT or KI and treated as indicated before VPS34 CII co-immunopurification via UVRAG-ZZ. Samples were analysed by Western blot as in D, revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII. Quantifications show mean of 4 independent experiments +/-SEM, ***p<0.001 [one-tailed ANOVA].


Figure 6A ) HEK293A , VPS15 KO control and stably rescued VPS15 KOs ( sgA - B3 ) were starved for 1 hr before WIPI2 ( green ) , LC3 ( red ) and DNA ( Hoechst , blue ) were visualised . Quantification of WIPI2 puncta number per cell , mean + / - SEM , n = 5 . B ) Stably rescued VPS15 KO ( sgA - B3 ) , HEK293A and VPS15 KO control cells were starved for 1 hr with ( SB ) or without ( St ) 100nM Bafilomycin A1 , or cultured in full media ( F ) . Quantification shows LC3II / Actin , mean + / - SEM , n = 5 .

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Figure 6A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5.B) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5.


Figure 7A ) HEK293A were treated with VPS34 - IN1 ( 1μM ) alone or in the presence of MRT68921 ( 1μM ) for the indicated time . Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot . Identical samples were loaded in parallel for VPS34 , ATG13 , PRKAB2 analysis , indicated by dashed line . E ) HEK293A and VPS15 KO cells were fed , or starved for 2 hrs before fixation , and labelling with ULK1 ( green ) , ATG9A ( red ) and GM130 ( blue ) . Dashed boxes show magnified regions of interest .

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Figure 7A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line.E) HEK293A and VPS15 KO cells were fed, or starved for 2 hrs before fixation, and labelling with ULK1 (green), ATG9A (red) and GM130 (blue). Dashed boxes show magnified regions of interest.


Figure 8B ) HEK293A were transfected with siRNAs targeting ATG14 , UVRAG or p62 , or with non - targeting siRNA ( RISC - free , RF ) and cells were treated with VPS34 - IN1 ( IN1 ; 1μM ) for 0 , 4 or 18 hrs as indicated . Quantification of ATG13 pS318 / Total ATG13 , mean + / - SEM , n = 3 . Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis , indicated by dashed line . C ) Cells transfected as in B were treated with IN1 for 18 hours as indicated , fixed and labelled for ubiquitin ( green ) , FIP200 ( red ) and p62 ( blue ) . FIP200 - positive bodies were quantified from 4 independent experiments with mean + / - SEM plotted . Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested .

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Figure 8B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line.C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.


Figure 1Plasma corticosterone levels at baseline , 30 minutes after induction of stress by placing mice in a clean cage , and after 90 minutes upon return in the home cage . DEX mice received an injection of dexamethasone at 0 . 1 mg / kg body weight 150 minutes before collecting the first blood sample ( n = 5 - 6 animals per group ) . Flox and GRBATKO mice were injected with 0 . 02 mg / kg dexamethasone and the procedure described in ( A ) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in ( A ) ( n = 6 - 7 animals per group ) . GR mRNA levels in BAT of Flox and GRBATKO mice . Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage . Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure , controls received an injection with saline ( n = 5 - 6 animals per group ) . Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice ( n = 5 - 6 animals per group ) . Blood glucose levels ( n = 5 - 6 animals per group ) .

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Figure 1Plasma corticosterone levels at baseline, 30 minutes after induction of stress by placing mice in a clean cage, and after 90 minutes upon return in the home cage. DEX mice received an injection of dexamethasone at 0.1 mg/kg body weight 150 minutes before collecting the first blood sample (n = 5-6 animals per group). Flox and GRBATKO mice were injected with 0.02 mg/kg dexamethasone and the procedure described in (A) was followed to induce stress in order to detect potential differences between Flox and GRBATKO mice which could have been obscured by the higher concentration used in (A) (n = 6-7 animals per group).GR mRNA levels in BAT of Flox and GRBATKO mice. Organs were harvested in the stressed condition after placing mice for 30 minutes in a clean cage. Half of the animals received a dexamethasone injection 180 minutes before onset of the procedure, controls received an injection with saline (n = 5-6 animals per group).Serum corticosterone levels under stressed conditions in Flox and GRBATKO mice (n = 5-6 animals per group). Blood glucose levels (n = 5-6 animals per group).


Figure 2Knockdown of GR in BAT assessed by immunoblotting . Β - Tubulin was used as loading control . Quantification of immunoblot in ( A ) using Image Lab ( n = 6 animals per group ) . Body temperature over the course of the cold exposure , determined daily ( n = 6 animals per group ) . Body weight at the end of the cold exposure ( n = 6 animals per group ) . BAT weight at the end of the cold exposure ( n = 6 animals per group ) . Representative Hematoxilin and eosin staining image of BAT of cold - exposed Flox and GRBATKO mice . Scale bar 50 µm . Triglyceride content in BAT from Flox and GRBATKO animals ( n = 5 - 6 animals per group ) . Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals , indicating unchanged thermogenic marker gene expression . Color gradient ranges from 2 - fold higher expression in GRBATKO over Flox controls ( red ) to 0 . 1 - fold ( lower ) expression ( blue ) ( n = 4 animals per group ) . Volcano plot from microarray gene expression data as in ( I ) , with cut - off values for the adjusted p - value < 0 . 1 and fold change > 1 . 5 . Serum corticosterone levels ( n = 6 animals per group ) .

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Figure 2Knockdown of GR in BAT assessed by immunoblotting. Β-Tubulin was used as loading control. Quantification of immunoblot in (A) using Image Lab (n = 6 animals per group).Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group).Body weight at the end of the cold exposure (n = 6 animals per group).BAT weight at the end of the cold exposure (n = 6 animals per group).Representative Hematoxilin and eosin staining image of BAT of cold-exposed Flox and GRBATKO mice. Scale bar 50 µm.Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).Heatmap of mean gene expression fold changes in thermogenic marker genes based on BAT microarray analysis comparing GRBATKO vs Flox control animals, indicating unchanged thermogenic marker gene expression. Color gradient ranges from 2-fold higher expression in GRBATKO over Flox controls (red) to 0.1-fold (lower) expression (blue) (n = 4 animals per group).Volcano plot from microarray gene expression data as in (I), with cut-off values for the adjusted p-value <0.1 and fold change >1.5.Serum corticosterone levels (n = 6 animals per group).


Figure 3Body weight after 16 h fasting and 6 h chow refeeding at 22 ° C ( n = 6 animals per group ) Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h ( n = 6 animals per group ) . Blood glucose ( n = 6 per group ) . Serum insulin and corticosterone levels determined by ELISA ( n = 6 animals per group ) . Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding ( n = 6 animals per group ) .

