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 ) .

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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

B-MEASURED_VAR O O 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 and GRBATKO animals . Scale bar 100 µm .

O O O O O O O O O O O B-CONTROLLED_VAR O 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 animals per group ) .

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

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


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

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

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

O B-MEASURED_VAR O O 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 and GRBATKO animals after 24 weeks HFD ( n = 10 - 13 animals per group ) .

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

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 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 .

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 O O 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 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

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

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 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

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 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 .

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

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

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

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 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-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 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 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 .

O O O B-MEASURED_VAR 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 B-CONTROLLED_VAR 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-MEASURED_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 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 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 the presence or absence of OmrA or OmrB . B - An ssDNA - oligo ( Hfq _ BS that base - pairs specifically to the Hfq binding site on dgcM mRNA ( nt 42 - 66 ) was preannealed to the mRNA prior to OmrA / B addition and translation . OmpA - 3xFLAG was included as internal control . Data information : Quantification of DgcM translation rates , relative to that of the unaffected OmpA control , is shown below the gels . The ratio of DgcM / OmpA signal was set to unity for the lane lacking sRNA in each set of three combinations ( - sRNA , + OmrA , + OmrB ) , indicated by " 1 . 00 * " . Effects due to OmrA / OmrB addition can be seen by comparing lanes 2 and 3 to lane 1 in each set .

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

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 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 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 analysis of total RNA isolated from the same cultures at the same time point as used for flow cytometry , probed for OmrA ( strains containing pOmrA ) or OmrB ( strains containing pOmrB ) . 5S rRNA was used as loading control .

O O 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 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 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 .

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

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

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

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 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 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

(A) Average number of LGG-1::GFP-containing puncta in eat-2(ad1116) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34 or pha-4 dsRNA their entire lives. p ≤ 0.0001 for bec-1, vps-34 and pha-4 RNAi treatments compared to control, respectively, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details.


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

O O 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 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 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 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 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 .

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

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

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 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 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 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 ) animals fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . Mean lifespan was 27 . 9 d for control , 19 . 9 d for bec - 1 RNAi , and 22 . 1 d for vps - 34 RNAi , all pair - wise comparisons to control , p < 0 . 0001 , Log - rank ( Mantel - Cox ) test . This experiment was performed two times . Please see Table 3 for additional data . ( D ) Survival curves of N2 wild - type animals ( WT ) fed either control bacteria or bacteria expressing bec - 1 or vps - 34 dsRNA during adulthood at 20 ° C . These assays were performed at the same time as the rab - 10 lifespan analysis shown in Figure 5C . Mean lifespan was 22 . 9 d for control , 23 . 6 d for bec - 1 RNAi , and 24 . 5 d for vps - 34 RNAi , pair - wise comparison to control , p = 0 . 057 and p = 0 . 16 , respectively , Log - rank ( Mantel - Cox ) test . Please see Table 1 for additional data .

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

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

(A) Average number of LGG-1::GFP-containing puncta in daf-2(e1370) progeny of animals fed either control bacteria or bacteria expressing bec-1, vps-34, or pha-4 dsRNA for their entire lives. p < 0.0001 for bec-1 and vps-34 RNAi-bacteria compared to control RNAi-bacteria, respectively, p = 0.17 for pha-4 RNAi-bacteria compared to control RNAi-bacteria, unpaired, two-tailed t-test. n, total number of seam cells observed. Error bars: ±SEM. See Figure 1 for details. Feeding daf-2 mutants for several generations with pha-4 dsRNA sharply decreased the number of eggs laid (data not shown). The mean lifespan of daf-2(e1370) animals was shortened 12.5% by pha-4 RNAi ([28] and data not shown), and we measured an 11% decrease in puncta in daf-2(e1370) animals fed pha-4 RNAi. Even though this decrease was not statistically significant it remains possible that it relates to the small difference seen in lifespan.


