G , H . Staticity scores for different surface features ( R + N , MV ) ( G ) for segmented IgM - BCR clusters assigned to different surface features ( H ) in unstimulated Ag - stimulated . Violin plot solid lines represent median dotted lines represent quartiles .

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

G, H. Staticity scores for different surface features (R+N, MV) (G) and for segmented IgM-BCR clusters assigned to different surface features (H) in unstimulated and Ag-stimulated cells. Violin plot solid lines represent the median and dotted lines represent the quartiles.


A . Phalloidin - Alexa555 of fixed permeabilized Ramos B expressing EGFP - CaaX IgM - BCR with or without 30 min 2 µM LatA treatment .

O O O 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. Phalloidin-Alexa555 staining of fixed and permeabilized Ramos B cells expressing EGFP-CaaX and IgM-BCR with or without 30 min 2 µM LatA treatment.


D . Surface - masked EGFP - CaaX IgM - BCR signal overlay of segmented elevations ( yellow ) with surface - masked IgM - BCR signal in treated with LatA LatA + Ag ( 50 ng / ml NIP - 15 - BSA ) . E . Time frames from a LatA + Ag showing directed movement of Ag - MC towards elevations ( yellow ) . Each Ag - MC shown with a different colored arrow .

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

D. Surface-masked EGFP-CaaX and IgM-BCR signal and overlay of segmented elevations (yellow) with the surface-masked IgM-BCR signal in cells treated with LatA and LatA+Ag (50 ng/ml NIP-15-BSA). E. Time frames from a LatA+Ag cell showing directed movement of Ag-MC towards the elevations (yellow). Each Ag-MC shown with a different colored arrow.


F . IgM - BCR enrichment within segmented clusters in LatA Lat + Ag . G . Cluster density at surface features . of IgM - BCR cluster density in terms of cluster per surface feature volume for segmented IgM - BCR clusters ( left panel , local contrast cutoff = 1x ) for Ag - MC ( right panel , local contrast cutoff = 3x ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IgM-BCR intensity enrichment within all the segmented clusters in LatA and Lat+Ag cells. G. Cluster density at surface features. Quantification of the IgM-BCR cluster density in terms of cluster count per surface feature volume for all segmented IgM-BCR clusters (left panel, local contrast cutoff = 1x) and for Ag-MC (right panel, local contrast cutoff = 3x). H


I . Surface - masked EGFP - CaaX IgM - BCR signal for a LatA LatA + Ag sum projections of their segmented IgM - BCR clusters ( duration : 37 . 5 s ) . IgM - BCR clusters are color - coded ( green yellow ) according to their assigned surface region .

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

I. Surface-masked EGFP-CaaX and IgM-BCR signal for a LatA and LatA+Ag cell and the sum projections of their segmented IgM-BCR clusters (duration: 37.5 s). IgM-BCR clusters are color-coded (green and yellow) according to their assigned surface region.


D ) expressing empty vector ( EV ) , or PRKAA1 - myc , PRKAB2 PRKAG2 - FLAG ( AMPK ) alone or with myc - Ulk1 WT ( ULK ) were cultured in full medium ( F ) , EBSS ( St ) , or serum - free medium ( SFM ) alone or in presence of 991 ( 1µM ) or MRT68921 ( 1μM ) for 90 minutes as indicated . 2 % Loading control pull down samples were analysed by Western blot .

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

D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.


A ) VPS15 - ZZ was co - expressed with Ulk1 Ulk2 wild type ( WT ) or kinase inactive ( KI ) before by Western blot . An electrophoretic mobility shift in VPS15 - ZZ was noted with WT only .

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

A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only.


A ) Lysates from 3 control clones 7 VPS15 CRISPR clones were analysed by Western blot . LC3 p62 accumulation as well as depletion of VPS34 CI components was observed in 6 sgA - derived clones .

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

A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones.


B ) Controls 6 VPS15 CRISPR KO clones were starved for 1 hr , fixed imaged for WIPI2 ( green ) . Depletion of VPS15 was associated with a block in WIPI2 formation large vacuoles ( red asterisks ) . of WIPI2 spots , mean + / - , n = 3 .

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

B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3.


D ) Wild type ( WT ) , 6SA or 6SE VPS15 - HA , or empty vector ( EV ) , were expressed in VPS15 KO or HEK293A . were starved for 1 hr distributions of WIPI2 ( green ) , HA ( red ) LC3 ( blue ) were assessed , mean + / - , n = 4 .

