( C ) Cross - Linking and Immunoprecipitation experiment ( CLIP ) was performed on cell extracts from normoxic and hypoxic ( 1 % O2 , 24h ) UV irradiated WT or eIF4EHP KO S2 cells . eIF4EHP was immunoprecipitated with eIF4EHP antibody - coated protein G sepharose beads before Ldh and Rpl32 mRNA detection by RT - qPCR in total extract ( input ) or immunoprecipitation eluate . mRNA enrichment levels in eluate as compared to input from 3 independent experiments were calculated as described Data represent mean + / - SD . Statistical significance was tested by two - way ANOVA followed by Sidak ' s multiple comparisons test , * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross-Linking and Immunoprecipitation experiment (CLIP) was performed on cell extracts from normoxic and hypoxic (1% O2, 24h) UV irradiated WT or eIF4EHP KO S2 cells. eIF4EHP was immunoprecipitated with eIF4EHP antibody-coated protein G sepharose beads before Ldh and Rpl32 mRNA detection by RT-qPCR in total extract (input) or immunoprecipitation eluate. mRNA enrichment levels in eluate as compared to input from 3 independent experiments were calculated as described Data represent mean +/- SD. Statistical significance was tested by two-way ANOVA followed by Sidak's multiple comparisons test, * P<0.05.


( A ) Female or male flies with chromosomal insertions of an UAS _ eIF4EHP ( BDSC 36876 ) RNAi construct were crossed with male or female Tub > Gal4 / Tm3 ( BDSC 5138 ) driver flies . Two hours after mating , embryos were transferred to 6 % O2 ( lowest tolerated O2 concentration ) or kept in normoxia until eclosion . Tub - Gal4 / UAS - eIF4EHP _ RNAi and Tm3 / UAS - eIF4EHP _ RNAi F1 individuals were counted based on the presence ( control ) or absence ( eIF4EHP KD ) of the Tm3 SB marker and results were expressed as % of total eclosions for each condition . Data represent mean + / - SD fro 2 independent biological replicates . Statistical significance was tested by two - way ANOVA followed by Sidak ' s multiple comparisons test . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Female or male flies with chromosomal insertions of an UAS_eIF4EHP (BDSC 36876) RNAi construct were crossed with male or female Tub>Gal4/Tm3 (BDSC 5138) driver flies. Two hours after mating, embryos were transferred to 6% O2 (lowest tolerated O2 concentration) or kept in normoxia until eclosion. Tub-Gal4/UAS-eIF4EHP_RNAi and Tm3/UAS-eIF4EHP_RNAi F1 individuals were counted based on the presence (control) or absence (eIF4EHP KD) of the Tm3 SB marker and results were expressed as % of total eclosions for each condition. Data represent mean +/-SD fro 2 independent biological replicates. Statistical significance was tested by two-way ANOVA followed by Sidak's multiple comparisons test. **** P<0.0001; *** P<0.001; ** P<0.01; * P<0.05.


( B ) Mobility of age - matched Tub - Gal4 / UAS - eIF4EHP _ RNAi ( eIF4EHPRNAi ) , TM3 / UAS - eIF4EHP _ RNAi ( control ) ( upper panels ) or Tub - Gal4 / UAS - mCherry _ RNAi ( m - cherryRNAi ) ( lower panel ) females was assessed by negative geotaxis assay in normoxic conditions ( 21 % O2 ) or 40 min . and 48h after an initial exposure to 1 % O2 for 24h . Flies climbing past threshold within 120s were counted and expressed as % of total flies . Each assay was performed in triplicate with 20 flies . Data are mean + / - SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mobility of age-matched Tub-Gal4/UAS-eIF4EHP_RNAi (eIF4EHPRNAi), TM3/UAS-eIF4EHP_RNAi (control) (upper panels) or Tub-Gal4/UAS-mCherry_RNAi (m-cherryRNAi) (lower panel) females was assessed by negative geotaxis assay in normoxic conditions (21% O2) or 40 min. and 48h after an initial exposure to 1% O2 for 24h. Flies climbing past threshold within 120s were counted and expressed as % of total flies. Each assay was performed in triplicate with 20 flies. Data are mean +/- SD.