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Figure 3Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group)Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group).Blood glucose (n = 6 per group).Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).


Figure 4Body weight gain over a 24 - week period of high - fat feeding ( n = 13 - 14 animals per group ) . Panel shows experimental design for Figures 4 - 6 . Oxygen consumption over a 90 - hour period . Grays columns indicate dark phase ( n = 6 - 8 mice per group ) . Average oxygen consumption specified for light phase , dark phase , and total ( n = 6 - 8 animals per genotype ) . Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight ( n = 6 - 8 animals per group ) . Locomotor activity over a 90 - hour period ( n = 6 - 8 animals per group ) .

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Figure 4Body weight gain over a 24-week period of high-fat feeding (n = 13-14 animals per group). Panel shows experimental design for Figures 4-6.Oxygen consumption over a 90-hour period. Grays columns indicate dark phase (n = 6-8 mice per group). Average oxygen consumption specified for light phase, dark phase, and total (n = 6-8 animals per genotype).Total oxygen consumption plotted against body weight indicating that lower energy expenditure in GRBATKO animals is independent of body weight (n = 6-8 animals per group).Locomotor activity over a 90-hour period (n = 6-8 animals per group).


Figure 5Intraperitoneal GTT after 19 weeks of HFD - feeding , glucose injections at 1 . 5 mg per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve calculated from ( A ) ( n = 11 - 13 animals per group ) . Plasma insulin levels during GTT ( n = 11 - 13 animals per group ) . Intraperitoneal ITT after 20 weeks of high - fat diet intervention . Insulin was adjusted to 1 mU per g of body weight ( n = 11 - 13 animals per group ) . Area under the curve determined from ( D ) ( n = 11 - 13 animals per group ) . Plasma insulin levels after a 5 - hour fast , before injecting insulin ( n = 11 - 13 animals per group ) .

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Figure 5Intraperitoneal GTT after 19 weeks of HFD-feeding, glucose injections at 1.5 mg per g of body weight (n = 11-13 animals per group). Area under the curve calculated from (A) (n = 11-13 animals per group).Plasma insulin levels during GTT (n = 11-13 animals per group).Intraperitoneal ITT after 20 weeks of high-fat diet intervention. Insulin was adjusted to 1 mU per g of body weight (n = 11-13 animals per group). Area under the curve determined from (D) (n = 11-13 animals per group).Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).


Figure 6Body weight in Flox and GRBATKO after 24 weeks of high - fat feeding ( n = 10 - 13 animals per group ) . BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks ( n = 10 - 13 animals per group ) . GR levels in BAT after 24 weeks high - fat feeding ( n = 10 - 13 mice per group ) . Representative H & E stained sections of BAT from Flox and GRBATKO animals . Scale bar 100 µm . Triglyceride content in BAT per gram of wet tissue ( n = 10 - 13 animals per group ) . qRT - PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high - fat feeding ( n = 9 - 13 animals per group ) . Representative immunohistochemistry for UCP1 in BAT . Scale bar 100 µm . Serum insulin levels determined by ELISA ( n = 10 - 13 animals per group ) . Blood glucose in Flox and GRBATKO animals after 24 weeks HFD ( n = 10 - 13 animals per group ) .

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Figure 6Body weight in Flox and GRBATKO after 24 weeks of high-fat feeding (n = 10-13 animals per group).BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks (n = 10-13 animals per group).GR levels in BAT after 24 weeks high-fat feeding (n=10-13 mice per group).Representative H&E stained sections of BAT from Flox and GRBATKO animals. Scale bar 100 µm.Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).qRT-PCR analysis of thermogenic marker gene expression in BAT of Flox and GRBATKO mice after high-fat feeding (n = 9-13 animals per group).Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.Serum insulin levels determined by ELISA (n = 10-13 animals per group).Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).


Figure 1B - Congo red plates with colonies of E . coli strain MC4100 relA + and its target mutation csgD : M4 - ompR : mut2 derivative , harboring sRNA - producing plasmids pOmrA , pOmrB , or an empty vector ( pControl ) . Incubation at 28 ° C for 48 h .

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Figure 1B - Congo red plates with colonies of E. coli strain MC4100 relA+ and its target mutation csgD:M4-ompR:mut2 derivative, harboring sRNA-producing plasmids pOmrA, pOmrB, or an empty vector (pControl). Incubation at 28°C for 48 h.


Figure 2B - Relative fluorescence / OD600 of cells of E . coli strain MC4100 relA + ∆ omrAB or MC4100 relA + ∆ omrAB ∆ hfq harboring the translational fusion plasmid pDgcM : : GFP in combination with plasmids expressing OmrA , OmrB , or an empty control vector , after 16 h of growth at 37 ° C . Error bars SD , N = 3 . Average fluorescence intensities ( a . u . ) / OD600 used to calculate ratios Note that absolute dgcM : : gfp expression values are ≈ 3 - 4 - fold lower in the ∆ hfq strainC - Western blot analysis of cell lysates from strains MC4100 relA + ∆ omrAB harboring plasmids pOmrA , pOmrB , or mutant variants as indicated , in combination with a plasmid expressing C - terminal FLAG - tagged dgcM or dgcM _ M2 . As a loading control , part of a protein - stained gel is shown below .

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Figure 2B - Relative fluorescence/OD600 of cells of E. coli strain MC4100 relA+ ∆omrAB or MC4100 relA+ ∆omrAB ∆hfq harboring the translational fusion plasmid pDgcM::GFP in combination with plasmids expressing OmrA, OmrB, or an empty control vector, after 16 h of growth at 37°C. Error bars SD, N=3. Average fluorescence intensities (a.u.)/ OD600 used to calculate ratios Note that absolute dgcM::gfp expression values are ≈3-4-fold lower in the ∆hfq strainC - Western blot analysis of cell lysates from strains MC4100 relA+ ∆omrAB harboring plasmids pOmrA, pOmrB, or mutant variants as indicated, in combination with a plasmid expressing C-terminal FLAG-tagged dgcM or dgcM_M2. As a loading control, part of a protein-stained gel is shown below.


Figure 3A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA ( 210 nt from + 1 ) by OmrA and OmrB . Inclusion of 30S ribosomes , tRNAfmet , OmrA , and OmrB is indicated . The unrelated IstR - 1 sRNA is a negative control . The 30S / tRNAfmet - dependent reverse transcription block ( toeprint ) , is indicated by an arrow . Sequencing reactions were loaded next to the primer extension products ( UCGA ) . The AUG start codon is indicated by asterisks . B - In vitro translation of dgcM : : 3xflag mRNA in the presence or absence of OmrA / OmrB , with or without purified Hfq protein , was carried out As an internal control , the non - target ompA : : 3xflag mRNA was included . The Western blot shows DgcM - 3xFLAG and OmpA - 3xFLAG protein levels .