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

D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4.


D ) VPS34 CI components expressed with Ulk1 1 - 427 WT or KI as indicated . After starvation for 1 hr in presence or absence of MRT68921 ( ULK inhibitor ) , were lysed VPS34 CI coimmunoprecipitated via ATG14 - ZZ . VPS15 S861 phosphorylation was increased in presence of active Ulk1 . VPS15 pS861 / total VPS15 was quantified , mean + / - , n = 3 .

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

D) 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 Ulk1 1 - 427 WT or KI 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 + / - , * * * p < 0 . 001 [ one - ANOVA ] .

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

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].


A ) HEK293A , VPS15 KO control stably rescued VPS15 KOs ( sgA - B3 ) were starved for 1 hr before WIPI2 ( green ) , LC3 ( red ) ( Hoechst , blue ) were visualised . of WIPI2 number per , mean + / - , n = 5 .

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

A) 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 VPS15 KO control were starved for 1 hr with ( SB ) or without ( St ) 100nM Bafilomycin A1 , or cultured in full media ( F ) . shows LC3II / Actin , mean + / - , n = 5 .

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

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.


A ) HEK293A were treated with VPS34 - IN1 ( 1μM ) alone or in presence of MRT68921 ( 1μM ) for indicated time . Untreated VPS15 KOs were included as a positive control lysates analysed by Western blot . Identical samples were loaded in parallel for VPS34 , ATG13 , PRKAB2 , indicated by dashed .

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

A) 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 VPS15 KO were fed , or starved for 2 hrs before fixation , labelling with ULK1 ( green ) , ATG9A ( red ) GM130 ( blue ) . Dashed boxes show magnified regions of interest .

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

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.


B ) HEK293A were transfected with siRNAs targeting ATG14 , UVRAG or p62 , or with non - targeting siRNA ( RISC - free , RF ) were treated with VPS34 - IN1 ( IN1 ; 1μM ) for 0 , 4 or 18 hrs as indicated . of ATG13 pS318 / Total ATG13 , mean + / - , n = 3 . Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot , indicated by dashed .

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

B) 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 ) transfected as in B were treated with IN1 for 18 hours as indicated , fixed labelled for ubiquitin ( green ) , FIP200 ( red ) p62 ( blue ) . FIP200 - positive bodies were quantified from 4 independent experiments with mean + / - plotted . transfected with ATG14 siRNA generated significantly more FIP200 bodies per than other conditions tested .

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

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.


Plasma corticosterone at baseline , 30 minutes after induction of stress by placing mice in a clean cage , after 90 minutes upon return in home cage . DEX mice received an injection of dexamethasone at 0 . 1 mg / kg 150 minutes before collecting first blood sample ( n = 5 - 6 per group ) . Flox GRBATKO mice were injected with 0 . 02 mg / kg dexamethasone procedure described in ( A ) was followed to induce stress in order to detect potential differences between Flox GRBATKO mice which could have been obscured by higher used in ( A ) ( n = 6 - 7 per group ) .

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

Plasma 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 in BAT of Flox GRBATKO mice . Organs were harvested in stressed condition after placing mice for 30 minutes in a clean cage . Half of received a dexamethasone injection 180 minutes before onset of procedure , controls received an injection with saline ( n = 5 - 6 per group ) .

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

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 under stressed conditions in Flox GRBATKO mice ( n = 5 - 6 per group ) . Blood glucose ( n = 5 - 6 per group ) .

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

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).


Knockdown of GR in BAT assessed by immunoblotting . Β - Tubulin was used as loading control . of immunoblot in ( A ) using Image Lab ( n = 6 per group ) .

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

Knockdown 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).


over course of cold exposure , determined daily ( n = 6 per group ) .

Body temperature over the course of the cold exposure, determined daily (n = 6 animals per group).


at end of cold exposure ( n = 6 per group ) .

Body weight at the end of the cold exposure (n = 6 animals per group).


BAT at end of cold exposure ( n = 6 per group ) .

BAT weight at the end of the cold exposure (n = 6 animals per group).


Representative Hematoxilin eosin image of BAT of cold - exposed Flox GRBATKO mice . Scale bar 50 µm .

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

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 GRBATKO ( n = 5 - 6 per group ) .