( I ) Representative high - resolution confocal images of C . elegans expressing mitochondria - targeted GFP in the body wall muscle cells ( scale bar = 10 μm ) . Quantification of mitochondrial morphology ( elongation ) is reported in the right panel ( one - way ANOVA with Holm - Sidak ' s correction , n = 37 - 47 animals from 3 independent experiments ) . Data information : , middle line represents the median , and the upper and lower lines represent the upper and lower quartiles , respectively . Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(I) Representative high-resolution confocal images of C. elegans expressing mitochondria-targeted GFP in the body wall muscle cells (scale bar= 10 μm). Quantification of mitochondrial morphology (elongation) is reported in the right panel (one-way ANOVA with Holm-Sidak's correction, n=37-47 animals from 3 independent experiments). Data information: , middle line represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( J ) Representative western blot analysis and densitometry of zcIs13 [ hsp - 6p : : GFP ] expression in control , gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E animals ( one - way ANOVA with Holm - Sidak ' s correction , n = 3 biological replicates ) . Data information : , bars represent mean ± SEM Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(J) Representative western blot analysis and densitometry of zcIs13[hsp-6p::GFP] expression in control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E animals (one-way ANOVA with Holm-Sidak's correction, n=3 biological replicates). Data information: , bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( M ) Immunoblot and statistical analysis of zcIs13 [ hsp - 6p : : GFP ] expression in control and nuo - 6 ( qm200 ) animals fed with empty vector ( C RNAi ) and cest - 2 . 2 RNAi bacteria ( unpaired t - test , n = 3 biological replicates ) . Data information : bars represent mean ± SEM Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(M) Immunoblot and statistical analysis of zcIs13[hsp-6p::GFP] expression in control and nuo-6(qm200) animals fed with empty vector (C RNAi) and cest-2.2 RNAi bacteria (unpaired t-test, n=3 biological replicates). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( A ) Representative images of 5 - day - old wt , gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant animals stained with Oil Red O ( ORO ) ( scale bar = full image - 100 μm ; hindgut magnification - 20 μm ) . ( B ) Quantification of hindgut ( left panel ) , foregut ( center panel ) and whole body ( right panel ) of 5 - day - old wt , gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes stained with ORO ( one - way ANOVA with Tukey ' s multiple comparison test , n = 18 - 26 animals ) . ( C ) Quantification of mean intensity of ORO staining in the hindgut of wt , gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes at different time points , starting from L4 stage ( one - way ANOVA with Dunnett ' s post hoc correction , n = 18 - 26 animals ) . ( D ) Mean intensity of ORO staining in the whole body of 5 - day - old wt , gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes plotted against body area . Data information : , bars represent mean ± SEM points represent mean ± SEM . Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with Oil Red O (ORO) (scale bar= full image-100 μm; hindgut magnification-20 μm). (B) Quantification of hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes stained with ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals). (C) Quantification of mean intensity of ORO staining in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals). (D) Mean intensity of ORO staining in the whole body of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes plotted against body area. Data information: , bars represent mean ±SEM points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( E ) Representative images of wt , cest - 2 . 2 O / E and cest - 2 . 2 ( lf ) animals stained with ORO . Arrows indicate mislocalized lipid droplets . ( F ) Quantification ( mean intensity ± SEM ) of ORO staining in hindgut ( left panel ) , foregut ( center panel ) and whole body ( right panel ) of 5 - day - old nematodes ( one - way ANOVA with Dunnett ' s post hoc correction , n = 11 - 17 animals ) . Data information : , bars represent mean ± SEM Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of wt, cest-2.2 O/E and cest-2.2 (lf) animals stained with ORO. Arrows indicate mislocalized lipid droplets. (F) Quantification (mean intensity ± SEM) of ORO staining in hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals). Data information: , bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( G ) Representative images of 5 - day - old wt , gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant animals stained with LipidTOX ( scale bar = 100 μm ) . ( H ) Statistical analysis of whole - body LipidTOX intensity of 5 - day - old nematodes ( ordinary one - way ANOVA with Tukey ' s multiple comparisons test , n = 25 - 37 animals ) . Data information : bars represent mean ± SEM ; Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with LipidTOX (scale bar= 100 μm). (H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( I - J ) Representative images of nematodes stained with LipidTOX ( I ) ( scale bar = 100 μm ) , with quantification ( J ) ( mean intensity ± SEM ) of LipidTOX staining . Data information : bars represent mean ± SEM Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(I-J) Representative images of nematodes stained with LipidTOX (I) (scale bar= 100 μm), with quantification (J) (mean intensity ± SEM) of LipidTOX staining. Data information: bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( K ) Representative thin layer chromatography ( TLC ) plate of samples from 5 - day - old gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes . ( L ) Densitometry of TLC - separated free fatty acids ( FFA ) across three biological replicates in 5 - day - old mutant animals . Data information : bars represent mean ± SEM ; Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(K) Representative thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes. (L) Densitometry of TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals. Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( N ) Sum of TLC - separated FFA of 5 - day old animals ( n = 5 biological replicates ) . ( O ) Quantification of individual FFA detected after TLC separation in 5 - day - old gas - 1 ( fc21 ) and gas - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes ( n = 5 biological replicates , n . d . = not detected ) . Data information : bars represent mean ± SEM ; Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(N) Sum of TLC-separated FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after TLC separation in 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


A , B . Immunoblotting were performed to validate Mettl3 or Mettl14 expression levels in CT26 and B16 cells as indicated . Gapdh served as a 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 O O O

A, B. Immunoblotting were performed to validate Mettl3 or Mettl14 expression levels in CT26 and B16 cells as indicated. Gapdh served as a loading control.


Tumor volume was monitored for control and Mettl3 or Mettl14 - depleted tumors with treatment as indicated in CT26 colon cancer Data are mean ± s . e . m of the indicated number of mice in each group . n , the numbers of mice . * P < 0 . 05 ; * * * P < 0 . 001 by Student ' s t - tests .