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Figure 3A - Toeprinting showing inhibition of initiation complex formation on a truncated dgcM mRNA (210 nt from +1) by OmrA and OmrB. Inclusion of 30S ribosomes, tRNAfmet, OmrA, and OmrB is indicated. The unrelated IstR-1 sRNA is a negative control. The 30S/ tRNAfmet-dependent reverse transcription block (toeprint), is indicated by an arrow. Sequencing reactions were loaded next to the primer extension products (UCGA). The AUG start codon is indicated by asterisks.B - In vitro translation of dgcM::3xflag mRNA in the presence or absence of OmrA/ OmrB, with or without purified Hfq protein, was carried out As an internal control, the non-target ompA::3xflag mRNA was included. The Western blot shows DgcM-3xFLAG and OmpA-3xFLAG protein levels.


Figure 4A - 5 ' end - labeled dgcM mRNA ( 10 nM ) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram . When added , Hfq hexamers were at 50 nM , the sRNAs OmrA and OmrB at 250 nM . The accessibility of each nucleotide was probed with water ( lanes 1 - 4 , negative control for RNA integrity ) , RNase T1 , or lead ( II ) acetate . Lane T1 : RNase T1 under denaturing conditions . Lane OH : alkaline ladders . Binding sites of Hfq ( green arrows and boxes ) , and site 1 ( red arrows and boxes ) and 2 ( blue arrows and boxes ) of the sRNAs , are highlighted . Note that site 2 is bound differently by OmrA and OmrB ( blue boxes on gel ) . Black boxes indicate Hfq - dependent T1 cleavage enhancements .

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Figure 4A - 5' end-labeled dgcM mRNA (10 nM) was subjected to probing All components added to the labeled RNA are indicated above the autoradiogram. When added, Hfq hexamers were at 50 nM, the sRNAs OmrA and OmrB at 250 nM. The accessibility of each nucleotide was probed with water (lanes 1-4, negative control for RNA integrity), RNase T1, or lead(II) acetate. Lane T1: RNase T1 under denaturing conditions. Lane OH: alkaline ladders. Binding sites of Hfq (green arrows and boxes), and site 1 (red arrows and boxes) and 2 (blue arrows and boxes) of the sRNAs, are highlighted. Note that site 2 is bound differently by OmrA and OmrB (blue boxes on gel). Black boxes indicate Hfq-dependent T1 cleavage enhancements.


Figure 5Mobility shift assay with end - labeled dgcM RNA ( 210 nt species from + 1 ) in the presence or absence of unlabeled OmrA or OmrB . When indicated , single - stranded DNA oligos were pre - annealed to site 1 ( dgcM _ site1 ) , site 2 ( dgcM _ site2 ) , or both , before addition of sRNAs . The additions for each sample are indicated above the Figure , and the identities of the nucleic acid complexes are indicated on the left for guidance .

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Figure 5Mobility shift assay with end-labeled dgcM RNA (210 nt species from +1) in the presence or absence of unlabeled OmrA or OmrB. When indicated, single-stranded DNA oligos were pre-annealed to site 1 (dgcM_site1), site 2 (dgcM_site2), or both, before addition of sRNAs. The additions for each sample are indicated above the Figure, and the identities of the nucleic acid complexes are indicated on the left for guidance.


Figure 6Western blots showing DgcM - 3xFLAG translated in vitro in the presence or absence of OmrA or OmrB . A - The inclusion of purified wild - type or mutant Hfq proteins is indicated , and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set . Western blots showing DgcM - 3xFLAG translated in vitro in the presence or absence of OmrA or OmrB . B - An ssDNA - oligo ( Hfq _ BS that base - pairs specifically to the Hfq binding site on dgcM mRNA ( nt 42 - 66 ) was preannealed to the mRNA prior to OmrA / B addition and translation . OmpA - 3xFLAG was included as internal control . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set .

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

Figure 6Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. A - The inclusion of purified wild-type or mutant Hfq proteins is indicated, and the translation assay was conducted The different Hfq interaction site mutant proteins are indicated. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.Western blots showing DgcM-3xFLAG translated in vitro in the presence or absence of OmrA or OmrB. B - An ssDNA-oligo (Hfq_BS that base-pairs specifically to the Hfq binding site on dgcM mRNA (nt 42-66) was preannealed to the mRNA prior to OmrA/B addition and translation. OmpA-3xFLAG was included as internal control. Data information: Quantification of DgcM translation rates, relative to that of the unaffected OmpA control, is shown below the gels. The ratio of DgcM/ OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations (-sRNA, +OmrA, +OmrB), indicated by "1.00*". Effects due to OmrA/ OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set.


Figure 7A - Histograms show flow cytometry analysis of exponentially growing cells of E . coli strain MC4100 relA - , and its mutant derivatives hfq - F42A ( hfq - prox ) , hfq - Y25D ( hfq - dist ) , hfq - R16S _ R17A _ R19A _ K47E ( hfq - rim ) , ∆ hfq , harboring the translational fusion plasmid pDgcM : : GFP in combination with pOmrA , pOmrB , or a control vector . Data was obtained on a MACSQuant VYB instrument . B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry , probed for OmrA ( strains containing pOmrA ) or OmrB ( strains containing pOmrB ) . 5S rRNA was used as loading control .

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

Figure 7A - Histograms show flow cytometry analysis of exponentially growing cells of E. coli strain MC4100 relA-, and its mutant derivatives hfq-F42A (hfq-prox), hfq-Y25D (hfq-dist), hfq-R16S_R17A_R19A_K47E (hfq-rim), ∆hfq, harboring the translational fusion plasmid pDgcM::GFP in combination with pOmrA, pOmrB, or a control vector. Data was obtained on a MACSQuant VYB instrument.B - Northern blot analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry, probed for OmrA (strains containing pOmrA) or OmrB (strains containing pOmrB). 5S rRNA was used as loading control.


Figure 1LGG - 1 : : GFP - positive puncta labeling autophagic membranes [ 22 ] were counted in wild - type or in food - limited animals . ( A ) Micrographs of eat - 2 ( ad1116 ) L3 larvae expressing GFP - tagged lgg - 1 / LC3 . Arrow indicates autophagic focus . Magnification is indicated . ( B ) Average number of LGG - 1 : : GFP - containing puncta in eat - 2 ( ad1116 ) mutants and N2 wild - type animals ( WT ) , p < 0 . 0001 . ( C ) Average number of LGG - 1 : : GFP - containing puncta in N2 wild - type , food - restricted animals grown in liquid media ( WT , food limited ) and N2 wild - type animals grown in liquid with a higher concentration of bacteria ( WT , fully fed ) , p < 0 . 0001 ; see Methods . Between three and ten seam cells were counted in each of 20 - 40 animals using high - power microscopy and averaged . n , total number of seam cells observed . Error bars : ± SEM . p - Values were calculated as unpaired , two - tailed t - test . Animals were raised at 20 ° C . Please see Table S1 for quantification of all data .