( B ) Average number of LGG - 1 : : GFP - containing puncta in daf - 16 ( mu86 ) ; daf - 2 ( e1370 ) double mutants compared to daf - 2 ( e1370 ) animals ; p = 0 . 50 , unpaired , two - tailed t - test . N2 wild - type animals ( WT ) are shown for comparison . n , total number of seam cells observed . Error bars : ± SEM . See Figure 1 for details . The double mutant expressing the LGG - 1 reporter had a mean lifespan similar to non - transgenic daf - 16 ; daf - 2 double mutants ( data not shown , [ 65 ] ) . Please see Table S1 for quantification of all data .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the phenotypic traits depicted in A . Panels from left to right : the Ruler , Timer and Sizer models , and cortex Col - 0 data . Symbols in the three left - most panels represent simulated data of individual simulated root files ( gray triangles for Ruler , pink squares for Timer and red diamonds for Sizer , n = 30 each ) . Data from individual cortex files in Col - 0 is in green circles ( n = 30 ) . Dashed lines are minimum square linear fits . Pearson correlation coefficient ( r ) for each pair of data are indicated . p stands for the p - value using standard Pearson correlation test . Continuos lines as in Fig . 2 C - E but for the corresponding parameter values ( Table EV3 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and pRPS5a : BRI1 : YFP ; bri1 . Scale bar 1 cm .

O O O O O O O O O O O B-CONTROLLED_VAR O 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 - 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 ) .

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

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

O O O O 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 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 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 B-CONTROLLED_VAR 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 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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.


( B ) Fold - change ± standard error in baseline IL - 6 plasma levels at week 12 from RA patients treated with placebo ( n = 47 ) , baricitinib 1 - mg ( n = 23 ) , 2 - mg ( n = 24 ) , 4 - mg ( n = 23 ) , and 8 - mg ( n = 23 ) in the phase 2b randomized , placebo - controlled study NCT01469013 . p values are for comparisons of baricitinib 1 - mg , 2 - mg , 4 - mg , and 8 - mg change from baseline compared to the placebo change from baseline . Treatment effects were estimated using a mixed effects repeated measures model ( SAS Proc Mixed ) using log10 transformed IL - 6 levels with an unstructured covariance matrix .

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

(B) Fold-change ± standard error in baseline IL-6 plasma levels at week 12 from RA patients treated with placebo (n=47), baricitinib 1-mg (n=23), 2-mg (n=24), 4-mg (n=23), and 8-mg (n=23) in the phase 2b randomized, placebo-controlled study NCT01469013. p values are for comparisons of baricitinib 1-mg, 2-mg, 4-mg, and 8-mg change from baseline compared to the placebo change from baseline. Treatment effects were estimated using a mixed effects repeated measures model (SAS Proc Mixed) using log10 transformed IL-6 levels with an unstructured covariance matrix.


( A and B ) Binding affinities in Kd are shown for baricitinib ( A and B ) , tofacitinib , upadacitinib , AZD7762 ( positive control ) , and broad - spectrum tyrosine kinase inhibitors sunitinib and erlotinib ( B ) . Kd values were estimated from an 11 - point concentration response curve with fixed top parameters and a four - parameter logistic model . Kd , equilibrium affinity constant ( n = 3 )

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

(A and B) Binding affinities in Kd are shown for baricitinib (A and B), tofacitinib, upadacitinib, AZD7762 (positive control), and broad-spectrum tyrosine kinase inhibitors sunitinib and erlotinib (B). Kd values were estimated from an 11-point concentration response curve with fixed top parameters and a four-parameter logistic model. Kd, equilibrium affinity constant (n=3)


( C ) Protein and mRNA expression of relevant NAKs in primary human liver organoids ( n = 3 ) ( p < 0 . 05 ) . The immunofluorescence shows the juxtaposition of 1A9 ( signal from the SARS - CoV - 2 ) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain ( n = 1 ) . Colocalization of 1A9 and ACE2 is depicted with an 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 B-MEASURED_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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(C) Protein and mRNA expression of relevant NAKs in primary human liver organoids (n=3) (p<0.05). The immunofluorescence shows the juxtaposition of 1A9 (signal from the SARS-CoV-2) in red and ACE2 in green along with the spheroid nuclei in blue as a DAPI stain (n=1). Colocalization of 1A9 and ACE2 is depicted with an arrow.


