( C ) In vivo vacuole morphology was assayed by FM4 - 64 staining . Scale bar : 5 µm . ( D ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo vacuole morphology was assayed by FM4-64 staining. Scale bar: 5 µm. (D) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( E ) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8 - EGFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Scale bar : 5 µm . ( F ) Fusion activity : vacuole fusion was assayed by measuring the percentage of co - localization of the two Pho8 - EGFP and Pho8 - mCherry signals . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments .

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

(E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.


( A ) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and / or Nyv1 . 10 µg of the organelles were analyzed by SDS - PAGE and Western blotting using antibodies to Vti1 , Vam3 and Nyv1 .

O O O O O O O O O O O O O O O O O O O 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) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1.


( B ) Localization in vivo . The indicated strains were grown logarithmically in synthetic complete ( SC ) medium . After 16 h at 30 ° C , when the final OD of each culture was about 1 , the cells were observed by confocal fluorescence and differential interference ( DIC ) microscopy . Scale bar : 5 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 O O 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) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.


( A ) In vivo morphology . Cells expressing the indicated SNARE variants were grown in SC medium ( 16 h , 30 ° C ) , stained with FM4 - 64 and their relative morphology was assessed by fluorescence and DIC microscopy . Scale bar : 5 µm . The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( B ) and ( C ) SNARE activation on isolated vacuoles . Vacuoles were isolated from a strain co - expressing NYV1 - S9 - EGFP and VAM3 - S9 - mCitrine and from an isogenic wild - type . 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP - regenerating system and recombinant , purified Sec18 / NSF ( rSec18 , 50 µg / ml ) . After 10 min of incubation at 27 ° C , the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1 . Co - immunoprecipitated proteins analyzed by SDS - PAGE and Western blotting . The histograms provide quantifications of the band intensities as the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O 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) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.


( A ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

O 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) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( B ) Vacuole morphology was assessed for the indicated cells as in Fig . 3C . Scale bar : 5 µm .

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

(B) Vacuole morphology was assessed for the indicated cells as in Fig. 3C. Scale bar: 5 µm.


( C ) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated . Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [ 65 ] but ensures that trans - SNARE complexes can only form in one orientation , between the Q - SNARE in DKY6281 and the R - SNARE in BJ3505 . The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay . In parallel , identical samples were incubated either incubated on ice , which prevents fusion , or in the presence of 0 . 5 % Triton X - 100 , which allows fusion - independent access of the maturase Pep4 to pro - ALP and controls for the levels of these two reporter enzymes in the samples .

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

(C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples.


( D ) Using strains from C , lipid and content mixing ( left and right panel , respectively ) were performed in parallel in the presence or absence of ATP . Anti - Vam3 antibody ( 3 µg ) was added to some reactions in order to inhibit trans - SNARE pairing and fusion . Means and the s . d . are shown for three independent experiments .

O O O O O O 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

(D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( A ) Protein levels on isolated vacuoles from these strains were compared as in Fig . 3B .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (A) Protein levels on isolated vacuoles from these strains were compared as in Fig. 3B.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( B ) Vacuole morphology was assessed as in Fig . 3C . Scale bar : 5 µm . ( C ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( D ) Fusion activity : vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8 - mCFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments . Scale bar : 5 µm . ( E ) Vacuole fusion was assayed by measuring the percentage of co - localisation of the two Pho8 - mCFP and Pho8 - mCherry signals .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.


( A ) Yeast strain expressing a plasmid with Vph1 - GFP was grown in SC - URA medium for 16 h at 30 ° C . The mobility of Vph1 - GFP in the absence ( upper panels ) or presence ( lower panel ) of CPZ ( 150 µM ) was assessed by FRAP . ( B ) The histogram shows the GFP signals in the bleached area during the FRAP procedure . ( C ) The half - time is shown . Means and s . d . are shown for 20 cells of 3 independent experiments .

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

(A) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments.


( D ) Yeast cells were grown in SC medium for 16 h at 30 ° C . After staining with FM4 - 64 , cells were treated with CPZ ( 150 µM ) . Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition . Scale bar : 5 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O

(D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm.


( E ) Fusion activity . Vacuoles were isolated from BJ cells carrying Nyv1 - S9 - EGFP and DKY cells deleted for Nyv1 and carrying Vam3 - S9 - mCitrine or from wild - type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine ( CPZ , 150 µM ) and anti - Vam3 ( 3 µg ) . Content mixing was assayed via alkaline phosphatase activity . Means and s . d . are shown for three independent experiments .

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

(E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.


( A ) OCR of wt and gas - 1 mutant nematodes ( normalized to nematode size as given by the COPAS Biosort ( Time of Flight - TOF ) ) . One representative curve shown ( mean ± SEM ; n = 3 ) * * * * p value < 0 . 0001 ; Kruskal - Wallis Test , Dunn ' s post hoc correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( B ) age - 1 and age - 1 ; gas - 1 mutants animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( C ) daf - 16 and daf - 16 ; age - 1 ; gas - 1 mutant animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Volcano plot of differentially phosphorylated proteins in gas - 1 ( fc21 ) mutants compared to wt . Cutoffs : 20 % FDR and | log2 ( foldchange ) | > 0 . 345 ( in red ) .

O O O O O O O O O O O O O O O O O O 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) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red).


( F ) Lifespan analysis of mev - 1 ( kn1 ) nematodes compared to wt , age - 1 ( hx546 ) and age - 1 ( hx546 ) ; mev - 1 ( kn1 ) animals . ( G )

(F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G)


( G ) ATP measurements in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to wt ) , n = 4 . Error bars : Mean ± SEM , * p value < 0 . 05 , * * * p value = 0 . 001 , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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

(G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction.


( H ) OCR of gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to TOF ) . Left panel : One representative curve ( Mean ± SEM ) . Right panel : Basal respiration in wt , gas - 1 and age - 1 ; gas - 1 animals , pooled from 3 biological replicates , * * * * p value < 0 . 0001 , ns = non - significant , Kruskal - Wallis Test , Dunn ' s post hoc correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction.


( I ) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms . Scale bar : 2 μm . ( J ) Left panel : Percentage of FRAP . Each point : mean recovery of ~ 30 bleached regions ( 2 regions per animal , 15 animals per condition ) ± SEM . Right panel : values of the maximum ( total ) recovery per strain ± SEM , * * * p value < 0 . 001 , * * * * p value < 0 . 0001 , one - way ANOVA , Tukey ' s post - hoc correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction.


( K ) Representative lifespan of age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and fzo - 1 RNAi bacteria . Average median lifespans ± SEM from all replicates indicated beneath representative curves . For all panels : wt = wild type , a = age - 1 , g = gas - 1 , a ; g = age - 1 ; gas - 1 .

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

(K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.


( A ) Left panel : Venn Diagram of the number of genes significantly dysregulated in age - 1 ; gas - 1 versus wt ( green ) and gas - 1 versus wt ( red ) . Right panel : Scatter - plots of the expression of genes significantly downregulated ( left ) or upregulated ( right ) in gas - 1 and age - 1 ; gas - 1 mutants compared to wt . In black : expression levels of genes that are significantly up - or down - regulated in gas - 1 mutants compared to wt . In red : expression levels of the same genes in age - 1 ; gas - 1 mutants compared to wt .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Venn Diagram of the number of genes significantly dysregulated in age-1; gas-1 versus wt (green) and gas-1 versus wt (red). Right panel: Scatter-plots of the expression of genes significantly downregulated (left) or upregulated (right) in gas-1 and age-1; gas-1 mutants compared to wt. In black: expression levels of genes that are significantly up- or down-regulated in gas-1 mutants compared to wt. In red: expression levels of the same genes in age-1; gas-1 mutants compared to wt.