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

Figure 1LGG-1::GFP-positive puncta labeling autophagic membranes [22] were counted in wild-type or in food-limited animals.(A) Micrographs of eat-2(ad1116) L3 larvae expressing GFP-tagged lgg-1/LC3. Arrow indicates autophagic focus. Magnification is indicated.(B) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) mutants and N2 wild-type animals (WT), p < 0.0001.(C) Average number of LGG-1::GFP-containing puncta in N2 wild-type, food-restricted animals grown in liquid media (WT, food limited) and N2 wild-type animals grown in liquid with a higher concentration of bacteria (WT, fully fed), p < 0.0001; see Methods.Between three and ten seam cells were counted in each of 20-40 animals using high-power microscopy and averaged. n, total number of seam cells observed. Error bars: ±SEM. p-Values were calculated as unpaired, two-tailed t-test. Animals were raised at 20 °C. Please see Table S1 for quantification of all data.


Figure 2 ( A ) Survival curves of eat - 2 ( ad1116 ) animals fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan was 23 . 7 d for control and 19 . 6 d for bec - 1 RNAi , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed a total of six times , and bec - 1 RNAi shortened the lifespan of eat - 2 animals ∼ 15 % - 30 % . Please see Table 1 for additional data . ( B ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . These assays were performed concurrently with the eat - 2 mutant lifespan analysis in Figure 2A . Mean lifespan was 17 . 3 d for control and 18 . 9 d for bec - 1 RNAi . p = 0 . 045 , Log - rank ( Mantel - Cox ) test . Depletion of bec - 1 did not significantly change the lifespan of N2 or sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals in any of six experiments . Please see Table 1 for additional data . ( C ) Survival curves of eat - 2 ( ad1116 ) animals fed either control bacteria or bacteria expressing vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 6 d for control and 22 . 8 d for vps - 34 RNAi , p = 0 . 0003 , Log - rank ( Mantel - Cox ) test . This experiment was performed a total of four times . Please see Table 1 for additional data . ( D ) Survival curves of sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals ( WT ) fed either control bacteria or bacteria expressing vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the eat - 2 lifespan analysis shown in Figure 2C . Mean lifespan was 21 . 5 d for control and 23 . 3 d for bec - 1 RNAi . p = 0 . 14 , Log - rank ( Mantel - Cox ) test . Depletion of vps - 34 did not significantly change the lifespan of N2 or sterile fer - 15 ( b26 ) ; fem - 1 ( hc17 ) animals in each of six different experiments . Please see Table 1 for additional data .

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

Figure 2(A) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan was 23.7 d for control and 19.6 d for bec-1 RNAi, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed a total of six times, and bec-1 RNAi shortened the lifespan of eat-2 animals ∼15%-30%. Please see Table 1 for additional data.(B) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. These assays were performed concurrently with the eat-2 mutant lifespan analysis in Figure 2A. Mean lifespan was 17.3 d for control and 18.9 d for bec-1 RNAi. p = 0.045, Log-rank (Mantel-Cox) test. Depletion of bec-1 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in any of six experiments. Please see Table 1 for additional data.(C) Survival curves of eat-2(ad1116) animals fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.6 d for control and 22.8 d for vps-34 RNAi, p = 0.0003, Log-rank (Mantel-Cox) test. This experiment was performed a total of four times. Please see Table 1 for additional data.(D) Survival curves of sterile fer-15(b26); fem-1(hc17) animals (WT) fed either control bacteria or bacteria expressing vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the eat-2 lifespan analysis shown in Figure 2C. Mean lifespan was 21.5 d for control and 23.3 d for bec-1 RNAi. p = 0.14, Log-rank (Mantel-Cox) test. Depletion of vps-34 did not significantly change the lifespan of N2 or sterile fer-15(b26); fem-1(hc17) animals in each of six different experiments. Please see Table 1 for additional data.


Figure 3 ( A ) Average number of LGG - 1 : : GFP - containing puncta in eat - 2 ( ad1116 ) progeny of animals fed either control bacteria or bacteria expressing bec - 1 , vps - 34 or pha - 4 dsRNA their entire lives . p ≤ 0 . 0001 for bec - 1 , vps - 34 and pha - 4 RNAi treatments compared to control , respectively , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . ( B ) Average number of LGG - 1 : : GFP - containing puncta in rab - 10 ( ok1494 ) progeny of animals fed either control bacteria or bacteria expressing pha - 4 dsRNA their entire lives ; p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . Please see Figure 1 for details and Table S1 for quantification of data . Feeding mutants for several generations with pha - 4 dsRNA , inhibited development and sharply decreased the number of eggs laid ( [ 46 ] , data not shown ) . However , the loss of many embryos was unlikely to bias our findings , since many progeny of daf - 2 mutants treated with pha - 4 RNAi also died but those that did not exhibited a robust autophagy phenotype ( Figure 6 ) .

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

Figure 3(A) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34 or pha-4 dsRNA their entire lives. p ≤ 0.0001 for bec-1, vps-34 and pha-4 RNAi treatments compared to control, respectively, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details.(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) progeny of animals fed either control bacteria or bacteria expressing pha-4 dsRNA their entire lives; p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details and Table S1 for quantification of data. Feeding mutants for several generations with pha-4 dsRNA, inhibited development and sharply decreased the number of eggs laid ([46], data not shown). However, the loss of many embryos was unlikely to bias our findings, since many progeny of daf-2 mutants treated with pha-4 RNAi also died but those that did not exhibited a robust autophagy phenotype (Figure 6).