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

Tumor volume was monitored for control and Mettl3 or Mettl14-depleted tumors with treatment as indicated in CT26 colon cancer Data are mean ± s.e.m of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * * P < 0.001 by Student's t-tests.


Tumor volume was monitored for control and Mettl3 or Mettl14 - depleted tumors with treatment as indicated in B16 melanoma , respectively . Data are mean ± s . e . m of the indicated number of mice in each group . n , the numbers of mice . * P < 0 . 05 ; * * * P < 0 . 001 by Student ' s t - tests .

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

Tumor volume was monitored for control and Mettl3 or Mettl14-depleted tumors with treatment as indicated in B16 melanoma, respectively. Data are mean ± s.e.m of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * * P < 0.001 by Student's t-tests.


E , F . Survival analysis of control tumors and those with depleted genes were recorded as indicated in CT26 colon cancer and B16 melanoma , respectively . Data are mean ± s . e . m . of the indicated number of mice in each group . n , the numbers of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F. Survival analysis of control tumors and those with depleted genes were recorded as indicated in CT26 colon cancer and B16 melanoma, respectively. Data are mean ± s.e.m. of the indicated number of mice in each group. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


A . Percentage of tumor - infiltrating T cells , Treg and NK cells were identified by flow cytometry from CT26 tumors as indicated . Each spot represents one mouse . * P < 0 . 05 ; * * P < 0 . 01 by Student ' s t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentage of tumor-infiltrating T cells, Treg and NK cells were identified by flow cytometry from CT26 tumors as indicated. Each spot represents one mouse. * P < 0.05; * * P < 0.01 by Student's t-tests.


B . Representative images of CD8 by IHC staining . Tissue sections from BALB / c mice bearing the indicated knockout of genes with treatment of PD1 antibody . Scale bars , 50 μm .

O O O O O O O O O O O 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. Representative images of CD8 by IHC staining. Tissue sections from BALB/c mice bearing the indicated knockout of genes with treatment of PD1 antibody. Scale bars, 50 μm.


C . Percentage of Granzyme B - expressing CD8 + T cells from control and Mettl3 or Mettl14 deficient CT26 tumors . Each spot represents one mouse . * P < 0 . 05 ; * * P < 0 . 01 by Student ' s t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Percentage of Granzyme B-expressing CD8+ T cells from control and Mettl3 or Mettl14 deficient CT26 tumors. Each spot represents one mouse. * P < 0.05; * * P < 0.01 by Student's t-tests.


Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8 - depleting antibody and PD - 1 antibody or PD - 1 as indicated . Tumor volume was measured over time points . n , the numbers of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - tests .

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

Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1 as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8 - depleting antibody and PD - 1 antibody or PD - 1 / GVAX as indicated . Tumor volume was measured over time points . n , the numbers of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - tests .

O O 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 O O O

Mice bearing control and Mettl3 or Mettl14 null tumors were treated with CD8-depleting antibody and PD-1 antibody or PD-1/GVAX as indicated. Tumor volume was measured over time points. n, the numbers of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


IFNγ production in serum ( F ) from BALB / c mice by ELISA . The results are representatives of at least three independent experiments . n , the numbers of mice . Data are mean ± s . e . m . * P < 0 . 05 by Student ' s t - tests .

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

IFNγ production in serum (F) from BALB/c mice by ELISA. The results are representatives of at least three independent experiments. n, the numbers of mice. Data are mean ± s.e.m. * P < 0.05 by Student's t-tests.


IFNγ production in Intratumor ( G ) from BALB / c mice by ELISA . The results are representatives of at least three independent experiments . n , the numbers of mice . Data are mean ± s . e . m . * P < 0 . 05 by Student ' s t - tests .

IFNγ production in Intratumor (G) from BALB/c mice by ELISA. The results are representatives of at least three independent experiments. n, the numbers of mice. Data are mean ± s.e.m. * P < 0.05 by Student's t-tests.


A . Volcano plot of differentially expressed genes obtained by DESeq2 analysis in Mettl3 or Mettl14 null tumors compared to control tumors . Significantly upregulated or downregulated genes are plotted in red and blue points , respectively . n . s , non - significant .

O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot of differentially expressed genes obtained by DESeq2 analysis in Mettl3 or Mettl14 null tumors compared to control tumors. Significantly upregulated or downregulated genes are plotted in red and blue points, respectively. n.s, non-significant.


F . Representative genes with m6A sites generated by integrative genomics viewer . Data is representative of duplicates with similar results . Red represents reads coverage of IP sample and blue represents reads coverage of input sample . Rectangular cyan shade represents the m6A peaks located on transcripts .

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 B-MEASURED_VAR O O O O O

F. Representative genes with m6A sites generated by integrative genomics viewer. Data is representative of duplicates with similar results. Red represents reads coverage of IP sample and blue represents reads coverage of input sample. Rectangular cyan shade represents the m6A peaks located on transcripts.


G . m6A enrichment of Stat1 and Irf1 was examined by m6A RIP - qPCR in control , Mettl3 , or Mettl14 depleted CT26 tumors as indicated . Ctla4 functioned as a m6A negative control ( Wang et al . , 2019 ) Data are mean ± s . d . * * P < 0 . 01 by Student ' s t - tests .