( C ) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative

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

(C) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves


( D ) Volcano plot of TMT / iTRAQ - based quantitative proteomic analysis . In red , proteins significantly dysregulated in age - 1 ; gas - 1 compared to gas - 1 ( FDR < 20 % ; | log2 ( fold change ) | > 0 . 47 ) . Dashed lines indicate cutoffs . Proteins involved in carbohydrate ( green ) and lipid ( blue ) metabolism are labeled .

(D) Volcano plot of TMT/iTRAQ-based quantitative proteomic analysis. In red, proteins significantly dysregulated in age-1; gas-1 compared to gas-1 (FDR< 20%; |log2 (fold change)| >0.47). Dashed lines indicate cutoffs. Proteins involved in carbohydrate (green) and lipid (blue) metabolism are labeled.


( H ) Left panel : Glucose quantification in wt , gas - 1 and age - 1 ; gas - 1 nematode lysates with NMR , n = 7 , ns = non - significant , Mann Whitney U test . Right panel : Quantification of total carbohydrates in the indicated strains with phenol - sulfuric assay . Error bars : mean ± SEM , 2 biological replicates ( 5 technical replicates each ) , normalized to wt and number of animals used . * * p value < 0 . 01 , ns = non - significant , Mann - Whitney U test .

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 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

(H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.


( C ) Immunoblot analysis of phospho - AAK - 2 in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( actin loading control ) . Numbers correspond to band densitometries across 3 biological replicates .

(C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates.


( D ) Representative lifespan of age - 1 ; gas - 1 double mutant and age - 1 ; aak - 2 ; gas - 1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves .

O O O O O O O O O O O O O O O O O O O O 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) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves.


( E ) Lifespan assay of gas - 1 mutant animals compared to gas - 1 ; AAK - 2 overexpressing ( O / E ) nematodes and their wt counterparts . Inset : immunoblot analysis of phospho - AAK - 2 in gas - 1 ( g ) and gas - 1 ; AAK - 2 O / E ( g ; a O / E ) animals ( actin loading control ) , showing the overexpressed protein ( lower band ) and the endogenous one ( upper band ) .

(E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band).


( F ) Left panel : Immunoblot analysis of phospho - KIN - 1 substrates in young adult nematodes . Numbers correspond to band densitometries across 2 biological replicates . Right panel : immunoblot analysis of phospho - KIN - 1 substrates in age - 1 ; gas - 1 animals grown on bacteria carrying an empty vector ( EV ) or expressing kin - 1 RNAi . Two different kin - 1 RNAi clones ( g1 , b5 ) were used in this experiment . Numbers correspond to band densitometries of the presented immunoblot . Dashed rectangles : band corresponding to downregulated KIN - 1 phospho - substrate .

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

(F) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate.


( G ) Representative lifespan assay of wt and age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and kin - 1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves .

(G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves.


ATP measurements in : ( H ) age - 1 ( hx546 ) ; gas - 1 ( fc21 ) ( a ; g ) and age - 1 ( hx546 ) ; aak - 2 ( ok524 ) ; gas - 1 ( fc21 ) ( a ; aa ; g ) mutant nematodes ;

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ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes;


ATP measurements in : ( I ) age - 1 ; gas - 1 ( EV ) ( a ; g ( EV ) ) and age - 1 ; gas - 1 ; kin - 1 ( RNAi ) ( a ; g ; kin - 1 ( RNAi ) ) . Error bars : Mean ± SEM , n = 4 , * p value < 0 . 05 , Mann Whitney U test .

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ATP measurements in: (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test.


( J ) Left panel : curves represent FRAP in age - 1 ; gas - 1 animals grown on RNAi from larval L1 ( or from egg for fzo - 1 RNAi ) to L4 stage . Each point : mean recovery of bleached regions ( 2 - 5 regions per animal , 10 - 20 animals per condition ) ± SEM . Right panel : Maximum ( total ) recovery per condition are shown , ± SEM . * p value < 0 . 05 , * * p value < 0 . 01 , * * * * p value < 0 . 0001 , Kruskal - Wallis test , Dunn ' s post - hoc correction .

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(J) Left panel: curves represent FRAP in age-1; gas-1 animals grown on RNAi from larval L1 (or from egg for fzo-1 RNAi) to L4 stage. Each point: mean recovery of bleached regions (2-5 regions per animal, 10-20 animals per condition) ± SEM. Right panel: Maximum (total) recovery per condition are shown, ± SEM. *p value< 0.05, **p value< 0.01, ****p value< 0.0001, Kruskal-Wallis test, Dunn's post-hoc correction.


A ) Volcano plot of lipidomic profiling in gas - 1 versus age - 1 ; gas - 1 mutants . Red stars : significantly altered lipids in age - 1 ; gas - 1 versus gas - 1 ( 5 % FDR ; n = 5 ) .

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A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5).


B ) NMR - 1D targeted metabolic profiling in age - 1 ; gas - 1 mutants ( normalized to gas - 1 ) . Dashed lines indicate fold change cutoff ( | log2 ( fold change ) | < 1 . 23 ) . Red bars : not significant , Light blue bars : FDR < 10 % , | log2 ( fold change ) | < 1 . 23 , Dark blue bars : FDR < 10 % , | log2 ( fold change ) | > 1 . 23 . Error bar : mean + SEM , n = 7 .

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B) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7.


D ) Left panel : qRT - PCR of xdh - 1 ( normalized to wt ) . Error bars : Mean ± SEM , n = 6 . * * * * p value < 0 . 0001 , * p value < 0 . 05 , ns = non - significant , Kruskal - Wallis with Dunn ' s post hoc test . Right : xanthine oxidase activity levels normalized to animal number . Error bars : mean ± SEM , n = 2 , ns = non - significant , Mann Whitney U test .

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D) Left panel: qRT-PCR of xdh-1 (normalized to wt). Error bars: Mean ± SEM, n=6. ****p value< 0.0001, *p value< 0.05, ns= non-significant, Kruskal-Wallis with Dunn's post hoc test. Right: xanthine oxidase activity levels normalized to animal number. Error bars: mean ± SEM, n=2, ns= non-significant, Mann Whitney U test.


F ) Immunoblot analysis of phospho - Akt ( S473 ) , pan Akt , phospho - PRAS40 ( T246 ) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0 . 2 % DMSO ( - ) or AMP ( 2 μM ) , ENPF ( 2 μM ) and PTX ( 36 μM ) ( + ) for 24 h ( actin loading control ) .

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F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control).


( G ) Immunoblot analysis of phospho - PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated ( actin loading control ) . For all immunoblot panels : numbers correspond to band densitometries across 2 - 3 biological replicates .

O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of phospho-PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated (actin loading control). For all immunoblot panels: numbers correspond to band densitometries across 2-3 biological replicates.


( A ) Effect of methylxanthines on wt lifespan . Average median lifespans ± SEM from all replicates indicated beneath representative curve .