Figure 4 ( A ) Average number of LGG - 1 : : GFP - containing puncta in let - 363 / TOR RNAi - arrested animals compared to N2 wild - type animals ( WT ) grown on control ( vector - only ) bacteria , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . We were not able to detect increased LGG - 1 : : GFP puncta in long - lived let - 363 ( RNAi ) adults ; however , one generation of daf - 2 RNAi , our positive control , did not significantly increase the number of foci in adults either ( data not shown ) . Please see Table S1 for quantification of all data . ( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + ) compared to N2 wild - type animals ( WT ) , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Please see Table S1 for quantification of all data . daf - 15 encodes the TOR - binding partner Raptor . ( C ) Survival curves of daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + , strain DR412 ) fed either control bacteria or bacteria expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan : daf - 15 / + animals grown on control RNAi - bacteria : 25 . 1 d , daf - 15 / + animals on bec - 1 RNAi : 20 . 8 d , p = 0 . 0008 , Log - rank ( Mantel - Cox ) test . The lifespan of daf - 15 / + animals grown on bec - 1 RNAi - bacteria during adulthood was measured again , yielding similar results ; the lifespan of daf - 15 / + animals was also measured three times following whole - life RNAi exposure . In these experiments , bec - 1 RNAi generally shortened the mean lifespan of daf - 15 / + animals to a greater extent than it shortened the lifespan of wild type . ( We also attempted to perform double - RNAi experiments , in which animals were cultured on a 50 : 50 mixture of let - 363 / TOR and bec - 1 [ or control ] RNAi bacteria . Although the trends we saw were in the expected direction , the effects produced by half - strength RNAi were small and not statistically significant [ data not shown ] . ) Please see Table 2 for additional data . ( D ) Survival curves of wild - type animals derived from strain DR412 ( WT ) fed either control bacteria or bacteria expressing bec - 1 dsRNA throughout their whole life at 20 ° C . These assays were performed concurrently with the daf - 15 / + lifespan analysis shown in Figure 4C . WT grown on control RNAi - bacteria : 21 . 4 d , WT on bec - 1 RNAi - bacteria : 21 . 1 d ( p = 0 . 34 ) , p between daf - 15 / + and WT grown on control RNAi - bacteria , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . Please see Table 2 for additional data .

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

Figure 4(A) Average number of LGG-1::GFP-containing puncta in let-363/TOR RNAi-arrested animals compared to N2 wild-type animals (WT) grown on control (vector-only) bacteria, p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. We were not able to detect increased LGG-1::GFP puncta in long-lived let-363(RNAi) adults; however, one generation of daf-2 RNAi, our positive control, did not significantly increase the number of foci in adults either (data not shown). Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+) compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data. daf-15 encodes the TOR-binding partner Raptor.(C) Survival curves of daf-15(m81)/unc-24(e138) heterozygotes (daf-15/+, strain DR412) fed either control bacteria or bacteria expressing bec-1 dsRNA during adulthood at 20 °C. Mean lifespan: daf-15/+ animals grown on control RNAi-bacteria: 25.1 d, daf-15/+animals on bec-1 RNAi: 20.8 d, p = 0.0008, Log-rank (Mantel-Cox) test. The lifespan of daf-15/+ animals grown on bec-1 RNAi-bacteria during adulthood was measured again, yielding similar results; the lifespan of daf-15/+ animals was also measured three times following whole-life RNAi exposure. In these experiments, bec-1 RNAi generally shortened the mean lifespan of daf-15/+ animals to a greater extent than it shortened the lifespan of wild type. (We also attempted to perform double-RNAi experiments, in which animals were cultured on a 50:50 mixture of let-363/TOR and bec-1 [or control] RNAi bacteria. Although the trends we saw were in the expected direction, the effects produced by half-strength RNAi were small and not statistically significant [data not shown].) Please see Table 2 for additional data.(D) Survival curves of wild-type animals derived from strain DR412 (WT) fed either control bacteria or bacteria expressing bec-1 dsRNA throughout their whole life at 20 °C. These assays were performed concurrently with the daf-15/+ lifespan analysis shown in Figure 4C. WT grown on control RNAi-bacteria: 21.4 d, WT on bec-1 RNAi-bacteria: 21.1 d (p = 0.34), p between daf-15/+ and WT grown on control RNAi-bacteria, p < 0.0001, Log-rank (Mantel-Cox) test. Please see Table 2 for additional data.


Figure 5 ( A ) Average number of LGG - 1 : : GFP - containing puncta in N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing daf - 2 ( as a control ) or rab - 10 dsRNA for two generations . p < 0 . 0001 for either daf - 2 or rab - 10 RNAi treatment compared to control RNAi treatment , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Please see Table S1 for quantification of all data . ( B ) Average number of LGG - 1 : : GFP - containing puncta in rab - 10 ( ok1494 ) mutants compared to N2 wild - type animals ( WT ) , p < 0 . 0001 , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . Please see Figure 1 for details . Please see Table S1 for quantification of all data . ( C ) Survival curves of rab - 10 ( ok1494 ) animals fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 9 d for control , 19 . 9 d for bec - 1 RNAi , and 22 . 1 d for vps - 34 RNAi , all pair - wise comparisons to control , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed two times . Please see Table 3 for additional data . ( D ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the rab - 10 lifespan analysis shown in Figure 5C . Mean lifespan was 22 . 9 d for control , 23 . 6 d for bec - 1 RNAi , and 24 . 5 d for vps - 34 RNAi , pair - wise comparison to control , p = 0 . 057 and p = 0 . 16 , respectively , Log - rank ( Mantel - Cox ) test . Please see Table 1 for additional data .

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

Figure 5(A) Average number of LGG-1::GFP-containing puncta in N2 wild-type animals (WT) fed either control bacteria or bacteria expressing daf-2 (as a control) or rab-10 dsRNA for two generations. p < 0.0001 for either daf-2 or rab-10 RNAi treatment compared to control RNAi treatment, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Please see Table S1 for quantification of all data.(B) Average number of LGG-1::GFP-containing puncta in rab-10(ok1494) mutants compared to N2 wild-type animals (WT), p < 0.0001, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. Please see Figure 1 for details. Please see Table S1 for quantification of all data.(C) Survival curves of rab-10(ok1494) animals fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. Mean lifespan was 27.9 d for control, 19.9 d for bec-1 RNAi, and 22.1 d for vps-34 RNAi, all pair-wise comparisons to control, p < 0.0001, Log-rank (Mantel-Cox) test. This experiment was performed two times. Please see Table 3 for additional data.(D) Survival curves of N2 wild-type animals (WT) fed either control bacteria or bacteria expressing bec-1 or vps-34 dsRNA during adulthood at 20 °C. These assays were performed at the same time as the rab-10 lifespan analysis shown in Figure 5C. Mean lifespan was 22.9 d for control, 23.6 d for bec-1 RNAi, and 24.5 d for vps-34 RNAi, pair-wise comparison to control, p = 0.057 and p = 0.16, respectively, Log-rank (Mantel-Cox) test. Please see Table 1 for additional data.