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

Triglyceride content in BAT from Flox and GRBATKO animals (n = 5-6 animals per group).


of mean gene expression fold changes in thermogenic marker genes on BAT microarray comparing GRBATKO vs Flox control , 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 per group ) .

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

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 adjusted p - value < 0 . 1 fold change > 1 . 5 .

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

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 ( n = 6 per group ) .

Serum corticosterone levels (n = 6 animals per group).


after 16 h fasting 6 h chow refeeding at 22 ° C ( n = 6 per group )

Body weight after 16 h fasting and 6 h chow refeeding at 22°C (n = 6 animals per group)


assessed after 16 h fasting subsequent refeeding for 6 h ( n = 6 per group ) .

Body temperature assessed after 16 h fasting and subsequent refeeding for 6 h (n = 6 animals per group).


Serum insulin corticosterone determined by ELISA ( n = 6 per group ) .

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

Serum insulin and corticosterone levels determined by ELISA (n = 6 animals per group).


Serum NEFA triglycerides in Flox GRBATKO mice after fasting refeeding ( n = 6 per group ) .

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

Serum NEFA and triglycerides concentration in Flox and GRBATKO mice after fasting and refeeding (n = 6 animals per group).


gain over a 24 - week period of high - fat feeding ( n = 13 - 14 per group ) . Panel shows experimental design for Figures 4 - 6 .

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

Body 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 phase ( n = 6 - 8 mice per group ) . Average oxygen consumption specified for phase , phase , total ( n = 6 - 8 per genotype ) .

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

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 indicating that lower energy expenditure in GRBATKO is independent of ( n = 6 - 8 per group ) .

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

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 per group ) .

Locomotor activity over a 90-hour period (n = 6-8 animals per group).


Intraperitoneal GTT after 19 of HFD - feeding , glucose injections at 1 . 5 mg per g of ( n = 11 - 13 per group ) . under curve calculated from ( A ) ( n = 11 - 13 per group ) .

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

Intraperitoneal 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 during GTT ( n = 11 - 13 per group ) .

Plasma insulin levels during GTT (n = 11-13 animals per group).


Intraperitoneal ITT after 20 of high - fat diet intervention . Insulin was adjusted to 1 mU per g of ( n = 11 - 13 per group ) . under curve determined from ( D ) ( n = 11 - 13 per group ) .

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

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 after a 5 - hour fast , before injecting insulin ( n = 11 - 13 per group ) .

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

Plasma insulin levels after a 5-hour fast, before injecting insulin (n = 11-13 animals per group).


in Flox GRBATKO after 24 of high - fat feeding ( n = 10 - 13 per group ) .

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

Body weight in Flox and GRBATKO after 24 weeks of high-fat feeding (n = 10-13 animals per group).


BAT iWAT in Flox GRBATKO mice fed HFD for 24 ( n = 10 - 13 per group ) .

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

BAT and iWAT weight in Flox and GRBATKO mice fed HFD for 24 weeks (n = 10-13 animals per group).


GR in BAT after 24 high - fat feeding ( n = 10 - 13 mice per group ) .

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

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 GRBATKO . Scale bar 100 µm .

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

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 per group ) .

Triglyceride content in BAT per gram of wet tissue (n = 10-13 animals per group).


qRT - PCR of thermogenic marker gene expression in BAT of Flox GRBATKO mice after high - fat feeding ( n = 9 - 13 per group ) .

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

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 .

Representative immunohistochemistry for UCP1 in BAT. Scale bar 100 µm.


Serum insulin determined by ELISA ( n = 10 - 13 per group ) .

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

Serum insulin levels determined by ELISA (n = 10-13 animals per group).


Blood glucose in Flox GRBATKO after 24 HFD ( n = 10 - 13 per group ) .

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

Blood glucose in Flox and GRBATKO animals after 24 weeks HFD (n = 10-13 animals per group).


B - Congo red plates with colonies of E . coli MC4100 relA + 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 .

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

B - 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.


B - Relative / OD600 of of E . coli MC4100 relA + ∆ omrAB or MC4100 relA + ∆ omrAB ∆ hfq harboring 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 intensities ( a . u . ) / OD600 used to calculate ratios Note that absolute dgcM : : gfp expression values are ≈ 3 - 4 - fold lower in ∆ hfq

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

B - 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 strain


C - Western blot of 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 - stained gel is shown below .

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

C - 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.