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(A) Effect of methylxanthines on wt lifespan. Average median lifespans ± SEM from all replicates indicated beneath representative curve.


( B ) Locomotory activity of 10 - day - old gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines . Values represent arbitrary units from 3 biological replicates with 4 technical replicates each , 35 worms per technical replicate . Error bars : mean ± SEM , * * p value < 0 . 01 , * * * p value < 0 . 001 , one - way ANOVA , Dunnett ' s post - hoc correction .

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(B) Locomotory activity of 10-day-old gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines. Values represent arbitrary units from 3 biological replicates with 4 technical replicates each, 35 worms per technical replicate. Error bars: mean ± SEM, **p value< 0.01, ***p value< 0.001, one-way ANOVA, Dunnett's post-hoc correction.


( C ) Effect of methylxanthines on the survival of gas - 1 mutants . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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(C) Effect of methylxanthines on the survival of gas-1 mutants. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Pharyngeal pumps / minute in young adult gas - 1 mutant nematodes treated with vehicle or methylxanthines . Error bars : mean ± SEM , n = 18 . * * * * p value < 0 . 0001 , one - way ANOVA , Dunnett ' s post - hoc correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pharyngeal pumps/minute in young adult gas-1 mutant nematodes treated with vehicle or methylxanthines. Error bars: mean ± SEM, n=18. ****p value< 0.0001, one-way ANOVA, Dunnett's post-hoc correction.


( E - F ) Immunoblot analysis of ( E ) KIN - 1 phospho - substrates in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) .

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(E-F) Immunoblot analysis of (E) KIN-1 phospho-substrates in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).


( E - F ) Immunoblot analysis of ( F ) phospho - AAK - 2 in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin 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 O O O O O O O O

(E-F) Immunoblot analysis of (F) phospho-AAK-2 in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).


( G ) Left panel : FRAP in vehicle or PTX - treated gas - 1 animals . Each point : mean recovery of 14 bleached regions ( 2 regions per animal , 7 animals per condition ) ± SEM . Right panel : maximum ( total ) recovery per condition ± SEM . * * p value < 0 . 01 unpaired t test . For all immunoblot panels : numbers correspond to band densitometries across biological replicates and for all lifespan curves : the legend shows Mean median lifespan ± SEM across all biological replicates .

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

(G) Left panel: FRAP in vehicle or PTX-treated gas-1 animals. Each point: mean recovery of 14 bleached regions (2 regions per animal, 7 animals per condition) ± SEM. Right panel: maximum (total) recovery per condition ± SEM. **p value< 0.01 unpaired t test. For all immunoblot panels: numbers correspond to band densitometries across biological replicates and for all lifespan curves: the legend shows Mean median lifespan ± SEM across all biological replicates.


A . Root tip of Arabidopsis with distinct growth zones marked : meristematic ( MZ , green ) , transition ( TZ , blue ) , elongation ( EZ , pink ) and differentiation ( DZ , yellow ) . Double arrow indicates the first expanded cortex cell considered as a start point of the TZ , which encompasses epidermal cells smaller than 45 µm . Epidermal cells reaching a length of more than 45 µm prior termination of the elongation are in the EZ . Scale bar 25 µm .

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A. Root tip of Arabidopsis with distinct growth zones marked: meristematic (MZ, green), transition (TZ, blue), elongation (EZ, pink) and differentiation (DZ, yellow). Double arrow indicates the first expanded cortex cell considered as a start point of the TZ, which encompasses epidermal cells smaller than 45 µm. Epidermal cells reaching a length of more than 45 µm prior termination of the elongation are in the EZ. Scale bar 25 µm.


B . CMTs visualized by the MAP4 - GFP reporter ( left ) and scheme of the CMT orientations ( right ) in epidermal cells of distinct growth zones ( TZ , EZ and DZ ) . Individual cells monitored at two time points ( 0 and 60 min ) . Dark blue arrows mark the cell expansion direction . Scale bar 10 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 O O O O O O 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. CMTs visualized by the MAP4-GFP reporter (left) and scheme of the CMT orientations (right) in epidermal cells of distinct growth zones (TZ, EZ and DZ). Individual cells monitored at two time points (0 and 60 min). Dark blue arrows mark the cell expansion direction. Scale bar 10 µm.


D . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . CMT plus - end growth rates ( μm / min ) measured by tracking the EB1b - GFP marker for 20 - 30 min in epidermal root cells of three growth zones . In the boxplots , the center lines show medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . * * P < 0 . 01 , * * * * P < 0 . 0001by the Student ' s t - test . The number of CMT plus - end events ( EB1b - GFP ) tracked per cell was 20 - 100 / min , of which the growth rate average is represented as a single dot ; 3 - 5 cells per root growth zone , 3 - 5 roots analyzed per biological replicate in 3 independent experiments . On the right , single trajectories of EB1b - GFP signal tracked over 60 sec in root epidermal cells at different growth zones . Scale bar 0 . 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. CMT plus-end growth rates (μm/min) measured by tracking the EB1b-GFP marker for 20-30 min in epidermal root cells of three growth zones. In the boxplots, the center lines show medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. ** P < 0.01, **** P < 0.0001by the Student's t-test. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot; 3-5 cells per root growth zone, 3-5 roots analyzed per biological replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones. Scale bar 0.5 µm.


E . Z - stack maximum image projection of the EB1b - GFP plus - end reporter tracked over 30 sec , in epidermal cells at different growth zones . Scale bars 10 µm .

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E. Z-stack maximum image projection of the EB1b-GFP plus-end reporter tracked over 30 sec, in epidermal cells at different growth zones. Scale bars 10 µm.


F . CMTs visualized by MAP4 - GFP in root epidermal cells at the TZ , the EZ and the DZ prior ( 0 ) and 60 min after treatment with 1 µm oryzalin . Scale bar 10 µm .

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F. CMTs visualized by MAP4-GFP in root epidermal cells at the TZ, the EZ and the DZ prior (0) and 60 min after treatment with 1 µm oryzalin. Scale bar 10 µm.


G . Quantification of the MAP4 - GFP CMTs reporter signal in wild - type root epidermal cells at the TZ , the EZ and the DZ zone treated with mock ( DMSO , white box ) , oryzalin ( 1 µM , light grey box ) and cytokinin ( CK ; 10 µM 6 - benzylaminopurine , BAP ) and oryzalin ( 1 µM ) ( dark grey box ) . For double treatments , roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds . Boxplots represent ratio between mean fluorescence intensity ( arbitrary units ) measured in epidermal cells at 60 and 0 min .

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G. Quantification of the MAP4-GFP CMTs reporter signal in wild-type root epidermal cells at the TZ, the EZ and the DZ zone treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and cytokinin (CK; 10 µM 6-benzylaminopurine, BAP) and oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min.


A . Arabidopsis root expressing the dual reporter TCSn : : dndT - tNOS DR5v2 : 3nGFP that is sensitive to cytokinin ( CK ) and auxin . CK ( red ) , auxin ( green ) and overlay of both signals detected in nuclei of cells at the root tip ( top , scale bar 50 µm ) . Green , pink , and blue arrowheads point at epidermal cells located in the meristematic ( MZ ) , transition ( TZ ) and elongation ( EZ ) zones , respectively . Magnification of the TZ shown ( bottom , scale bar 10 µm ) .