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

G. m6A enrichment of Stat1 and Irf1 was examined by m6A RIP-qPCR in control, Mettl3, or Mettl14 depleted CT26 tumors as indicated. Ctla4 functioned as a m6A negative control (Wang et al., 2019) Data are mean ± s.d. * * P < 0.01 by Student's t-tests.


H . Immunoblots of p - Stat1 ( phosphorylated ) , Stat1 and Irf1 were carried out in the indicated tumors in triplicate with Gapdh as a 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 O O O O

H. Immunoblots of p-Stat1 (phosphorylated), Stat1 and Irf1 were carried out in the indicated tumors in triplicate with Gapdh as a loading control.


I . Tumor growth from CT26 cells with Mettl3 , Mettl14 , Mettl3 / Stat1 , Mettl3 / Irf1 , Mettl14 / Stat1 or Mettl14 / Irf1 - depleted genes and control under treatment of PD - 1 antibody as indicated . n , the numbers of mice . Data are mean ± s . e . m of the indicated number of mice in each group .

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

I. Tumor growth from CT26 cells with Mettl3, Mettl14, Mettl3/Stat1, Mettl3/Irf1, Mettl14/Stat1 or Mettl14/Irf1-depleted genes and control under treatment of PD-1 antibody as indicated. n, the numbers of mice. Data are mean ± s.e.m of the indicated number of mice in each group.


A . Cellular proliferation analysis of Mettl3 or Mettl14 depleted and control CT26 cells treated with indicated combinations of cytokines for 48h . The mean ± SD of three replicates is shown . * P < 0 . 05 ; * * P < 0 . 01 by Student ' s t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cellular proliferation analysis of Mettl3 or Mettl14 depleted and control CT26 cells treated with indicated combinations of cytokines for 48h. The mean ± SD of three replicates is shown. * P < 0.05; * * P < 0.01 by Student's t-tests.


B . BALB / c mice bearing Mettl3 or Mettl14 deficient and control tumors were treated with IFNγ - blocking antibody and PD - 1 antibody as indicated . Tumor size was measured over time . n , the numbers of mice . Data are mean ± s . e . m of the indicated number of mice . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BALB/c mice bearing Mettl3 or Mettl14 deficient and control tumors were treated with IFNγ -blocking antibody and PD-1 antibody as indicated. Tumor size was measured over time. n, the numbers of mice. Data are mean ± s.e.m of the indicated number of mice. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


C . Quantitative RT - PCR was performed to identify transcriptional changes of the IFN - γ response gene expression ( n = 3 ) . Data are shown as the relative fold change ( RFC , color coded bar ) .

O O O O O O O O O O O O O O O O O O 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. Quantitative RT-PCR was performed to identify transcriptional changes of the IFN-γ response gene expression (n=3). Data are shown as the relative fold change (RFC, color coded bar).


D , E . mRNA stability of Stat1 and Irf1 were measured by qRT - PCR in tumor cells treated with IFN - γ and actinomycin D . Mean ± SD of n = 3 . * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - tests .

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

D, E. mRNA stability of Stat1 and Irf1 were measured by qRT-PCR in tumor cells treated with IFN-γ and actinomycin D. Mean ± SD of n=3. * * P < 0.01; * * * P < 0.001 by Student's t-tests.


F . Validation the effect of knock out of Ythdf1 - 3 using western blotting , Gapdh served as a 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

F. Validation the effect of knock out of Ythdf1-3 using western blotting, Gapdh served as a loading control.


G . qPCR analysis of Stat1 and Irf1 ' s expression in the indicated depletion of CT26 cells with / without stimulation of IFNγ . Mean ± SD of n = 3 . * P < 0 . 05 by Student ' s t - tests .

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

G. qPCR analysis of Stat1 and Irf1's expression in the indicated depletion of CT26 cells with/without stimulation of IFNγ. Mean ± SD of n=3. * P < 0.05 by Student's t-tests.


H . Western blot analysis of Mettl3 and Mettl14 in overexpressed CT26 cells . Gapdh served as a loading control for each .

H. Western blot analysis of Mettl3 and Mettl14 in overexpressed CT26 cells. Gapdh served as a loading control for each.


I , J . qPCR analysis of the mRNA stability of Stat1 and Irf1 in the indicated CT26 cells treated with IFNγ and actinomycin D . Mean ± SD of n = 3 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 by Student ' s t - tests .

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

I, J. qPCR analysis of the mRNA stability of Stat1 and Irf1 in the indicated CT26 cells treated with IFNγ and actinomycin D. Mean ± SD of n=3. * P < 0.05; * * P < 0.01; * * * P < 0.001 by Student's t-tests.


A , B . The protein level of STAT1 was negatively correlated with METTL3 and METTL14 in human pMMR - MSI - L CRC colon tissue ( r2 = - 3 . 2477 for METTL3 , r2 = - 2 . 7491 for METTL14 ) . Each dot represents one tumor tissue .

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

A, B. The protein level of STAT1 was negatively correlated with METTL3 and METTL14 in human pMMR-MSI-L CRC colon tissue (r2=-3.2477 for METTL3, r2=-2.7491 for METTL14). Each dot represents one tumor tissue.