A - Toeprinting showing inhibition of initiation formation on a truncated dgcM mRNA ( 210 nt from + 1 ) by OmrA OmrB . Inclusion of 30S ribosomes , tRNAfmet , OmrA , OmrB is indicated . unrelated IstR - 1 sRNA is a negative control . 30S / tRNAfmet - dependent reverse transcription block ( toeprint ) , is indicated by an arrow . Sequencing reactions were loaded next to primer extension products ( UCGA ) . AUG start codon is indicated by asterisks .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 presence or absence of OmrA / OmrB , with or without purified Hfq , was carried out As an internal control , non - target ompA : : 3xflag mRNA was included . Western blot shows DgcM - 3xFLAG OmpA - 3xFLAG .

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

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.


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

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

A - 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.


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

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

Mobility 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.


Western blots showing DgcM - 3xFLAG translated in vitro in presence or absence of OmrA or OmrB . A - inclusion of purified wild - type or mutant Hfq is indicated , translation was conducted different Hfq interaction site mutant are indicated . Data information : of DgcM translation rates , relative to that of unaffected OmpA control , is shown below gels . of DgcM / OmpA signal was set to unity for 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 3 to lane 1 in each set .

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

Western 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 presence or absence of OmrA or OmrB . B - An ssDNA - oligo ( Hfq _ BS that base - pairs specifically to Hfq binding site on dgcM mRNA ( nt 42 - 66 ) was preannealed to mRNA prior to OmrA / B addition translation . OmpA - 3xFLAG was included as internal control . Data information : of DgcM translation rates , relative to that of unaffected OmpA control , is shown below gels . of DgcM / OmpA signal was set to unity for 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 3 to lane 1 in each set .

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

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.


A - Histograms show flow cytometry of exponentially growing of E . coli MC4100 relA - , its mutant derivatives hfq - F42A ( hfq - prox ) , hfq - Y25D ( hfq - dist ) , hfq - R16S _ R17A _ R19A _ K47E ( hfq - rim ) , ∆ hfq , harboring translational fusion plasmid pDgcM : : GFP in combination with pOmrA , pOmrB , or a control vector . Data was obtained on a MACSQuant VYB instrument .

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

A - 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 of total isolated from same cultures at 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 O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

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.


LGG - 1 : : GFP - positive labeling autophagic membranes [ 22 ] were counted in wild - type or in food - limited . ( A ) 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 in eat - 2 ( ad1116 ) mutants N2 wild - type ( WT ) , p < 0 . 0001 . ( C ) Average number of LGG - 1 : : GFP - containing in N2 wild - type , food - restricted grown in liquid media ( WT , food limited ) N2 wild - type grown in liquid with a higher of ( WT , fully fed ) , p < 0 . 0001 ; see Methods . Between three ten seam were counted in each of 20 - 40 using high - power microscopy averaged . n , total number of seam observed . Error bars : ± . p - Values were calculated as unpaired , two - t - . were raised at 20 ° C . Please see Table S1 for of data .

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

LGG-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.


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

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

(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.


( A ) Average number of LGG - 1 : : GFP - containing in eat - 2 ( ad1116 ) progeny of fed either control or expressing bec - 1 , vps - 34 or pha - 4 dsRNA their entire lives . p ≤ 0 . 0001 for bec - 1 , vps - 34 pha - 4 RNAi treatments compared to control , respectively , unpaired , two - t - . n , total number of seam observed . Error bars : ± . See Figure 1 for details .

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

(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 in rab - 10 ( ok1494 ) progeny of fed either control or expressing pha - 4 dsRNA their entire lives ; p < 0 . 0001 , unpaired , two - t - . n , total number of seam observed . Error bars : ± . Please see Figure 1 for details Table S1 for of data . Feeding mutants for several generations with pha - 4 dsRNA , inhibited development sharply decreased number of eggs laid ( [ 46 ] , data not shown ) . However , loss of many 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 O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

(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).


( A ) Average number of LGG - 1 : : GFP - containing in let - 363 / TOR RNAi - arrested compared to N2 wild - type ( WT ) grown on control ( vector - only ) , p < 0 . 0001 , unpaired , two - t - . n , total number of seam observed . Error bars : ± . See Figure 1 for details . We were not able to detect increased LGG - 1 : : GFP in long - lived let - 363 ( RNAi ) adults ; however , one generation of daf - 2 RNAi , our positive control , did not significantly increase number of foci in adults either ( data not shown ) . Please see Table S1 for of data . ( B ) Average number of LGG - 1 : : GFP - containing in daf - 15 ( m81 ) / unc - 24 ( e138 ) heterozygotes ( daf - 15 / + ) compared to N2 wild - type ( WT ) , p < 0 . 0001 , unpaired , two - t - . n , total number of seam observed . Error bars : ± . See Figure 1 for details . Please see Table S1 for of data . daf - 15 encodes TOR - binding partner Raptor .