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A. Arabidopsis root expressing the dual reporter TCSn::dndT-tNOS DR5v2:3nGFP that is sensitive to cytokinin (CK) and auxin. CK (red), auxin (green) and overlay of both signals detected in nuclei of cells at the root tip (top, scale bar 50 µm). Green, pink, and blue arrowheads point at epidermal cells located in the meristematic (MZ), transition (TZ) and elongation (EZ) zones, respectively. Magnification of the TZ shown (bottom, scale bar 10 µm).


B . Relative fluorescence intensity of TCSn : : ntdT : tNOS signal ( red ) and DR5v2 : 3nGFP signal ( green ) measured in epidermal cells along the longitudinal root growth axis . Cell number 1 corresponds to a meristematic cell placed at position - 4 before the beginning of TZ ( as marked by green arrow at 2A ) . Mean ± s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 by Student ' s t - test , n = 18 roots .

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B. Relative fluorescence intensity of TCSn::ntdT:tNOS signal (red) and DR5v2:3nGFP signal (green) measured in epidermal cells along the longitudinal root growth axis. Cell number 1 corresponds to a meristematic cell placed at position -4 before the beginning of TZ (as marked by green arrow at 2A). Mean ± s.d., * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by Student's t-test, n= 18 roots.


C - F . CMTs visualized with MAP4 - GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK ( C ) , CK and oryzalin ( D ) , auxin ( E ) and CK and auxin ( F ) treatments , and in DZ cells at time point 0 and 60 min after auxin treatment ( E ) . As CK and auxin sources 10 µM BAP and 0 . 1 µM NAA were used , respectively , and 1 µM oryzalin . For the double ( CK / Oryzalin and CK / Auxin ) treatments , roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds . Scale bar 10 µm .

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C-F. CMTs visualized with MAP4-GFP in root epidermal cells of the TZ and the EZ at time point 0 and 60 min after CK (C), CK and oryzalin (D), auxin (E) and CK and auxin (F) treatments, and in DZ cells at time point 0 and 60 min after auxin treatment (E). As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively, and 1 µM oryzalin. For the double (CK/Oryzalin and CK/Auxin) treatments, roots were pretreated with CK for 60 min prior to transfer to medium supplemented with both compounds. Scale bar 10 µm.


H . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . Z - stack maximum image projections of EB1b - GFP tracked over 30 sec in epidermal cells without ( mock , DMSO ) and with CK , auxin or CK and auxin treatment ( 60 min ) . Scale bars 10 µm . I . CMT plus - end growth rates ( µm / min ) measured by tracking of EB1b - GFP reporter over 20 - 30 min in epidermal cells of different growth zones treated for 60 min with mock ( DMSO ) , or media supplemented with CK ( 30 or 60 min ) , auxin and CK plus auxin . In the boxplots , the center lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . The number of CMT plus - end events ( EB1b - GFP ) tracked per cell was 20 - 100 / min , of which the growth rate average is represented as a single dot . Statistical significance evaluated by two - way ANOVA ( n = 3 - 5 cells per root growth zone with 3 - 5 roots per replicate in 3 independent experiments . On the right , single trajectories of EB1b - GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions . Scale bar 0 . 5 µm . Hormone concentrations and treatment conditions ( H , I ) were as described ( C , E and F ) .

O O O O O O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm. I. CMT plus-end growth rates (µm/min) measured by tracking of EB1b-GFP reporter over 20-30 min in epidermal cells of different growth zones treated for 60 min with mock (DMSO), or media supplemented with CK (30 or 60 min), auxin and CK plus auxin. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot. Statistical significance evaluated by two-way ANOVA (n = 3-5 cells per root growth zone with 3-5 roots per replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones and treatment conditions. Scale bar 0.5 µm. Hormone concentrations and treatment conditions (H, I) were as described (C, E and F).


H . Analysis of the CMT plus - end growth with the EB1b - GFP reporter . Z - stack maximum image projections of EB1b - GFP tracked over 30 sec in epidermal cells without ( mock , DMSO ) and with CK , auxin or CK and auxin treatment ( 60 min ) . Scale bars 10 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 O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O

H. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. Z-stack maximum image projections of EB1b-GFP tracked over 30 sec in epidermal cells without (mock, DMSO) and with CK, auxin or CK and auxin treatment (60 min). Scale bars 10 µm.


A - D . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant at the transition ( TZ ) and the elongation ( EZ ) zones at time point 0 and 60 min after mock ( DMSO ) ( A ) , cytokinin ( CK ) ( B ) , auxin ( C ) and CK plus auxin ( D ) treatments . As CK and auxin sources 10 µM BAP and 0 . 1 µM NAA were used , respectively . For the double CK plus auxin treatment roots were pretreated for 60 min with CK , whereafter they were transferred to medium supplemented with both compounds . Scale bar 10 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

A-D. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant at the transition (TZ) and the elongation (EZ) zones at time point 0 and 60 min after mock (DMSO) (A), cytokinin (CK) (B), auxin (C) and CK plus auxin (D) treatments. As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively. For the double CK plus auxin treatment roots were pretreated for 60 min with CK, whereafter they were transferred to medium supplemented with both compounds. Scale bar 10 µm.


F , G . Analysis of the CMT plus - end growth with the EB1b - GFP reporter in cre1 - 12 . F . Z - stack maximum image projection of EB1b - GFP tracked for 30 seconds ( on the left ) and single trajectories of EB1b - GFP signal tracked over 60 sec ( on the right ) in root epidermal cells of the EZ in the wild type and cre1 - 12 treated with CK for 60 min . Scale bars 10 µm and 0 . 5 µm , respectively . G . CMT plus - end growth rates ( µm / min ) quantified from the EB1b - GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock ( DMSO ) or CK . In the boxplots , the center lines show the medians ; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software ; whiskers span minimum to maximum values , individual data points are represented by dots . ( ns = non - significant , * * * P < 0 . 001 by Student ' s t - test , n = 7 - 9 cells with 7 roots per condition in 3 independent experiments ) . Concentrations and treatment conditions ( F , G ) were as for ( A , B ) .

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

F, G. Analysis of the CMT plus-end growth with the EB1b-GFP reporter in cre1-12. F. Z-stack maximum image projection of EB1b-GFP tracked for 30 seconds (on the left) and single trajectories of EB1b-GFP signal tracked over 60 sec (on the right) in root epidermal cells of the EZ in the wild type and cre1-12 treated with CK for 60 min. Scale bars 10 µm and 0.5 µm, respectively. G. CMT plus-end growth rates (µm/min) quantified from the EB1b-GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock (DMSO) or CK. In the boxplots, the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. (ns = non-significant, *** P < 0.001 by Student's t-test, n= 7-9 cells with 7 roots per condition in 3 independent experiments). Concentrations and treatment conditions (F, G) were as for (A, B).