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 O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


A Phenotypes of WT , dja6 , dja5 , dja6 dja5 and RNAi seedlings on MS medium with 2 % sucrose . Scale bars = 0 . 5 cm .

O O O O O O 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 Phenotypes of WT, dja6, dja5, dja6 dja5 and RNAi seedlings on MS medium with 2% sucrose. Scale bars = 0.5 cm.


B , C Phenotypes and chlorophyll fluorescence images of 6 - week - old WT , dja6 dja5 and complemented plants ( B ) on MS medium with 2 % sucrose , scale bars = 0 . 5 cm ; WT , dja6 , dja5 and RNAi mutants ( C ) grown on soil , scale bars = 1 cm . Fluorescences in ( B ) and ( C ) were measured with the FluorCam700MF and visualized using a pseudocolor index as indicated on the right .

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 O O O O O O O O O O O O O O O O O O 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,C Phenotypes and chlorophyll fluorescence images of 6-week-old WT, dja6 dja5 and complemented plants (B) on MS medium with 2% sucrose, scale bars = 0.5 cm; WT, dja6, dja5 and RNAi mutants (C) grown on soil, scale bars = 1 cm. Fluorescences in (B) and (C) were measured with the FluorCam700MF and visualized using a pseudocolor index as indicated on the right.


D Chlorophyll content of total leaves in 6 - week - old WT , dja6 , dja5 , dja6 dja5 and RNAi seedlings . FW , fresh weight . Data are the means ± SEM ( n = 7 biological replicates ) .

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

D Chlorophyll content of total leaves in 6-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings. FW, fresh weight. Data are the means ± SEM (n = 7 biological replicates).


E The ultrastructure of plastids from WT , dja6 , dja5 , dja6 dja5 and RNAi mutants . T , thylakoids . Scale bars = 0 . 2 μm .

E The ultrastructure of plastids from WT, dja6, dja5, dja6 dja5 and RNAi mutants. T, thylakoids. Scale bars = 0.2 μm.


A , B Total protein was extracted from 4 - week - old WT , dja6 , dja5 , dja6 dja5 and RNAi seedlings , respectively . WT and dja6 dja5 used in ( A ) were grown on MS medium with 2 % sucrose , while WT , dja6 , dja5 and RNAi mutants used in ( B ) were grown on soil . 10 μg protein was loaded , except for detection with anti - SUFE1 and anti - SUFA antiserum , for which 25 μg were loaded . Specific bands were identified by immunoblotting and by their molecular weight , and are indicated by asterisks . Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right , respectively . The Fe - S types of Fe - S proteins are shown in parentheses . Actin served as controls to normalize protein levels . For each protein , three independent biological replicates were performed and a representative one is shown .

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

A, B Total protein was extracted from 4-week-old WT, dja6, dja5, dja6 dja5 and RNAi seedlings, respectively. WT and dja6 dja5 used in (A) were grown on MS medium with 2% sucrose, while WT, dja6, dja5 and RNAi mutants used in (B) were grown on soil. 10 μg protein was loaded, except for detection with anti-SUFE1 and anti-SUFA antiserum, for which 25 μg were loaded. Specific bands were identified by immunoblotting and by their molecular weight, and are indicated by asterisks. Designations of photosynthetic protein complexes and their diagnostic components are labeled on the left and right, respectively. The Fe-S types of Fe-S proteins are shown in parentheses. Actin served as controls to normalize protein levels. For each protein, three independent biological replicates were performed and a representative one is shown.


A - C Metal concentrations in the WT , dja6 dja5 and RNAi mutants . Fe concentrations in total leaves ( A and B ) and total chloroplast ( C ) from 4 - week - old WT , dja6 dja5 and RNAi mutants were quantified by inductively coupled plasma mass spectrometry , and are shown as means ± SEM ( n = 3 biological replicates ) of μg g - 1 dry weight ( DW ) . WT and RNAi mutants used in ( A and C ) were grown on soil , while WT and dja6 dja5 seedlings used in ( B ) were grown on MS medium with 2 % sucrose . Similar results were obtained in two additional independent biological experiments . Asterisks indicate significant differences from the value of WT ( two - sample Student ' s t - test ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Metal concentrations in the WT, dja6 dja5 and RNAi mutants. Fe concentrations in total leaves (A and B) and total chloroplast (C) from 4-week-old WT, dja6 dja5 and RNAi mutants were quantified by inductively coupled plasma mass spectrometry, and are shown as means ± SEM (n = 3 biological replicates) of μg g-1 dry weight (DW). WT and RNAi mutants used in (A and C) were grown on soil, while WT and dja6 dja5 seedlings used in (B) were grown on MS medium with 2% sucrose. Similar results were obtained in two additional independent biological experiments. Asterisks indicate significant differences from the value of WT (two-sample Student's t-test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).