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

(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 / + , DR412 ) fed either control or expressing bec - 1 dsRNA during adulthood at 20 ° C . Mean lifespan : daf - 15 / + grown on control RNAi - : 25 . 1 d , daf - 15 / + on bec - 1 RNAi : 20 . 8 d , p = 0 . 0008 , Log - rank ( Mantel - Cox ) . lifespan of daf - 15 / + grown on bec - 1 RNAi - during adulthood was measured again , yielding similar results ; lifespan of daf - 15 / + was also measured three times following whole - life RNAi exposure . In these experiments , bec - 1 RNAi generally shortened mean lifespan of daf - 15 / + to a greater extent than it shortened lifespan of wild type . ( We also attempted to perform double - RNAi experiments , in which were cultured on a 50 : 50 mixture of let - 363 / TOR bec - 1 [ or control ] RNAi . Although trends we saw were in expected direction , effects produced by half - strength RNAi were small not statistically significant [ data not shown ] . ) Please see Table 2 for additional data . ( D ) Survival curves of wild - type derived from DR412 ( WT ) fed either control or expressing bec - 1 dsRNA throughout their whole life at 20 ° C . These assays were performed concurrently with daf - 15 / + lifespan shown in Figure 4C . WT grown on control RNAi - : 21 . 4 d , WT on bec - 1 RNAi - : 21 . 1 d ( p = 0 . 34 ) , p between daf - 15 / + WT grown on control RNAi - , p < 0 . 0001 , Log - rank ( Mantel - Cox ) . Please see Table 2 for additional data .

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

(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.


( A ) Average number of LGG - 1 : : GFP - containing in N2 wild - type ( WT ) fed either control or 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 - t - . n , total number of seam observed . Error bars : ± . See Figure 1 for details . Please see Table S1 for of data . ( B ) Average number of LGG - 1 : : GFP - containing in rab - 10 ( ok1494 ) mutants compared to N2 wild - type ( WT ) , p < 0 . 0001 , unpaired , two - t - . n , total number of seam observed . Error bars : ± . Please see Figure 1 for details . Please see Table S1 for of data .

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

(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 ) fed either control or 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 , 22 . 1 d for vps - 34 RNAi , pair - wise comparisons to control , p < 0 . 0001 , Log - rank ( Mantel - Cox ) . This experiment was performed two times . Please see Table 3 for additional data . ( D ) Survival curves of N2 wild - type ( WT ) fed either control or expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at same time as rab - 10 lifespan shown in Figure 5C . Mean lifespan was 22 . 9 d for control , 23 . 6 d for bec - 1 RNAi , 24 . 5 d for vps - 34 RNAi , pair - wise comparison to control , p = 0 . 057 p = 0 . 16 , respectively , Log - rank ( Mantel - Cox ) . Please see Table 1 for additional data .

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

(C) Survival 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.


( A ) Average number of LGG - 1 : : GFP - containing in daf - 2 ( e1370 ) progeny of fed either control or expressing bec - 1 , vps - 34 , or pha - 4 dsRNA for their entire lives . p < 0 . 0001 for bec - 1 vps - 34 RNAi - compared to control RNAi - , respectively , p = 0 . 17 for pha - 4 RNAi - compared to control RNAi - , unpaired , two - t - . n , total number of seam observed . Error bars : ± . See Figure 1 for details . Feeding daf - 2 mutants for several generations with pha - 4 dsRNA sharply decreased number of eggs laid ( data not shown ) . mean lifespan of daf - 2 ( e1370 ) was shortened 12 . 5 % by pha - 4 RNAi ( [ 28 ] data not shown ) , we measured an 11 % decrease in in daf - 2 ( e1370 ) fed pha - 4 RNAi . Even though this decrease was not statistically significant it remains possible that it relates to small difference seen in lifespan .