H , I . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant . Cells at the TZ , the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin ( 1 µM ) ( H ) , and CK plus oryzalin ( I ) . As CK source 10 µM BAP was used and 1 µM oryzalin . For double ( CK and oryzalin ) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds . Scale bar 10 µm . J . Quantification of the MAP4 - GFP CMTs reporter signal in cre1 - 12 root epidermal cells at different root zones treated with mock ( DMSO , white box ) , oryzalin ( 1 µM , light grey box ) and CK ( 10 µM BAP ) plus oryzalin ( 1 µM ) ( dark grey box ) . For double treatments , roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds . Boxplots represent ratio between mean fluorescence intensity ( arbitrary units ) measured in epidermal cells at 60 and 0 min . The center lines show the medians and the box limits indicate the 25th and 75th percentiles ; whiskers span the minimal to maximal values , individual data points are represented by dots . Ratio close to 1 ( segmented line ) corresponds to the unchanged MAP - GFP signal for 60 min . ( * P < 0 . 05 , * * * * P < 0 . 0001 by Student ' s t - test , n = 3 - 10 cells per root growth zone with 5 - 8 roots per condition in 4 independent replicates ) .

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

H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm. J. Quantification of the MAP4-GFP CMTs reporter signal in cre1-12 root epidermal cells at different root zones treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and CK (10 µM BAP) plus oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min. The center lines show the medians and the box limits indicate the 25th and 75th percentiles; whiskers span the minimal to maximal values, individual data points are represented by dots. Ratio close to 1 (segmented line) corresponds to the unchanged MAP-GFP signal for 60 min. (* P < 0.05, **** P < 0.0001 by Student's t-test, n= 3-10 cells per root growth zone with 5-8 roots per condition in 4 independent replicates).


H , I . CMTs visualized with MAP4 - GFP in root epidermal cells of the cre1 - 12 mutant . Cells at the TZ , the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin ( 1 µM ) ( H ) , and CK plus oryzalin ( I ) . As CK source 10 µM BAP was used and 1 µM oryzalin . For double ( CK and oryzalin ) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds . Scale bar 10 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 O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm.


A . Relative root growth of wild - type ( Col - 0 ) ( grey lines ) and 35S : : ARR1 ∆ DDK - GR ( purple lines ) seedlings grown on mock ( DMSO ; continuous line ) or with dexamethasone ( DEX 10 µM ; dashed line ) supplemented medium . Five days old seedlings ( Day 0 ) were monitored for 3 days . Mean ± s . d . ; * * P < 0 . 01 , * * * * P < 0 . 0001 by Student ' s t - test , referred to wild - type - mock . n = 12 - 14 roots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on mock (DMSO; continuous line) or with dexamethasone (DEX 10 µM; dashed line) supplemented medium. Five days old seedlings (Day 0) were monitored for 3 days. Mean ± s.d.; ** P < 0.01, **** P < 0.0001 by Student's t-test, referred to wild-type - mock. n = 12-14 roots.


B . Immunostaining of α - tubulin in epidermal cells of the transition ( TZ ) and the elongation ( EZ ) zone of 35S : : ARR1 ∆ DDK - GR roots after non - , 3h or 16h of treatment with dexamethasone ( DEX 10 µM ) . Scale bar 10 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 O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

B. Immunostaining of α-tubulin in epidermal cells of the transition (TZ) and the elongation (EZ) zone of 35S::ARR1∆DDK-GR roots after non-, 3h or 16h of treatment with dexamethasone (DEX 10 µM). Scale bar 10 µm.


D , E . Analysis of root sensitivity to oryzalin in wild - type ( Col - 0 ) and 35S : : ARR1 ∆ DDK - GR . Seedlings were grown for 5 days on mock ( Murashige and Skoog ) medium and then transferred to medium supplemented with 1 µM oryzalin , CK with oryzalin ( 10 µM BAP and 1 µM oryzalin ) , with or without DEX 10 µM , for 3 days . For the double CK and oryzalin treatment , seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds . Representative images ; white arrowheads indicate root length at day of transfer . Scale bar 1 mm ( D ) . Quantifications calculated as percentage ( % ) of root tips exhibiting swelling ( dark grey bars ) , and weak and strong resistance to oryzalin ( grey and white bars , respectively ) . On the right representative images of root phenotype categories . n = 10 - 25 roots per treatment were evaluated . ( E ) .

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

D, E. Analysis of root sensitivity to oryzalin in wild-type (Col-0) and 35S::ARR1∆DDK-GR. Seedlings were grown for 5 days on mock (Murashige and Skoog) medium and then transferred to medium supplemented with 1 µM oryzalin, CK with oryzalin (10 µM BAP and 1 µM oryzalin), with or without DEX 10 µM, for 3 days. For the double CK and oryzalin treatment, seedlings were pretreated with 10 µM BAP for 60 min prior to transfer to medium supplemented with both compounds. Representative images; white arrowheads indicate root length at day of transfer. Scale bar 1 mm (D). Quantifications calculated as percentage (%) of root tips exhibiting swelling (dark grey bars), and weak and strong resistance to oryzalin (grey and white bars, respectively). On the right representative images of root phenotype categories. n=10-25 roots per treatment were evaluated. (E).


F . Relative root growth of wild - type ( Col - 0 ) ( grey lines ) and 35S : : ARR1 ∆ DDK - GR ( purple lines ) seedlings grown on oryzalin 1µM ( continuous line ) or on oryzalin 1µM and DEX 10 µM ( dashed line ) supplemented media monitored for 3 days . Day 0 , day of transfer . Mean ± s . d . ; n = 12 - 14 roots .

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

F. Relative root growth of wild-type (Col-0) (grey lines) and 35S::ARR1∆DDK-GR (purple lines) seedlings grown on oryzalin 1µM (continuous line) or on oryzalin 1µM and DEX 10 µM (dashed line) supplemented media monitored for 3 days. Day 0, day of transfer. Mean ± s.d.; n = 12-14 roots.


A . Time lapse imaging of Arabidopsis root expressing the plasma membrane marker ( PM ) pUB10 : : EYFP - NPSN12 ( wave line W131Y ) ( Geldner et al , 2009 ) . Root growth recorded for 2 min and overlay of root tip images at time point 0 ( magenta ) and 2 min ( cyan ) used for the calculation of the root growth rate ( RGR ) and the cell elongation rate ( CER ) . Scale bar 25 µm . B . RGR of wild type ( grey bars ) and cre1 - 12 ( blue bars ) treated for 1 and 4 hours with mock ( DMSO ) , CK ( 10 µM BAP ) or auxin ( 0 . 1 µM NAA ) . Mean ± s . d . , ( * P < 0 . 05and * * * * P < 0 . 0001 by the Student ' s t - test , n = 5 - 8 roots per condition in 3 independent replicates ) .

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

A. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm. B. RGR of wild type (grey bars) and cre1-12 (blue bars) treated for 1 and 4 hours with mock (DMSO), CK (10 µM BAP) or auxin (0.1 µM NAA). Mean ± s.d., (* P < 0.05and **** P < 0.0001 by the Student's t-test, n= 5-8 roots per condition in 3 independent replicates).


A . Time lapse imaging of Arabidopsis root expressing the plasma membrane marker ( PM ) pUB10 : : EYFP - NPSN12 ( wave line W131Y ) ( Geldner et al , 2009 ) . Root growth recorded for 2 min and overlay of root tip images at time point 0 ( magenta ) and 2 min ( cyan ) used for the calculation of the root growth rate ( RGR ) and the cell elongation rate ( CER ) . Scale bar 25 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Time lapse imaging of Arabidopsis root expressing the plasma membrane marker (PM) pUB10::EYFP-NPSN12 (wave line W131Y)(Geldner et al, 2009). Root growth recorded for 2 min and overlay of root tip images at time point 0 (magenta) and 2 min (cyan) used for the calculation of the root growth rate (RGR) and the cell elongation rate (CER). Scale bar 25 µm.