D Volcano plot of the result from quantitative transcriptome profiling in WT and dja6 dja5 mutant . Statistical analysis was performed using Student ' s t - test . Data on the X - axis represent the Log2 - ratio of FC ( fold change ) between the absolute abundances of transcripts identified from dja6 dja5 against WT . The Y - axis represents the Log10 of FDR ( false discovery rate ) . All DEGs ( differentially expressed genes ) with FDR < 0 . 01 and FC > 2 are marked in grey , while all iron - related DEGs with FDR < 0 . 01 and FC > 2 are marked in red . The iron - related DEGs labeled with red dots within the black boxes are referred to in the text . All data are based on three biological replicates .

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D Volcano plot of the result from quantitative transcriptome profiling in WT and dja6 dja5 mutant. Statistical analysis was performed using Student's t-test. Data on the X-axis represent the Log2-ratio of FC (fold change) between the absolute abundances of transcripts identified from dja6 dja5 against WT. The Y-axis represents the Log10 of FDR (false discovery rate). All DEGs (differentially expressed genes) with FDR < 0.01 and FC > 2 are marked in grey, while all iron-related DEGs with FDR < 0.01 and FC > 2 are marked in red. The iron-related DEGs labeled with red dots within the black boxes are referred to in the text. All data are based on three biological replicates.


D DJA6 can bind Fe2 + in chloroplast . Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs , respectively . The untransformed protoplasts and protoplasts transformed with non - fused EBFP proteins were used as controls . The protoplasts were further stained by FeRhoNoxTM - 1 ( red ) to visualize the cellular distribution of Fe2 + . Green , EBFP fluorescence signals ; magenta , chloroplast autofluorescence signals ; yellow , regions where the EBFP and FeRhoNoxTM - 1 fluorescence signals merged . The speckles which DJA6 - EBFP co - localizes with FeRhoNoxTM - 1 are indicated by white arrows . Scale bars = 5 μm .

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

D DJA6 can bind Fe2+ in chloroplast. Arabidopsis mesophyll protoplasts were transformed with plasmids that express the indicated gene constructs, respectively. The untransformed protoplasts and protoplasts transformed with non-fused EBFP proteins were used as controls. The protoplasts were further stained by FeRhoNoxTM-1 (red) to visualize the cellular distribution of Fe2+. Green, EBFP fluorescence signals; magenta, chloroplast autofluorescence signals; yellow, regions where the EBFP and FeRhoNoxTM-1 fluorescence signals merged. The speckles which DJA6-EBFP co-localizes with FeRhoNoxTM-1 are indicated by white arrows. Scale bars = 5 μm.


B Co - IP assays for the interactions of DJA6 with SUFE1 and SUFC . Total protein extracts of protoplasts co - transfected with designated constructs were subjected to immunoprecipitation , and then analyzed by immunoblotting with antibodies indicated on the right . Input ( left lane ) indicates that 100 μg total protein was loaded on the gel .

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B Co-IP assays for the interactions of DJA6 with SUFE1 and SUFC. Total protein extracts of protoplasts co-transfected with designated constructs were subjected to immunoprecipitation, and then analyzed by immunoblotting with antibodies indicated on the right. Input (left lane) indicates that 100 μg total protein was loaded on the gel.


G , H BiFC analyses for the interactions between truncated forms of DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts . Scale bars = 10 μm .

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G, H BiFC analyses for the interactions between truncated forms of DJA6 and SUFE1 or SUFC in Arabidopsis mesophyll protoplasts. Scale bars = 10 μm.


C Phenotypes and chlorophyll fluorescence images of 6 - week - old WT and 35Spro : SynDJA6 - FLAG / dja6 dja5 transgenic plants grown on soil . Scale bars = 1 cm .

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C Phenotypes and chlorophyll fluorescence images of 6-week-old WT and 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants grown on soil. Scale bars = 1 cm.


D Expression of FLAG - tagged SynDJA6 in 35Spro : SynDJA6 - FLAG / dja6 dja5 transgenic plants . Total leaf extracts ( 100 μg proteins ) from WT and transgenic seedlings were loaded . FLAG - tagged SynDJA6 is indicated by asterisk .

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D Expression of FLAG-tagged SynDJA6 in 35Spro:SynDJA6-FLAG/dja6 dja5 transgenic plants. Total leaf extracts (100 μg proteins) from WT and transgenic seedlings were loaded. FLAG-tagged SynDJA6 is indicated by asterisk.


A . Gentamicin survival assay showing bacteria captured by DC or B cells . Data information : Column bars in the figure represent the mean of at least three independent experiments . Error bars indicate the SD . Significant differences , analyzed by T test ( two samples ) or one way ANOVA ( more than two samples ) , are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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A. Gentamicin survival assay showing bacteria captured by DC or B cells. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Significant differences, analyzed by T test (two samples) or one way ANOVA (more than two samples), are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


B - C Confocal images of DCs decorated with HEL ( not coloured ) infected with L . moncytogenes ( in red ) , coincubated during 30 min ( B ) or 120 min ( C ) with noninfected B cells ( in blue ) isolated from MD4 mice . Actin is shown in cyan . In C , the orthogonal views show intracellular bacteria in B cell . Scale bars represent 5 μm . White arrow in B point to a bacterium at the B cell - DC contact area surrounded by actin . Arrowheads in C indicate the XZ and YZ axes .