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

(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 in daf - 16 ( mu86 ) ; daf - 2 ( e1370 ) double mutants compared to daf - 2 ( e1370 ) ; p = 0 . 50 , unpaired , two - t - . N2 wild - type ( WT ) are shown for comparison . n , total number of seam observed . Error bars : ± . See Figure 1 for details . double mutant expressing 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 of data .

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

(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.


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

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

C. 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


A . Cartoon of each terminal differentiation mechanism ( Ruler in gray , Timer in pink Sizer in red ) ( right ) juxtaposed confocal of an 8 - day - old WT seedling with zones being indicated . Colors of zones as in Fig . 1 . Note differences in subindex nomenclature compared with Fig . 1 . B . Pseudocode of algorithm used in each model .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 . of elongation zone LEZ versus its number of , NEZ . Panels from left to right : Ruler , Timer Sizer models , epidermal Col - 0 data . Symbols in three left - most panels represent simulated data of simulated root files ( gray triangles for Ruler , pink squares for Timer red diamonds for Sizer , n = 122 each ) . Data from epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 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 p - value using standard Pearson correlation . epidermis exhibits of number of in EZ with its , also with 1 / ln ( rEZ ) . epidermal mature does not have correlation with inverse of elongation factor . These features are only reproduced in Sizer model . For each model , simulated roots differ in threshold value for elongation termination , elongation rate , meristematic activity initial , according to a variability inferred from epidermal Col - 0 data ( Appendix Fig . S4B - F ) . parameter values are same for three models except for threshold for elongation termination , which is specific of each model has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . models have same dependence on threshold value for differentiation ( blue ) but each model has its own dependence on spatial profile of along EZ ( rEZ ) ( lines in gray for Ruler , black red for Timer red for Sizer ) . Timer model depends on elongation dynamics , in contrast with Ruler Sizer models , its relationships are not univocally defined by spatial profile of along EZ ( rEZ ) . For Timer model , continuous lines represent theoretical prediction obtained when either relong ( black ) or Rprod ( red ) changes . For boxplots , boxes represent interquartile range ( 25th - 75th percentiles , with median indicated by black horizontal ) of distribution , whiskers extend to minimum maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

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

C-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 . of mature ( EZ closest to DZ ) lmax versus 1 / rEZ . Panels from left to right : Ruler , Timer Sizer models , epidermal Col - 0 data . Symbols in three left - most panels represent simulated data of simulated root files ( gray triangles for Ruler , pink squares for Timer red diamonds for Sizer , n = 122 each ) . Data from epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 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 p - value using standard Pearson correlation . epidermis exhibits of number of in EZ with its , also with 1 / ln ( rEZ ) . epidermal mature does not have correlation with inverse of elongation factor . These features are only reproduced in Sizer model . For each model , simulated roots differ in threshold value for elongation termination , elongation rate , meristematic activity initial , according to a variability inferred from epidermal Col - 0 data ( Appendix Fig . S4B - F ) . parameter values are same for three models except for threshold for elongation termination , which is specific of each model has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . models have same dependence on threshold value for differentiation ( blue ) but each model has its own dependence on spatial profile of along EZ ( rEZ ) ( lines in gray for Ruler , black red for Timer red for Sizer ) . Timer model depends on elongation dynamics , in contrast with Ruler Sizer models , its relationships are not univocally defined by spatial profile of along EZ ( rEZ ) . For Timer model , continuous lines represent theoretical prediction obtained when either relong ( black ) or Rprod ( red ) changes . For boxplots , boxes represent interquartile range ( 25th - 75th percentiles , with median indicated by black horizontal ) of distribution , whiskers extend to minimum maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