( D , E ) Time course of days ( one , three , and six days ) post infection ( dpi ) using liver spheroids and SARS - CoV - 2 at 0 . 1 and 1 . 0 multiplicity of infection ( MOI ) ( n = 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) Time course of days (one, three, and six days) post infection (dpi) using liver spheroids and SARS-CoV-2 at 0.1 and 1.0 multiplicity of infection (MOI) (n=1).


( F ) Viability of spheroids was quantified by measuring intracellular ATP concentrations . Note that no significant ( n . s . ; Student ' s t - test ) decrease in cell viability was observed for any concentration tested ( n = 10 ) .

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

(F) Viability of spheroids was quantified by measuring intracellular ATP concentrations. Note that no significant (n.s.; Student's t-test) decrease in cell viability was observed for any concentration tested (n=10).


( H ) Viral load in infected ( MOI 0 . 1 ) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection ( * p < 0 . 05 ; Student ' s t - test ) . Data are mean ± standard error of mean ( n = 3 ) .

O O O 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

(H) Viral load in infected (MOI 0.1) spheroids as measured by qPCR after treatment with 400 nM and 800 nM of baricitinib 48 hours after infection (*p<0.05; Student's t-test). Data are mean ± standard error of mean (n=3).


( A ) Detection of SARS - CoV - 2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID - 19 Plus RealAmp Kit assay ( ELITechGroup S . p . A . , Turin , Italy ) . Three viral target genes , RdRp , N , and E , together with the housekeeping gene GAPDH were simultaneously amplified . Here the most sensitive target gene , N , is shown . Samples with Ct values > 40 were defined as negative . A dashed horizontal line indicates the cut - off . IL - 6 values are shown in the same graph . Time of baricitinib treatment is highlighted ( Patients A , B , and D for 10 days , Patient C for 12 days ) with total time of evaluation on the X - axis . Fever and cough symptoms are indicated . The bars in grey indicate that cough has improved but has not resolved .

O O O O O O O O O O O O O O O O O O O O O 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 O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Detection of SARS-CoV-2 in nasopharyngeal swabs and peripheral blood using the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup S.p.A., Turin, Italy). Three viral target genes, RdRp, N, and E, together with the housekeeping gene GAPDH were simultaneously amplified. Here the most sensitive target gene, N, is shown. Samples with Ct values > 40 were defined as negative. A dashed horizontal line indicates the cut-off. IL-6 values are shown in the same graph. Time of baricitinib treatment is highlighted (Patients A, B, and D for 10 days, Patient C for 12 days) with total time of evaluation on the X-axis. Fever and cough symptoms are indicated. The bars in grey indicate that cough has improved but has not resolved.


( B ) Levels of CD3 + , CD4 + , CD8 + , B cells ( CD19 + ) and NK cells ( CD3 - , CD16 + , CD56 + ) were determined using the AQUIOS CL Flow Cytometry System ( Beckman Coulter ) .

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

(B) Levels of CD3+, CD4+, CD8+, B cells (CD19+) and NK cells (CD3-, CD16+, CD56+) were determined using the AQUIOS CL Flow Cytometry System (Beckman Coulter).


( C ) Total serum IgG , IgA , and IgM levels are shown for all patients .

(C) Total serum IgG, IgA, and IgM levels are shown for all patients.


Detection of IgG antibodies against the S1 / S2 antigens of SARS - CoV - 2 . Time of baricitinib treatment is indicated for all patients ( patients A , B , and D for 10 days , patient C for 12 days ) .

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

Detection of IgG antibodies against the S1/S2 antigens of SARS-CoV-2. Time of baricitinib treatment is indicated for all patients (patients A, B, and D for 10 days, patient C for 12 days).