C - E . Analysis of CK and auxin effects on CER of epidermal cells . Epidermal cells of the TZ and the EZ in the wild type and cre1 - 12 with PMs visualized with the YFP - NPSN12 marker . Overlay of images of epidermal cells at time 0 ( magenta ) and 2 min ( cyan ) is presented . Scale bar 10 µm ( C ) . CER ( µm / min ) of root epidermal cells of the TZ ( D ) and the EZ ( E ) of the wild type ( grey bars ) and cre1 - 12 ( blue bars ) mutant . Hormone concentrations and conditions of treatment ( D , E ) were as described for ( C ) . Mean ± s . d . , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 by Student ' s t - test , n = 15 - 20 cells per root growth zone in 3 - 6 roots per condition , in 3 independent ) .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of CK and auxin effects on CER of epidermal cells. Epidermal cells of the TZ and the EZ in the wild type and cre1-12 with PMs visualized with the YFP-NPSN12 marker. Overlay of images of epidermal cells at time 0 (magenta) and 2 min (cyan) is presented. Scale bar 10 µm (C). CER (µm/min) of root epidermal cells of the TZ (D) and the EZ (E) of the wild type (grey bars) and cre1-12 (blue bars) mutant. Hormone concentrations and conditions of treatment (D, E) were as described for (C). Mean ± s.d., (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 by Student's t-test, n=15-20 cells per root growth zone in 3-6 roots per condition, in 3 independent).


A . MT plus - end growth rates ( µm / min ) in leukocytes expressing the EB3 - mCherry plus - end marker before ( dark grey ) and after ( light grey ) treatment with mock ( DMSO ) , CK ( 10 µM BAP or 10 µM trans - zeatin ) and auxin ( 0 . 1 µM NAA ) . MT plus - end growth rates were monitored for 10 min before and for 10 min after each treatment . The number of MT plus - end events ( EB3 - mCherry ) tracked per cell was 100 - 200 / min . Mean ± s . d . , ( * * * P < 0 . 001 by Student ' s t - test , n = 1 - 3 cells per condition in 4 independent replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry plus-end marker before (dark grey) and after (light grey) treatment with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin) and auxin (0.1 µM NAA). MT plus-end growth rates were monitored for 10 min before and for 10 min after each treatment. The number of MT plus-end events (EB3-mCherry) tracked per cell was 100-200/min. Mean ± s.d., (*** P < 0.001 by Student's t-test, n = 1-3 cells per condition in 4 independent replicate).


A . MT plus - end growth rates ( µm / min ) in leukocytes expressing the EB3 -

A. MT plus-end growth rates (µm/min) in leukocytes expressing the EB3-mCherry


C - E . Immunostaining of α - tubulin ( green ) in leukocytes mounted in media containing DAPI ( blue , nuclear marker ) and quantification of immunolabeled MTs . Cells were not or treated with 300 nM nocodazole ( N ) for 10 min ( C ) . Leukocytes pretreated for 30 min with mock ( DMSO ) , CK ( 10 µM BAP or 10 µM trans - zeatin ) , adenine ( 10 µM ) or auxin ( 10 µM NAA ) prior to a 10 - min treatment with 300 nM nocodazol ( D ) . Scale bar 50 µm ( C , D ) . E . Relative fluorescence intensity of MTs in leukocytes treated with nocodazole , or nocodazole and hormones as described for ( D ) compared to untreated ( black bar ) cells . Mean ± s . d . , ( P < 0 . 05 by two - way Anova , n = 18 - 33 cells per condition in 3 independent replicates ) .

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

C-E. Immunostaining of α-tubulin (green) in leukocytes mounted in media containing DAPI (blue, nuclear marker) and quantification of immunolabeled MTs. Cells were not or treated with 300 nM nocodazole (N) for 10 min (C). Leukocytes pretreated for 30 min with mock (DMSO), CK (10 µM BAP or 10 µM trans-zeatin), adenine (10 µM) or auxin (10 µM NAA) prior to a 10-min treatment with 300 nM nocodazol (D). Scale bar 50 µm (C, D). E. Relative fluorescence intensity of MTs in leukocytes treated with nocodazole, or nocodazole and hormones as described for (D) compared to untreated (black bar) cells. Mean ± s.d., (P < 0.05 by two-way Anova, n = 18-33 cells per condition in 3 independent replicates).


( A ) Immunofluorescence labeling of FTSJ1 - Flag ( red ) in HEK293T cells . The nucleus was stained by DAPI ( blue ) . Scale bar , 20 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

(A) Immunofluorescence labeling of FTSJ1-Flag (red) in HEK293T cells. The nucleus was stained by DAPI (blue). Scale bar, 20 μm.


( B ) Subcellular localization of FTSJ1 . Cytoplasmic ( C ) and nuclear ( N ) fractions were separated from HEK293T cells expressing FTSJ1 - Flag . α - Tubulin and Lamin A / C were used as indicators of the cytoplasmic and nuclear fractions , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Subcellular localization of FTSJ1. Cytoplasmic (C) and nuclear (N) fractions were separated from HEK293T cells expressing FTSJ1-Flag. α-Tubulin and Lamin A/C were used as indicators of the cytoplasmic and nuclear fractions, respectively.


( C ) Co - IP assays of the interaction between FTSJ1 and WDR6 in co - transfected HEK293T cells . Anti - HA IP ( top ) and anti - Flag IP ( middle ) were immunoblotted by anti - HA and anti - Flag antibodies , respectively . Bottom : cell lysates immunoblotted by anti - HA or anti - Flag antibodies , with GAPDH serving 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 O O O O O O O O O O O O O O O O O O O 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) Co-IP assays of the interaction between FTSJ1 and WDR6 in co-transfected HEK293T cells. Anti-HA IP (top) and anti-Flag IP (middle) were immunoblotted by anti-HA and anti-Flag antibodies, respectively. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.


( D ) Co - IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition .

(D) Co-IP assays of the interaction between FTSJ1 and WDR6 with RNase A addition.


B , C ) Co - IP assays of the interaction between FTSJ1 - Flag or truncated FTSJ1 mutants and WDR6 - HA in co - transfected HEK293T cells . Anti - Flag IP ( top ) were respectively immunoblotted by anti - HA and anti - Flag antibodies . Bottom : cell lysates immunoblotted by anti - HA or anti - Flag antibodies , with GAPDH serving as a loading control .

B, C) Co-IP assays of the interaction between FTSJ1-Flag or truncated FTSJ1 mutants and WDR6-HA in co-transfected HEK293T cells. Anti-Flag IP (top) were respectively immunoblotted by anti-HA and anti-Flag antibodies. Bottom: cell lysates immunoblotted by anti-HA or anti-Flag antibodies, with GAPDH serving as a loading control.


E ) IP assays of FTSJ1 - Asn221DIZPK . The empty vector was transfected as a negative control . ( F ) A list of top FTSJ1 - Asn221DIZPK putative interacting proteins identified by IP - MS in HEK293T cells .

O O O O O O O O O O O O O O O O O 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) IP assays of FTSJ1-Asn221DIZPK. The empty vector was transfected as a negative control. (F) A list of top FTSJ1-Asn221DIZPK putative interacting proteins identified by IP-MS in HEK293T cells.