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

C-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 between meristematic activity ( Rprod ) elongation rate ( relong ) . Panels from left to right : Ruler , Timer Sizer models , epidermal Col - 0 data . Symbols in three left - most panels represent simulated data of simulated root files ( gray triangles for Ruler , pink squares for Timer red diamonds for Sizer , n = 122 each ) . Data from epidermal files in Col - 0 is in green circles ( n = 122 from day 6 , 8 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 p - value using standard Pearson correlation . epidermis exhibits of number of in EZ with its , also with 1 / ln ( rEZ ) . epidermal mature does not have correlation with inverse of elongation factor . These features are only reproduced in Sizer model . For each model , simulated roots differ in threshold value for elongation termination , elongation rate , meristematic activity initial , according to a variability inferred from epidermal Col - 0 data ( Appendix Fig . S4B - F ) . parameter values are same for three models except for threshold for elongation termination , which is specific of each model has relative variability of 35 % ( Ruler ) , 7 % ( Timer ) 26 % ( Sizer ) ( Table EV3 ) . Parameter values selected such that no statistical significance is found between model ( for each model ) epidermal data in any of five phenotypic traits ( Wilcoxon - rank sum , p - value > 0 . 01 , Table EV4 , Appendix Fig . S4B - F ) . Continuous lines are theoretical predictions for each model ( Appendix Text S1E ) . models have same dependence on threshold value for differentiation ( blue ) but each model has its own dependence on spatial profile of along EZ ( rEZ ) ( lines in gray for Ruler , black red for Timer red for Sizer ) . Timer model depends on elongation dynamics , in contrast with Ruler Sizer models , its relationships are not univocally defined by spatial profile of along EZ ( rEZ ) . For Timer model , continuous lines represent theoretical prediction obtained when either relong ( black ) or Rprod ( red ) changes . For boxplots , boxes represent interquartile range ( 25th - 75th percentiles , with median indicated by black horizontal ) of distribution , whiskers extend to minimum maximal values excluding outliers . Outliers are those values more ( less ) than 3 / 2 of upper ( lower ) quartile .

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

C-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.


Simulation results are depicted in gray for Ruler , pink for Timer red for Sizer models ( n = 30 simulated root files , each ) . Data from cortex tissue files in Col - 0 is in green ( n = 30 , day 6 post germination ( Tables EV1 , EV2 ) ) . Parameter values for models ( Table EV3 ) selected such that no statistical significance is found between model ( for each model ) cortex data in any of five phenotypic traits depicted in A ( Wilcoxon - rank sum , p - value > 0 . 01 , Table EV4 ) . For each model , simulated roots differ in threshold value for elongation termination , elongation rate , meristematic activity initial ( Table EV3 ) . parameter values are same for three models except for threshold for elongation termination , which is specific of each model has relative variability of 40 % ( Ruler ) , 3 % ( Timer ) 32 % ( Sizer ) ( Table EV3 ) . A . Boxplots for five phenotypic traits : elongation factor rEZ , mature ( of EZ closest to DZ ) lmax , of elongation zone LEZ , number of in elongation zone NEZ root growth rate . For cortex Col - 0 data , first four phenotypic traits are measured in same root files ( n = 30 , day 6 post germination ) , while rootgrowth rate is measured on a different set of roots ( n = 22 , Methods ) . Ruler Timer models drive larger variability in root growth rate .

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

Simulation 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 phenotypic traits depicted in A . Panels from left to right : Ruler , Timer Sizer models , cortex Col - 0 data . Symbols in three left - most panels represent simulated data of simulated root files ( gray triangles for Ruler , pink squares for Timer red diamonds for Sizer , n = 30 each ) . Data from 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 p - value using standard Pearson correlation . Continuos lines as in Fig . 2 C - E but for corresponding parameter values ( Table EV3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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).


C - D : NEZ versus 1 / lnrEZ in presence or absence of meristem - elongation correlation , when Rprod is changed two - fold .

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

C-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 ( E , F ) of root growth ( G , H ) in presence ( E , G ) , or absence ( F , H ) of correlation , for different values of meristematic activity ( Rprod ) ( n = 35 ) . In cases data was computed using real experimental values for 1 / ln ( rEZ ) , 1 / ( 2ln ( rEZ ) ) 2 / ( ln ( rEZ ) ) hypothetical values for NEZ chosen to either preserve or not correlation . p < 0 . 005 * * * , p < 0 . 01 * * , p < 0 . 05 * Boxplot representation as in Fig . 2 .

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

E-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.


A . Eight - day - old seedlings of WT , bri1 pRPS5a : BRI1 : YFP ; bri1 . Scale bar 1 cm .

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

A. Eight-day-old seedlings of WT, bri1 and pRPS5a:BRI1:YFP;bri1. Scale bar 1 cm.


B . Root - measurements of 10 - day - old bri1 - 116 , bes1 - D pRP5SA : BRI : YFP ; bri1 - 116 seedlings compared to wild type . Symbols represent mean of more than 20 , from 3 independent experiments . Bars show standard deviation . Straight lines represent linear regression applied to each curve starting with day 4 post germination . In this way root growth rate can be extracted for each phenotype ( See Table EV7 ) .