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B-C Confocal images of DCs decorated with HEL (not coloured) infected with L.moncytogenes (in red), coincubated during 30 min (B) or 120 min (C) with noninfected B cells (in blue) isolated from MD4 mice. Actin is shown in cyan. In C, the orthogonal views show intracellular bacteria in B cell. Scale bars represent 5 μm. White arrow in B point to a bacterium at the B cell-DC contact area surrounded by actin. Arrowheads in C indicate the XZ and YZ axes.


G . Bacteria - infected and HEL - loaded DCs were co - incubated with B cells from MD4 mouse . After conjugate formation , BacB cells were re - isolated , and intracellular bacteria were detected by gentamicin survival assays at different times . Data were normalized compared to time 0 . As control of the bacterial fitness , in parallel , HeLa cells were directly infected .

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G. Bacteria-infected and HEL-loaded DCs were co-incubated with B cells from MD4 mouse. After conjugate formation, BacB cells were re-isolated, and intracellular bacteria were detected by gentamicin survival assays at different times. Data were normalized compared to time 0. As control of the bacterial fitness, in parallel, HeLa cells were directly infected.


H . WT B cells were allowed to form conjugates with Listeria - infected DC , after 30 min samples were treated with the indicated inhibitors ; Leupeptin + NH4Cl , pepstatin - A + E64D , lactacystin , or PBS for controls . One hour later gentamicin was added to kill extracellular bacteria and two hours later B cells were reisolated by negative selection , lysed and plated in agar - containing plates . CFUs were counted ; each colony represents one live intracellular bacterium inside B cells . Data information : Column bars in the figure represent the mean of at least three independent experiments . Error bars indicate the SD . Significant differences , analyzed by T test ( two samples ) or one way ANOVA ( more than two samples ) , are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. WT B cells were allowed to form conjugates with Listeria-infected DC, after 30 min samples were treated with the indicated inhibitors; Leupeptin + NH4Cl, pepstatin-A + E64D, lactacystin, or PBS for controls. One hour later gentamicin was added to kill extracellular bacteria and two hours later B cells were reisolated by negative selection, lysed and plated in agar-containing plates. CFUs were counted; each colony represents one live intracellular bacterium inside B cells. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Significant differences, analyzed by T test (two samples) or one way ANOVA (more than two samples), are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


CD80 ( A - B ) expression in B cells measured by flow cytometry on day 0 ( non - instructed ; naïve ) and day 2 after L . monocytogenes capture ( BacB ) . A show representative experiments , B show the mean of at least three independent experiments . Data information : Column bars in the figure represent the mean . Error bars indicate the SD . Significant differences , analyzed by T test are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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CD80 (A-B) expression in B cells measured by flow cytometry on day 0 (non-instructed; naïve) and day 2 after L. monocytogenes capture (BacB). A show representative experiments, B show the mean of at least three independent experiments. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


E - F . H - 2Kb expression in B cells measured by flow cytometry on day 0 ( naïve , pale grey ) and day 1 after L . monocytogenes capture ( BacB ; dark grey ) . E shows a representative experiment , F shows the mean of four independent experiments Data information : Column bars in the figure represent the mean . Error bars indicate the SD . Significant differences , analyzed by T test are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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E-F. H-2Kb expression in B cells measured by flow cytometry on day 0 (naïve, pale grey) and day 1 after L. monocytogenes capture (BacB; dark grey). E shows a representative experiment, F shows the mean of four independent experiments Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


G - H . Expression of OVAp - I in the context of H - 2Kb was detected by flow cytometry with anti - OVAp - I / H - 2Kb antibody , comparing BacB capturing Listeria - WT ( pale grey ) and Listeria - OVA ( dark grey ) . G shows a representative experiment , H shows the mean of two independent experiments . Data information : Column bars in the figure represent the mean . Error bars indicate the SD . Significant differences , analyzed by T test are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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G-H. Expression of OVAp-I in the context of H-2Kb was detected by flow cytometry with anti-OVAp-I/H-2Kb antibody, comparing BacB capturing Listeria-WT (pale grey) and Listeria-OVA (dark grey). G shows a representative experiment, H shows the mean of two independent experiments. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


I . Confocal images of Listeria - WT ( left panels ) or Listeria - OVA ( right panels ) BacB cells incubated with nai ̈ ve OTI CD8 + T cells previously stained with CellTraceViolet ( CV , red ) . Actin and CD3 fluorescence are shown on a " fire " scale and in cyan and white respectively in the merged images . Scale bars = 10 μm . * indicate CD8 + T cells . Arrow points to the IS . J . Quantification of actin accumulation at the IS on CD8 + T cells conjugated with Listeria - WT or Listeria - OVA BacB cells , analyzed using Synapse measures software from confocal images . Each dot corresponds to one IS . Three independent experiments were performed . Data information : Column bars in the figure represent the mean . Error bars indicate the SD . Significant differences , analyzed by T test are represented by asterisk ; * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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I. Confocal images of Listeria-WT (left panels) or Listeria-OVA (right panels) BacB cells incubated with naïve OTI CD8+ T cells previously stained with CellTraceViolet (CV, red). Actin and CD3 fluorescence are shown on a "fire" scale and in cyan and white respectively in the merged images. Scale bars = 10 μm. * indicate CD8+ T cells. Arrow points to the IS. J. Quantification of actin accumulation at the IS on CD8+ T cells conjugated with Listeria-WT or Listeria-OVA BacB cells, analyzed using Synapse measures software from confocal images. Each dot corresponds to one IS. Three independent experiments were performed. Data information: Column bars in the figure represent the mean. Error bars indicate the SD. Significant differences, analyzed by T test are represented by asterisk; *P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