Figure 6 ( A ) Average number of LGG - 1 : : GFP - containing puncta in daf - 2 ( e1370 ) progeny of animals fed either control bacteria or bacteria expressing bec - 1 , vps - 34 , or pha - 4 dsRNA for their entire lives . p < 0 . 0001 for bec - 1 and vps - 34 RNAi - bacteria compared to control RNAi - bacteria , respectively , p = 0 . 17 for pha - 4 RNAi - bacteria compared to control RNAi - bacteria , unpaired , two - tailed t - test . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . Feeding daf - 2 mutants for several generations with pha - 4 dsRNA sharply decreased the number of eggs laid ( data not shown ) . The mean lifespan of daf - 2 ( e1370 ) animals was shortened 12 . 5 % by pha - 4 RNAi ( [ 28 ] and data not shown ) , and we measured an 11 % decrease in puncta in daf - 2 ( e1370 ) animals fed pha - 4 RNAi . Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan . ( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 16 ( mu86 ) ; daf - 2 ( e1370 ) double mutants compared to daf - 2 ( e1370 ) animals ; p = 0 . 50 , unpaired , two - tailed t - test . N2 wild - type animals ( WT ) are shown for comparison . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . The double mutant expressing the LGG - 1 reporter had a mean lifespan similar to non - transgenic daf - 16 ; daf - 2 double mutants ( data not shown , [ 65 ] ) . Please see Table S1 for quantification of all data .

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

Figure 6(A) Average number of LGG-1::GFP-containing puncta in daf-2(e1370) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34, or pha-4 dsRNA for their entire lives. p < 0.0001 for bec-1 and vps-34 RNAi-bacteria compared to control RNAi-bacteria, respectively, p = 0.17 for pha-4 RNAi-bacteria compared to control RNAi-bacteria, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Feeding daf-2 mutants for several generations with pha-4 dsRNA sharply decreased the number of eggs laid (data not shown). The mean lifespan of daf-2(e1370) animals was shortened 12.5% by pha-4 RNAi ([28] and data not shown), and we measured an 11% decrease in puncta in daf-2(e1370) animals fed pha-4 RNAi. Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan.(B) Average number of LGG-1::GFP-containing puncta in daf-16(mu86); daf-2(e1370) double mutants compared to daf-2(e1370) animals; p = 0.50, unpaired, two-tailed t-test. N2 wild-type animals (WT) are shown for comparison. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. The double mutant expressing the LGG-1 reporter had a mean lifespan similar to non-transgenic daf-16; daf-2 double mutants (data not shown, [65]). Please see Table S1 for quantification of all data.


Figure 1C . Cell length profile as a function of the cell number position from the QC for three wild - type seedlings ( symbols , top ) at day 8 post germination and three simulated root files for the Sizer model ( symbols , bottom ) . Notice the use of a logarithmic scale in cell lengths , which visualizes the cell lengthlog10

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

Figure 1C. Cell length profile as a function of the cell number position from the QC for three wild-type seedlings (symbols, top) at day 8 post germination and three simulated root files for the Sizer model (symbols, bottom). Notice the use of a logarithmic scale in cell lengths, which visualizes the cell lengthlog10 rEZ


Figure 2A . Cartoon of each cell terminal differentiation mechanism ( Ruler in gray , Timer in pink and Sizer in red ) and ( right ) juxtaposed confocal images of an 8 - day - old WT seedling with the zones being indicated . Colors of zones as in Fig . 1 . Note the differences in subindex nomenclature compared with Fig . 1 . B . Pseudocode of the algorithm used in each model . C - E . Relationships between pairs of phenotypic traits . C . Length of the elongation zone LEZ versus its number of cells , NEZ . Panels from left to right : the Ruler , Timer and Sizer models , and epidermal Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 122 each ) . Data from individual epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 and 10 post germination ( Tables EV1 , EV2 ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . The epidermis exhibits of the number of cells in the EZ with its length , and also with 1 / ln ( rEZ ) . The epidermal mature cell length does not have correlation with the inverse of the elongation factor . These features are only reproduced in the Sizer model . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length , according to a variability inferred from the epidermal Col - 0 data ( Appendix Fig . S4B - F ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) and 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) and epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . All models have the same dependence on the threshold value for differentiation ( blue line ) but each model has its own dependence on the the spatial profile of cells along the EZ ( rEZ ) ( lines in gray for Ruler , black and red for Timer and red for Sizer ) . The Timer model depends on the cell elongation dynamics and , in contrast with the Ruler and Sizer models , its relationships are not univocally defined by the spatial profile of cells along the EZ ( rEZ ) . For the Timer model , the continuous lines represent the theoretical prediction obtained when either relong ( black line ) or Rprod ( red line ) changes . For all the boxplots , boxes represent the interquartile range ( 25th - 75th percentiles , with the median indicated by the black horizontal line ) of the distribution , whiskers extend to the minimum and maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile . C - E . Relationships between pairs of phenotypic traits . D . Length of mature cell ( EZ cell closest to DZ ) lmax versus 1 / rEZ . Panels from left to right : the Ruler , Timer and Sizer models , and epidermal Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 122 each ) . Data from individual epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 and 10 post germination ( Tables EV1 , EV2 ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . The epidermis exhibits of the number of cells in the EZ with its length , and also with 1 / ln ( rEZ ) . The epidermal mature cell length does not have correlation with the inverse of the elongation factor . These features are only reproduced in the Sizer model . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length , according to a variability inferred from the epidermal Col - 0 data ( Appendix Fig . S4B - F ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) and 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) and epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . All models have the same dependence on the threshold value for differentiation ( blue line ) but each model has its own dependence on the the spatial profile of cells along the EZ ( rEZ ) ( lines in gray for Ruler , black and red for Timer and red for Sizer ) . The Timer model depends on the cell elongation dynamics and , in contrast with the Ruler and Sizer models , its relationships are not univocally defined by the spatial profile of cells along the EZ ( rEZ ) . For the Timer model , the continuous lines represent the theoretical prediction obtained when either relong ( black line ) or Rprod ( red line ) changes . For all the boxplots , boxes represent the interquartile range ( 25th - 75th percentiles , with the median indicated by the black horizontal line ) of the distribution , whiskers extend to the minimum and maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile . C - E . Relationships between pairs of phenotypic traits . E . NEZ versus 1 / ln ( rEZ ) , which can be interpreted as the ratio between the meristematic activity ( Rprod ) and the elongation rate ( relong ) . Panels from left to right : the Ruler , Timer and Sizer models , and epidermal Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 122 each ) . Data from individual epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 and 10 post germination ( Tables EV1 , EV2 ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . The epidermis exhibits of the number of cells in the EZ with its length , and also with 1 / ln ( rEZ ) . The epidermal mature cell length does not have correlation with the inverse of the elongation factor . These features are only reproduced in the Sizer model . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length , according to a variability inferred from the epidermal Col - 0 data ( Appendix Fig . S4B - F ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) and 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) and epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . All models have the same dependence on the threshold value for differentiation ( blue line ) but each model has its own dependence on the the spatial profile of cells along the EZ ( rEZ ) ( lines in gray for Ruler , black and red for Timer and red for Sizer ) . The Timer model depends on the cell elongation dynamics and , in contrast with the Ruler and Sizer models , its relationships are not univocally defined by the spatial profile of cells along the EZ ( rEZ ) . For the Timer model , the continuous lines represent the theoretical prediction obtained when either relong ( black line ) or Rprod ( red line ) changes . For all the boxplots , boxes represent the interquartile range ( 25th - 75th percentiles , with the median indicated by the black horizontal line ) of the distribution , whiskers extend to the minimum and maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