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

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 of 8 - day - old wild type WT , bri1 - 116 pRP5SA : BRI : YFP ; bri1 - 116 roots stained with PI . Green lines label end of transition zone yellow lines label first root hair ( end of elongation zone ) . inset shows pRP5SA expression domain of an 8 - day - old pRP5SA : BRI : YFP ; bri1 - 116 seedling . Green labels end of meristematic zone , which coincides with end of pRP5SA expression domain . Scale bars correspond to 100 microns .

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

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 phenotypic traits used to assess mechanism of differentiation in bri1 - 116 mutant ( C , E , G ) pRP5SA : BRI : YFP ; bri1 - 116 ( D , F , H ) for epidermis ( left large plot ) cortex ( right inset plot ) . Each circle denotes data extracted from a single root ( n = 126 ( epidermis ) n = 25 ( cortex ) for bri1 - 116 n = 90 ( epidermis ) n = 17 ( cortex ) for pRP5SA : BRI : YFP ; bri1 - 116 , see Table EV1 , pooled from 6 , 8 10 day - old seedlings for epidermis from day 6 for cortex ( data in Table EV5 ) ) . In G , H , slope of linear regression is 2 . 13 ± 0 . 04 for bri1 - 116 2 . 53 ± 0 . 04 for pRP5SA : BRI : YFP ; bri1 - 116 . Pearson correlation coefficient r is indicated p - value ( p , using standard Person correlation ) . In E , bri1 - 116 epidermal data do not conform to normal distributions .

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

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.


Simulation results are depicted in gray for Ruler , pink for Timer red for Sizer models ( n = 28 each ) . Data from cortex tissue in Col - 0 grown with 1µM PAC is in green ( n = 28 , day 6 post germination ) . Parameter values for models ( Table EV3 ) selected such that no statistical significance is found between model ( for each model ) cortex data in any of five phenotypic traits depicted in A ( Wilcoxon - rank sum , p - value > 0 . 01 , Table EV4 ) . For each model , simulated roots differ in threshold value for elongation termination , elongation rates , meristematic activities initial . parameter values are same for three models except for threshold for elongation termination , which has relative variability of 16 % ( Ruler ) , 2 % ( Timer ) 14 % ( Sizer ) . A Boxplots for phenotypic traits : elongation factor rEZ , mature ( of EZ closest to DZ ) lmax , of elongation zone LEZ , number of in elongation zone NEZ root growth rate . For cortex data , first four phenotypic traits are measured in same root files ( n = 28 , day 6 post germination ) , while root growth rate is measured on a different set of roots ( n = 40 , Methods ) .

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

Simulation results are depicted in gray for Ruler, pink for Timer and red for Sizer models (n=28 each). Data from cortex tissue in Col-0 grown with 1µM PAC is in green (n=28, day 6 post germination). 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 rates, the meristematic activities and the initial cell length. The parameter values are the same for the three models except for the threshold for cell elongation termination, which has relative variability of 16% (Ruler), 2% (Timer) and 14% (Sizer). A Boxplots for 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 data, the first four phenotypic traits are all measured in the same root files (n=28, day 6 post germination), while the root growth rate is measured on a different set of roots (n=40, Methods).


B - D Relationships between pairs of phenotypic traits depicted in A . Panels from left to right : Ruler , Timer Sizer models , empirical cortex Col - 0 data . Symbols in three left - most panels represent simulated data . Continuous lines are theoretical predictions for each model . In contrast with Ruler Sizer models , theoretical Timer model relationships depend on which factor varies ( either elongation rate ( black ) or meristematic activity ( red ) ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient r for each pair of data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and empirical cortex Col-0 data. Symbols in the three left-most panels represent simulated data. Continuous lines are theoretical predictions for each model. In contrast with the Ruler and Sizer models, the theoretical Timer model relationships depend on which factor varies (either the elongation rate (black line) or the meristematic activity (red line)). Dashed lines are minimum square linear fits. Pearson correlation coefficient r for each pair of data.


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Relationships between pairs of the phenotypic traits depicted in A. Panels from left to right: the Ruler, Timer and Sizer models, and empirical cortex Col-0 data. Symbols in the three left-most panels represent simulated data. Continuous lines are theoretical predictions for each model. In contrast with the Ruler and Sizer models, the theoretical Timer model relationships depend on which factor varies (either the elongation rate (black line) or the meristematic activity (red line)). Dashed lines are minimum square linear fits. Pearson correlation coefficient r for each pair of data.