Mass chromatograms of the nucleosides , Cm ( Q1 / Q3 = 258 . 1 / 112 . 1 ) , Gm ( Q1 / Q3 = 298 . 1 / 152 . 1 ) and A ( Q1 / Q3 = 268 . 1 / 136 . 2 ) of tRNAPhe ( GAA ) isolated from WT , ftsj1 KO and wdr6 KO cells . Target peaks are indicated by black triangles . Cm , 2 ' - O - methylcytidine ; Gm , 2 ' - O - methylguanosine ; Q1 / Q3 : the mass of the precursor ion and the mass of the product ion .

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

Mass chromatograms of the nucleosides, Cm (Q1/Q3=258.1/112.1), Gm (Q1/Q3=298.1/152.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. Cm, 2'-O-methylcytidine; Gm, 2'-O-methylguanosine; Q1/Q3: the mass of the precursor ion and the mass of the product ion.


Mass chromatograms of the nucleosides m1G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) , m7G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) and m2G ( Q1 / Q3 = 298 . 1 / 166 . 1 ) , o2yW ( Q1 / Q3 = 541 . 2 / 409 . 0 ) , m5C ( Q1 / Q3 = 258 . 1 / 126 . 1 ) and A ( Q1 / Q3 = 268 . 1 / 136 . 2 ) of tRNAPhe ( GAA ) isolated from WT , ftsj1 KO and wdr6 KO cells . Target peaks are indicated by black triangles . m2G , 2 - methylguanosine ; D , dihydrouridine ; m22G , N2 , N2 - dimethylguanosine ; m5C , 5 - methylcytidine ; m7G , 7 - methylguanosine ; m5U , 5 - methyluridine ; m1A : 1 - methyladenosine ; ψ : pseudouridine ; o2yW , peroxywybutosine . Q1 / Q3 : the mass of the precursor ion and the mass of the product ion . Combined the retention time of standard product , we marked the o2yW in Figure 3G . Considering the change of this peak area was consistent with that of o2yW , we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled .

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

Mass chromatograms of the nucleosides m1G (Q1/Q3=298.1/166.1), m7G (Q1/Q3=298.1/166.1) and m2G (Q1/Q3=298.1/166.1), o2yW (Q1/Q3=541.2/409.0), m5C (Q1/Q3=258.1/126.1) and A (Q1/Q3=268.1/136.2) of tRNAPhe(GAA) isolated from WT, ftsj1 KO and wdr6 KO cells. Target peaks are indicated by black triangles. m2G, 2-methylguanosine; D, dihydrouridine; m22G, N2,N2-dimethylguanosine; m5C, 5-methylcytidine; m7G, 7-methylguanosine; m5U, 5-methyluridine; m1A: 1-methyladenosine; ψ: pseudouridine; o2yW, peroxywybutosine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. Combined the retention time of standard product, we marked the o2yW in Figure 3G. Considering the change of this peak area was consistent with that of o2yW, we speculated the other peak was generated by the intermediate product of o2yW with nature isotope labelled.


( B , C ) Quantification of the Cm / A and Gm / A of tRNAPhe ( GAA ) , △ ftsj1 _ tRNAPhe ( GAA ) , and m1G37 _ tRNAPhe ( GAA ) after incubation with FTSJ1 , WDR6 , and FTSJ1 - WDR6 by UPLC - MS / MS analysis , respectively . Error bars represent the standard deviation of three independent experiments . n . d . , not detected .

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(B, C) Quantification of the Cm/A and Gm/A of tRNAPhe(GAA), △ftsj1_tRNAPhe(GAA), and m1G37_tRNAPhe(GAA) after incubation with FTSJ1, WDR6, and FTSJ1-WDR6 by UPLC-MS/MS analysis, respectively. Error bars represent the standard deviation of three independent experiments. n.d., not detected.


( D ) UPLC - MS / MS analysis of 2 ' - O - methylation of m1G37 _ tRNAPhe ( GAA ) , - G34A , - G34C and G34U after incubation with FTSJ1 - WDR6 . 2 μL of Gm ( 1 ng / mL ) , Am ( 1 ng / mL ) , Cm ( 1 ng / mL ) and Um ( 5 ng / mL ) standards were injected to UPLC - MS / MS as control . cps , counts per second .

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(D) UPLC-MS/MS analysis of 2'-O-methylation of m1G37_tRNAPhe(GAA), -G34A, -G34C and G34U after incubation with FTSJ1-WDR6. 2 μL of Gm (1 ng/mL), Am (1 ng/mL), Cm (1 ng/mL) and Um (5 ng/mL) standards were injected to UPLC-MS/MS as control. cps, counts per second.


A ) The SAM binding affinity of FTSJ1 as measured by ITC .

A) The SAM binding affinity of FTSJ1 as measured by ITC.


( B The binding affinity of FTSJ1 alone or FTSJ1 - WDR6 for tRNA analyzed by the gel mobility shift assay .

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(B The binding affinity of FTSJ1 alone or FTSJ1-WDR6 for tRNA analyzed by the gel mobility shift assay.


C ) For the reaction in figure C , 0 . 2 μΜ GST , 0 . 2 μΜ GST - WDR6 , 1 μM FTSJ1 , the mixture of 1 μM FTSJ1 with 0 . 2 μΜ GST - WDR6 or 0 . 2 μΜ GST , 0 . 5 mM SAM and 250 nM tRNAPhe ( GAA ) transcript were incubated .

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C) For the reaction in figure C, 0.2 μΜ GST, 0.2 μΜ GST-WDR6, 1 μM FTSJ1, the mixture of 1 μM FTSJ1 with 0.2 μΜ GST-WDR6 or 0.2 μΜ GST, 0.5 mM SAM and 250 nM tRNAPhe(GAA) transcript were incubated.


The representative graphs for quantification of the expected Nm32 / A % and Nm34 / A % ratios of G37 - tRNAPhe ( GAA ) , tRNALeu ( UAA ) , tRNATrp ( CCA ) , tRNAArg ( ACG ) , tRNAArg ( UCG ) , and A37 - tRNAMet ( CAU ) , which were isolated from WT , ftsj1 KO , and wdr6 KO cells by UPLC - MS / MS analysis . Error bars represent the standard deviation of three independent experiments . p values were determined using two - tailed Student ' s t - test for paired samples . * * * p < 0 . 001 . n . s . , no significance . n . d . , not detected .

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

The representative graphs for quantification of the expected Nm32/A% and Nm34/A% ratios of G37-tRNAPhe(GAA), tRNALeu(UAA), tRNATrp(CCA), tRNAArg(ACG), tRNAArg(UCG), and A37-tRNAMet(CAU), which were isolated from WT, ftsj1 KO, and wdr6 KO cells by UPLC-MS/MS analysis. Error bars represent the standard deviation of three independent experiments. p values were determined using two-tailed Student's t-test for paired samples. ***p < 0.001. n.s., no significance. n.d., not detected.


( A , B ) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0 . 2 mg / mL paromomycin assayed by cell counting kit - 8 proliferation analysis .

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(A, B) The growth curve of WT and two ftsj1 KO HEK293T cell lines under normal culture or in the culture condition with 0.2 mg/mL paromomycin assayed by cell counting kit-8 proliferation analysis.


( C , D ) The growth curve of WT with or without overexpressing tRNAPhe ( GAA ) under normal culture or with 0 . 2 mg / mL paromomycin .