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

Figure 2A. Cartoon of each cell terminal differentiation mechanism (Ruler in gray, Timer in pink and Sizer in red) and (right) juxtaposed confocal images of an 8-day-old WT seedling with the zones being indicated. Colors of zones as in Fig.1. Note the differences in subindex nomenclature compared with Fig. 1. B. Pseudocode of the algorithm used in each model.C-E. Relationships between pairs of phenotypic traits. C. Length of the elongation zone LEZ versus its number of cells, NEZ. Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.C-E. Relationships between pairs of phenotypic traits. D. Length of mature cell (EZ cell closest to DZ) lmax versus 1/rEZ. Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.C-E. Relationships between pairs of phenotypic traits. E. NEZ versus 1/ln(rEZ), which can be interpreted as the ratio between the meristematic activity (Rprod) and the elongation rate (relong). Panels from left to right: the Ruler, Timer and Sizer models, and epidermal Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=122 each). Data from individual epidermal files in Col-0 is in green circles (n=122 from day 6, 8 and 10 post germination (Tables EV1, EV2)). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. The epidermis exhibits of the number of cells in the EZ with its length, and also with 1/ln(rEZ). The epidermal mature cell length does not have correlation with the inverse of the elongation factor. These features are only reproduced in the Sizer model. For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length, according to a variability inferred from the epidermal Col-0 data (Appendix Fig. S4B-F). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 35% (Ruler), 7% (Timer) and 26% (Sizer) (Table EV3). Parameter values selected such that no statistical significance is found between model (for each model) and epidermal data in any of five phenotypic traits (Wilcoxon-rank sum test, p-value >0.01, Table EV4, Appendix Fig. S4B-F). Continuous lines are theoretical predictions for each model (Appendix Text S1E). All models have the same dependence on the threshold value for differentiation (blue line) but each model has its own dependence on the the spatial profile of cells along the EZ (rEZ) (lines in gray for Ruler, black and red for Timer and red for Sizer). The Timer model depends on the cell elongation dynamics and, in contrast with the Ruler and Sizer models, its relationships are not univocally defined by the spatial profile of cells along the EZ (rEZ). For the Timer model, the continuous lines represent the theoretical prediction obtained when either relong (black line) or Rprod (red line) changes. For all the boxplots, boxes represent the interquartile range (25th-75th percentiles, with the median indicated by the black horizontal line) of the distribution, whiskers extend to the minimum and maximal values excluding outliers. Outliers are those values more (less) than 3/2 of upper (lower) quartile.


Figure 3Simulation results are depicted in gray for Ruler , pink for Timer and red for Sizer models ( n = 30 simulated root files , each ) . Data from individual cortex tissue files in Col - 0 is in green ( n = 30 , day 6 post germination ( Tables EV1 , EV2 ) ) . Parameter values for the models ( Table EV3 ) selected such that no statistical significance is found between model ( for each model ) and cortex data in any of the five phenotypic traits depicted in A ( Wilcoxon - rank sum test , p - value > 0 . 01 , Table EV4 ) . For each model , the simulated roots and cells differ in the threshold value for cell elongation termination , the cell elongation rate , the meristematic activity and the initial cell length ( Table EV3 ) . The parameter values are the same for the three models except for the threshold for cell elongation termination , which is specific of each model and has relative variability of 40 % ( Ruler ) , 3 % ( Timer ) and 32 % ( Sizer ) ( Table EV3 ) . A . Boxplots for five phenotypic traits : elongation factor rEZ , mature cell length ( length of the EZ cell closest to the DZ ) lmax , length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate . For the cortex Col - 0 data , the first four phenotypic traits are all measured in the same root files ( n = 30 , day 6 post germination ) , while the rootgrowth rate is measured on a different set of roots ( n = 22 , Methods ) . The Ruler and Timer models drive larger variability in the root growth rate . B - D . Relationships between pairs of the phenotypic traits depicted in A . Panels from left to right : the Ruler , Timer and Sizer models , and cortex Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 30 each ) . Data from individual cortex files in Col - 0 is in green circles ( n = 30 ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . Continuos lines as in Fig . 2 C - E but for the corresponding parameter values ( Table EV3 ) . B - D . Relationships between pairs of the phenotypic traits depicted in A . Panels from left to right : the Ruler , Timer and Sizer models , and cortex Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 30 each ) . Data from individual cortex files in Col - 0 is in green circles ( n = 30 ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . Continuos lines as in Fig . 2 C - E but for the corresponding parameter values ( Table EV3 ) . B - D . Relationships between pairs of the phenotypic traits depicted in A . Panels from left to right : the Ruler , Timer and Sizer models , and cortex Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 30 each ) . Data from individual cortex files in Col - 0 is in green circles ( n = 30 ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . Continuos lines as in Fig . 2 C - E but for the corresponding parameter values ( Table EV3 ) .