B . Mean of at least three experiments showing the % of naïve OTI CD8 + T cell proliferated after activation ( days 1 - 3 ) with BacB capturing Listeria - WT ( WT ) or Listeria OVA ( OVA ) . Non - activated , non - proliferative CD8 + T cells ( NP ) are shown as control . Data information : Column bars in the figure represent the mean of at least three independent experiments . Error bars indicate the SD . Statistical significant differences analyzed by one - way ANOVA is represented by * . * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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B. Mean of at least three experiments showing the % of naïve OTI CD8+ T cell proliferated after activation (days 1-3) with BacB capturing Listeria-WT (WT) or Listeria OVA (OVA). Non-activated, non-proliferative CD8+ T cells (NP) are shown as control. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


C - D . CD25 expression in OTI CD8 + T cells on day 1 ( light red ) , day 2 ( red ) and day 3 ( dark red ) after activation with Listeria - WT ( left panel ) or Listeria - OVA ( right panel ) BacB cells . FMO control is shown in grey and dash line . C , representative experient . D , Mean of at least three independent experiments . Data information : Column bars in the figure represent the mean of at least three independent experiments . Error bars indicate the SD . Statistical significant differences analyzed by one - way ANOVA is represented by * . * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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C-D. CD25 expression in OTI CD8+ T cells on day 1 (light red), day 2 (red) and day 3 (dark red) after activation with Listeria-WT (left panel) or Listeria-OVA (right panel) BacB cells. FMO control is shown in grey and dash line. C, representative experient. D, Mean of at least three independent experiments. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


G - H . Proliferation of OTI CD8 + T cells activated with Listeria - OVA BacB cells ( H - 2Kb ) that captured bacteria from infected H - 2Kb or H - 2Kk iBM - DCs . Non - activated naïve CD8 + T cells are shown ( thin grey line ) . B cells were isoated from WT mice . G shows a representative experiment , H shows the mean of the proliferation index from at least three independent experiments ( relative to H - 2Kk iBM - DCs donor cells ) . Data information : Column bars in the figure represent the mean of at least three independent experiments . Error bars indicate the SD . Statistical significant differences analyzed by one - way ANOVA is represented by * . * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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G-H. Proliferation of OTI CD8+ T cells activated with Listeria-OVA BacB cells (H-2Kb) that captured bacteria from infected H-2Kb or H-2Kk iBM-DCs. Non-activated naïve CD8+ T cells are shown (thin grey line). B cells were isoated from WT mice. G shows a representative experiment, H shows the mean of the proliferation index from at least three independent experiments (relative to H-2Kk iBM-DCs donor cells). Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


J - L . Intracellular expression , measured by flow cytometry , of the indicated proteins in non - activated , naïve , CD8 + T cells or in CD8 + T cells activated with either antiCD3 + antiCD28 antibodies ( αCD3 / CD28 ) , OVA - decorated splenocytes ( OVA - pI ) , or Listeria - OVA BacB cells ( BacB ) . The mean fluorescence intensity and the sd corresponding to three independent experiments is shown . Data information : Column bars in the figure represent the mean of at least three independent experiments . Error bars indicate the SD . Statistical significant differences analyzed by one - way ANOVA is represented by * . * P < 0 . 05 , * * * P < 0 . 0005 , non - significant differences are marked as ( ns ) .

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J-L. Intracellular expression, measured by flow cytometry, of the indicated proteins in non-activated, naïve, CD8+ T cells or in CD8+ T cells activated with either antiCD3 + antiCD28 antibodies (αCD3/CD28), OVA-decorated splenocytes (OVA-pI), or Listeria-OVA BacB cells (BacB). The mean fluorescence intensity and the sd corresponding to three independent experiments is shown. Data information: Column bars in the figure represent the mean of at least three independent experiments. Error bars indicate the SD. Statistical significant differences analyzed by one-way ANOVA is represented by *.*P<0.05, ***P<0.0005, non-significant differences are marked as (ns).


A - D Surface expression of H - 2Kb ( A - B ) and CD80 ( C - D ) in L . monocytogenes ( orange ) or E . coli ( purple ) BacB cells analyzed by flow cytometry . A and C panels show representative experiments , B and D panels show the mean of at least three independent experiments .

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A-D Surface expression of H-2Kb (A-B) and CD80 (C-D) in L. monocytogenes (orange) or E. coli (purple) BacB cells analyzed by flow cytometry. A and C panels show representative experiments, B and D panels show the mean of at least three independent experiments.