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(C, D) The growth curve of WT with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.


( E , F ) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe ( GAA ) under normal culture or with 0 . 2 mg / mL paromomycin .

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(E, F) The growth curve of ftsj1 KO cells with or without overexpressing tRNAPhe(GAA) under normal culture or with 0.2 mg/mL paromomycin.


( H , I ) The translation efficiency of F - luc with the 6 × UUC codons or 6 × UUU codons .

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(H, I) The translation efficiency of F-luc with the 6×UUC codons or 6×UUU codons.


( J , K ) The translation efficiency of F - luc with all UUC codons or all UUU codons .

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

(J, K) The translation efficiency of F-luc with all UUC codons or all UUU codons.


Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and the indicated combinations of empty HA - vector , HA - tagged FIP200 , ULK1 , or ATG13 before immunoprecipitation with anti - HA antibodies . Immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to Western blotting against anti - HA or Flag . Data information : the experiments were performed at least three times independently with similar results .

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

Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and the indicated combinations of empty HA-vector, HA-tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti-HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti-HA or Flag. Data information: the experiments were performed at least three times independently with similar results.


C - F . WT ( C ) , FIP200 knockout ( KO ) ( D ) , ULK1 / 2 knockout ( E ) MEF cells or ATG13 knockout 4T1 cells ( F ) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G ( IgG ) or anti - TRIM27 before blotting against the indicated endogenous forms of TRIM27 , FIP200 , ULK1 , ATG13 , and USP7 . Data information : the experiments were performed at least three times independently with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti-TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7. Data information: the experiments were performed at least three times independently with similar results.


G . Co - immunoprecipitation assays in HEK293T cells after co - transfection with Flag - TRIM27 and either full length ULK ( HA - ULK1 1 - 1051 ) or the indicated various truncated forms of ULK1 designated HA - ULK1 1 - 828 , HA - ULK1 1 - 600 , and HA - ULK1 1 - 500 ; AA , amino acids . Data information : , the experiments were performed at least three times independently with similar results .

G. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and either full length ULK (HA-ULK1 1-1051) or the indicated various truncated forms of ULK1 designated HA-ULK1 1-828, HA-ULK1 1-600, and HA-ULK1 1-500; AA, amino acids. Data information: , the experiments were performed at least three times independently with similar results.


H . Co - immunoprecipitation assays in HeLa cells stably expressing HA - ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated . Data information : the experiments were performed at least three times independently with similar results .

O O O O O O O O O O O O O O O O O O O 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. Co-immunoprecipitation assays in HeLa cells stably expressing HA-ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated. Data information: the experiments were performed at least three times independently with similar results.


HeLa cells stably expressing Flag - TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium . Anti - Flag immunoprecipitates ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against Flag and ULK1 . Data information : the experiments were performed at least three times independently with similar results .

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

HeLa cells stably expressing Flag-TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti-Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1. Data information: the experiments were performed at least three times independently with similar results.


Purified recombinant GST or GST - TRIM27 was incubated with recombinant HA - ULK1 and bound ULK1 analyzed by Western blotting . Data information : the experiments were performed at least three times independently with similar results .

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

Purified recombinant GST or GST-TRIM27 was incubated with recombinant HA-ULK1 and bound ULK1 analyzed by Western blotting. Data information: the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T cells after co - transfection with HA - ULK1 and His - Ub along with Flag - TRIM27 ( A ) Samples recovered with Ni - NTA ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against anti - HA , Flag or His as indicated . Data information : , the experiments were performed at least three times independently with similar results .

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

Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs ( shTRIM27 - 1 and - 2 ) ( B ) . Samples recovered with Ni - NTA ( top panel ) and input samples ( bottom panel ) were then subjected to blotting against anti - HA or His as indicated . Data information : , the experiments were performed at least three times independently with similar results .

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

Ubiquitination assays in HEK293T cells after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA or His as indicated. Data information: , the experiments were performed at least three times independently with similar results.


C . In vitro ubiquitination assays performed with the indicated combinations of recombinant GST , GST - TRIM27 , affinity - purified HA - ULK1 and His - Ub in the presence or absence of E1 , E2 , and ATP . The reactions were analyzed with anti - Ub , anti - HA , or anti - GST . Data information : the experiments were performed at least three times independently with similar results .

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

C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST. Data information: the experiments were performed at least three times independently with similar results.


Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs ( D ) Data information : , the experiments were performed at least three times independently with similar results .

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

Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D) Data information: , the experiments were performed at least three times independently with similar results.


Western blot analysis of HEK293T cells transfected with empty Flag vector ( - ) or Flag - TRIM27 after treatment with or without 10 μM MG132 for 4 hours ( E ) . Data information : the experiments were performed at least three times independently with similar results .

O 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

Western blot analysis of HEK293T cells transfected with empty Flag vector (-) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 hours (E). Data information: the experiments were performed at least three times independently with similar results.


F . Fluorescence microscopy image of MEFs with stable expression of mCherry - ULK1 and GFP - TRIM27 after treating cells with DMSO ( MOCK ) or 10 μM MG132 for 4 hours . Scale bar , 10 μm . Data information : the experiments were performed at least three times independently with similar results .

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F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 hours. Scale bar, 10 μm. Data information: the experiments were performed at least three times independently with similar results.


HA - ULK1 levels in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T cells with or without co - expression of Flag - TRIM27 ( G , H ) Western blots against HA - ULK1 and actin control ( G , were subjected to densitometric quantitation ( H , Data information : the experiments were performed at least three times independently with similar results . All the statistical data are presented as mean ± s . d . of three independent experiments . Statistical analysis was performed using two - tailed unpaired Student ' s t - tests ( H , * * P < 0 . 01 .

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HA-ULK1 levels in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) Western blots against HA-ULK1 and actin control (G, were subjected to densitometric quantitation (H, Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H , **P < 0.01.


endogenous ULK1 levels ( I , J ) in 50 μg / mL cycloheximide ( CHX ) - treated HEK293T cells or without TRIM27 RNAi ( I , J ) . and actin control I ) were subjected to densitometric quantitation J ) . Data information : the experiments were performed at least three times independently with similar results . All the statistical data are presented as mean ± s . d . of three independent experiments . Statistical analysis was performed using two - tailed unpaired Student ' s t - tests J ) , * * P < 0 . 01 .

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endogenous ULK1 levels (I, J) in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells or without TRIM27 RNAi (I, J). and actin control I) were subjected to densitometric quantitation J). Data information: the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests J), **P < 0.01.


Ubiquitination assays were performed in HEK293T cells after co - transfection with full length HA - ULK1 or the indicated truncated constructs ( K ) in the presence or absence of Flag - TRIM27 . Data information : , the experiments were performed at least three times independently with similar results .

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

Ubiquitination assays were performed in HEK293T cells after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) in the presence or absence of Flag-TRIM27. Data information: , the experiments were performed at least three times independently with similar results.


Ubiquitination assays were performed in HEK293T cells after co - transfection with double ( K568 / K571R ) or single ( K568R , K571R ) lysine substitution mutants ( L ) in the presence or absence of Flag - TRIM27 . Data information : the experiments were performed at least three times independently with similar results .

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

Ubiquitination assays were performed in HEK293T cells after co-transfection with double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27. Data information: the experiments were performed at least three times independently with similar results.