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

Figure 3Simulation results are depicted in gray for Ruler, pink for Timer and red for Sizer models (n=30 simulated root files, each). Data from individual cortex tissue files in Col-0 is in green (n=30, day 6 post germination (Tables EV1, EV2)). Parameter values for the models (Table EV3) selected such that no statistical significance is found between model (for each model) and cortex data in any of the five phenotypic traits depicted in A (Wilcoxon-rank sum test, p-value >0.01, Table EV4). For each model, the simulated roots and cells differ in the threshold value for cell elongation termination, the cell elongation rate, the meristematic activity and the initial cell length (Table EV3). The parameter values are the same for the three models except for the threshold for cell elongation termination, which is specific of each model and has relative variability of 40% (Ruler), 3% (Timer) and 32% (Sizer) (Table EV3). A. Boxplots for five phenotypic traits: elongation factor rEZ, mature cell length (length of the EZ cell closest to the DZ) lmax, length of the elongation zone LEZ , number of cells in the elongation zone NEZ and root growth rate. For the cortex Col-0 data, the first four phenotypic traits are all measured in the same root files (n=30, day 6 post germination), while the rootgrowth rate is measured on a different set of roots (n=22, Methods). The Ruler and Timer models drive larger variability in the root growth rate.B-D. Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and cortex Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=30 each). Data from individual cortex files in Col-0 is in green circles (n=30). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. Continuos lines as in Fig. 2 C-E but for the corresponding parameter values (Table EV3).B-D. Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and cortex Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=30 each). Data from individual cortex files in Col-0 is in green circles (n=30). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. Continuos lines as in Fig. 2 C-E but for the corresponding parameter values (Table EV3).B-D. Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and cortex Col-0 data. Symbols in the three left-most panels represent simulated data of individual simulated root files (gray triangles for Ruler, pink squares for Timer and red diamonds for Sizer, n=30 each). Data from individual cortex files in Col-0 is in green circles (n=30). Dashed lines are minimum square linear fits. Pearson correlation coefficient (r) for each pair of data are indicated. p stands for the p-value using standard Pearson correlation test. Continuos lines as in Fig. 2 C-E but for the corresponding parameter values (Table EV3).


Figure 4C - D : NEZ versus 1 / lnrEZ in the presence or absence of the meristem - elongation correlation , when Rprod is changed two - fold . E - H : Theoretical distribution of mature cells length ( E , F ) and of root growth ( G , H ) in the presence ( E , G ) , or absence ( F , H ) of the correlation , for different values of the meristematic activity ( Rprod ) ( n = 35 ) . In all cases data was computed using the real experimental values for 1 / ln ( rEZ ) , 1 / ( 2ln ( rEZ ) ) and 2 / ( ln ( rEZ ) ) and hypothetical values for NEZ chosen to either preserve or not the correlation . p < 0 . 005 * * * , p < 0 . 01 * * , p < 0 . 05 * Boxplot representation as in Fig . 2 .

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

Figure 4C-D: NEZ versus 1/lnrEZ in the presence or absence of the meristem-elongation correlation, when Rprod is changed two-fold.E-H: Theoretical distribution of mature cells length (E,F) and of root growth (G,H) in the presence (E,G), or absence (F,H) of the correlation, for different values of the meristematic activity (Rprod) (n=35). In all cases data was computed using the real experimental values for 1/ln(rEZ), 1/(2ln(rEZ)) and 2/(ln(rEZ)) and hypothetical values for NEZ chosen to either preserve or not the correlation. p<0.005 ***, p<0.01 **, p<0.05 * Boxplot representation as in Fig. 2.


Figure 5A . Eight - day - old seedlings of WT , bri1 and pRPS5a : BRI1 : YFP ; bri1 . Scale bar 1 cm . B . Root - length measurements of 10 - day - old bri1 - 116 , bes1 - D and pRP5SA : BRI : YFP ; bri1 - 116 seedlings compared to the wild type . Symbols represent the mean of more than 20 plants , from 3 independent experiments . Bars show standard deviation . Straight lines represent the linear regression applied to each curve starting with day 4 post germination . In this way the root growth rate can be extracted for each phenotype ( See Table EV7 ) . C . Confocal images of 8 - day - old wild type WT , bri1 - 116 and pRP5SA : BRI : YFP ; bri1 - 116 roots stained with PI . Green lines label the end of the transition zone and the yellow lines label the first root hair ( end of elongation zone ) . The inset shows pRP5SA expression domain of an 8 - day - old pRP5SA : BRI : YFP ; bri1 - 116 seedling . Green line labels the end of the meristematic zone , which coincides with the end of pRP5SA expression domain . Scale bars correspond to 100 microns . C - H . Main relationships as in Fig . 2C - E between experimental values of the phenotypic traits used to assess the mechanism of differentiation in bri1 - 116 mutant ( C , E , G ) and pRP5SA : BRI : YFP ; bri1 - 116 line ( D , F , H ) for the epidermis ( left large plot ) and cortex ( right inset plot ) . Each circle denotes data extracted from a single root ( n = 126 ( epidermis ) and n = 25 ( cortex ) for bri1 - 116 and n = 90 ( epidermis ) and n = 17 ( cortex ) for pRP5SA : BRI : YFP ; bri1 - 116 , see Table EV1 , pooled from 6 , 8 and 10 day - old seedlings for epidermis and from day 6 for cortex ( data in Table EV5 ) ) . In G , H , the slope of the linear regression is 2 . 13 ± 0 . 04 for bri1 - 116 and 2 . 53 ± 0 . 04 for pRP5SA : BRI : YFP ; bri1 - 116 . Pearson correlation coefficient r is indicated and p - value ( p , using standard Person correlation test ) . In E , bri1 - 116 epidermal data do not conform to normal distributions .

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

Figure 5A. Eight-day-old seedlings of WT, bri1 and pRPS5a:BRI1:YFP;bri1. Scale bar 1 cm.B. Root-length measurements of 10-day-old bri1-116, bes1-D and pRP5SA:BRI:YFP;bri1-116 seedlings compared to the wild type. Symbols represent the mean of more than 20 plants, from 3 independent experiments. Bars show standard deviation. Straight lines represent the linear regression applied to each curve starting with day 4 post germination. In this way the root growth rate can be extracted for each phenotype (See Table EV7).C. Confocal images of 8-day-old wild type WT, bri1-116 and pRP5SA:BRI:YFP;bri1-116 roots stained with PI. Green lines label the end of the transition zone and the yellow lines label the first root hair (end of elongation zone). The inset shows pRP5SA expression domain of an 8-day-old pRP5SA:BRI:YFP;bri1-116 seedling. Green line labels the end of the meristematic zone, which coincides with the end of pRP5SA expression domain. Scale bars correspond to 100 microns.C-H. Main relationships as in Fig. 2C-E between experimental values of the phenotypic traits used to assess the mechanism of differentiation in bri1-116 mutant (C,E,G) and pRP5SA:BRI:YFP;bri1-116 line (D,F,H) for the epidermis (left large plot) and cortex (right inset plot). Each circle denotes data extracted from a single root (n=126 (epidermis) and n=25 (cortex) for bri1-116 and n=90 (epidermis) and n=17 (cortex) for pRP5SA:BRI:YFP;bri1-116, see Table EV1, pooled from 6, 8 and 10 day-old seedlings for epidermis and from day 6 for cortex (data in Table EV5)). In G, H, the slope of the linear regression is 2.13±0.04 for bri1-116 and 2.53±0.04 for pRP5SA:BRI:YFP;bri1-116. Pearson correlation coefficient r is indicated and p-value (p, using standard Person correlation test). In E, bri1-116 epidermal data do not conform to normal distributions.