( A ) Levels of D - dimer , CRP , ferritin , white blood cells , and neutrophils are shown for all patients .

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

(A) Levels of D-dimer, CRP, ferritin, white blood cells, and neutrophils are shown for all patients.


( B ) Levels of aspartate aminotransferase , alanine aminotransferase , and bilirubin are shown for all patients . ALT , alanine aminotransferase ; AST , aspartate

(B) Levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin are shown for all patients. ALT, alanine aminotransferase; AST, aspartate aminotransferase


( C ) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time . Day one CT scan shows ground glass opacity ( arrows ) sub - pleurally in the lower lobes bilaterally ( early stage one according to Pan et al ( Pan , Ye et al . , 2020 ) . Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles ( arrows ) in the site of the early lesions ( absorption stage four according to Pan et al ( Pan et al . , 2020 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chest CT scan for Patient C on day one and 19 from symptom onset showing clinical improvement over time. Day one CT scan shows ground glass opacity (arrows) sub-pleurally in the lower lobes bilaterally (early stage one according to Pan et al (Pan, Ye et al., 2020). Day 19 CT scan shows that consolidation was gradually absorbed with evident residual fibrosis and emphysema bubbles (arrows) in the site of the early lesions (absorption stage four according to Pan et al (Pan et al., 2020).


B . The 5 ' UTR of rne is bound by RNase E and non - genomically encoded A - tails are maximally recovered - 9nt from the rne start codon . Known stem loop strucutres ( HP1 - 3 ) and the ribosomal binding site ( RBS ) are shaded grey .

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B. The 5'UTR of rne is bound by RNase E and non-genomically encoded A-tails are maximally recovered -9nt from the rne start codon. Known stem loop strucutres (HP1-3) and the ribosomal binding site (RBS) are shaded grey.


C . RNase E binding and oligoadenylation of the pdlB - yigL dicistronic transcript . The reported RNase E cleavage site ( red dashed line ) and SgrS binding site ( grey shading ) are indicated .

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C. RNase E binding and oligoadenylation of the pdlB-yigL dicistronic transcript. The reported RNase E cleavage site (red dashed line) and SgrS binding site (grey shading) are indicated.


D . Length of non - genomically encoded oligo ( A ) tails recovered from RNase E bound reads .

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D. Length of non-genomically encoded oligo(A) tails recovered from RNase E bound reads.


E . Position of Hfq binding and oliogadenylation relative to RNase E binding peaks . The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak . A detailed description of the data processing is presented in the Appended Supplementary Methods .

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E. Position of Hfq binding and oliogadenylation relative to RNase E binding peaks. The cumulative position of Hfq and oligoadenylation peaks were determined relative to RNase E binding peaks for 672 RNase E binding sites that were within 1kb of an Hfq binding peak. A detailed description of the data processing is presented in the Appended Supplementary Methods.


B . Hfq binding ( blue ) , RNase E binding ( grey ) , and oligoadenylation of the MicA binding site in the ompA mRNA . The MicA seed sequence is shaded blue .

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B. Hfq binding (blue), RNase E binding (grey), and oligoadenylation of the MicA binding site in the ompA mRNA. The MicA seed sequence is shaded blue.


C . Interaction strength ( Gibbs free energy ) of all RNA - RNA interactions recovered by RNase E - CLASH ( solid line ) and for randomly paired hybrid read halves ( dashed line ) .

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C. Interaction strength (Gibbs free energy) of all RNA-RNA interactions recovered by RNase E-CLASH (solid line) and for randomly paired hybrid read halves (dashed line).


D . Interaction strength of sRNA - mRNA hybrid read halves ( solid line ) and randomly paired sRNA and mRNA read halves ( dashed line ) .

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D. Interaction strength of sRNA-mRNA hybrid read halves (solid line) and randomly paired sRNA and mRNA read halves (dashed line).


E . Position of sRNA - mRNA interactions relative to the mRNA start codon .

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E. Position of sRNA-mRNA interactions relative to the mRNA start codon.