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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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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 .

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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 ) .

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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 .

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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 .

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

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

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

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 .

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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 .

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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 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

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 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

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 ) .

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

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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 ) .

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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 -

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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 ) .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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

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

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

(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 .

O O O O O O O O O O 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

(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 .

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

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

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 .

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(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 B-CONTROLLED_VAR 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

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

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 .

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

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

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 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

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

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

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 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

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 .

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

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 B-CONTROLLED_VAR 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

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 B-CONTROLLED_VAR 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.


M . Ubiquitination of HA - ULK1 in cells co - expressing Flag - TRIM27 with either His - Ub WT , His - Ub K48 , or His - Ub K63 , respectively . Data information : the experiments were performed at least three times independently with similar results .

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

M. Ubiquitination of HA-ULK1 in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively. Data information: the experiments were performed at least three times independently with similar results.


N . In vitro ubiquitination assays against affinity - purified HA - ULK1 WT or the HA - ULK1 K568 / K571R mutant in the presence or absence of GST - 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 B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

N. In vitro ubiquitination assays against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27. Data information: the experiments were performed at least three times independently with similar results.


O . ULK1 / 2 knockout MEFs stably expressing the indicated HA - ULK1 constructs were subjected to Western blot analysis against HA or actin before ( 0h ) and after treatment with CHX for 12 hours . 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 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. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0h) and after treatment with CHX for 12 hours. Data information: the experiments were performed at least three times independently with similar results.


A . Western blot analysis of autophagy marker proteins ( p62 and Ser318 phosphorylated ATG13 ) in WT HeLa cells and two independent TRIM27 KO clones . ULK1 and TRIM27 levels were measured , along with actin as a loading control .

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A. Western blot analysis of autophagy marker proteins (p62 and Ser318 phosphorylated ATG13) in WT HeLa cells and two independent TRIM27 KO clones. ULK1 and TRIM27 levels were measured, along with actin as a loading control.


B . TRIM27 KO HeLa cells were reconstituted with Flag - TRIM27 and Western blotting was performed as per ( A ) .

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B. TRIM27 KO HeLa cells were reconstituted with Flag-TRIM27 and Western blotting was performed as per (A).


C . LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 ( BAF A1 ) .

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C. LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 (BAF A1).


D . Western blot analysis of indicated proteins in MEFs generated from Trim27 + / + or Trim27 - / - mice .

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D. Western blot analysis of indicated proteins in MEFs generated from Trim27 +/+ or Trim27 -/- mice.


E - H . Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27 + / + or Trim27 - / - mice cultured in fed ( E ) or starved conditions ( G ) . Quantification of LC3B puncta Fed conditions ( F ) , starved conditions ( H ) ( n = 50 cells per condition ) . Data are mean ± s . d . in F and H . Scale bar , 10 μm . Data information : ( F , H , Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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E-H. Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27+/+ or Trim27-/- mice cultured in fed (E) or starved conditions (G). Quantification of LC3B puncta Fed conditions (F), starved conditions (H) (n = 50 cells per condition). Data are mean ± s.d. in F and H. Scale bar, 10 μm. Data information: (F, H, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


I - J . Western blot analysis of autophagy markers in the kidneys of three - month - old Trim27 + / + and Trim27 - / - mice . Each lane represents a different mouse ( I ) . Quantification of p62 , LC3 - II / LC3 - I ratio , and LC3 - II / actin ratio in I ( J ) . Data are mean ± s . e . m . for three mice per genotype . Data information : J , Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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I-J. Western blot analysis of autophagy markers in the kidneys of three-month-old Trim27 +/+ and Trim27-/- mice. Each lane represents a different mouse (I). Quantification of p62, LC3-II/LC3-I ratio, and LC3-II/actin ratio in I (J). Data are mean ± s.e.m. for three mice per genotype. Data information: J, Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


K . Microscopy analysis of GFP - LC3 puncta in MEFs generated from indicated genotype mice in normal or starvation conditions . Scale bar , 10 μm . L - M . Quantitation of data presented in ( K ) shows mean number of GFP - LC3 puncta per cell ± s . d . ( n = 50 cells per condition ) . Data information : L and M ) Statistical analysis was performed using two - tailed unpaired Student ' s t - tests , * P < 0 . 05 , * * * P < 0 . 001 .

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K. Microscopy analysis of GFP-LC3 puncta in MEFs generated from indicated genotype mice in normal or starvation conditions. Scale bar, 10 μm. L-M. Quantitation of data presented in (K) shows mean number of GFP-LC3 puncta per cell ± s.d. (n = 50 cells per condition). Data information: L and M) Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.


A . Ubiquitination analysis of HA - ULK1 in HeLa derivatives co - expressing His - Ub with or without TRIM27 knockout , the cells were treated with starvation for the indicated time .

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A. Ubiquitination analysis of HA-ULK1 in HeLa derivatives co-expressing His-Ub with or without TRIM27 knockout, the cells were treated with starvation for the indicated time.


B . Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout . The cells were treated with starvation for the indicated time . The lysates were analyzed using anti - ULK1 , anti - TRIM27 , or anti - Actin .

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B. Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout. The cells were treated with starvation for the indicated time. The lysates were analyzed using anti-ULK1, anti-TRIM27, or anti-Actin.


C . HA - ULK1 , HA - ULK1 K568 / K571R were stably expressed in ULK1 knockout HeLa cells , the cells were treated with starvation , the lysates were analyzed by western blot using anti - HA , or anti - Actin .

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C. HA-ULK1, HA-ULK1 K568/K571R were stably expressed in ULK1 knockout HeLa cells, the cells were treated with starvation, the lysates were analyzed by western blot using anti-HA, or anti-Actin.


E . Co - immunoprecipitation of Flag - TRIM27 with HA - ULK1 in HEK293T cells with or without STK38L knockdown , the cells were treated with starvation . Flag - TRIM27 was immunoprecipitated using anti - Flag , the bound HA - ULK1 was detected using anti - HA .

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E. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 in HEK293T cells with or without STK38L knockdown, the cells were treated with starvation. Flag-TRIM27 was immunoprecipitated using anti-Flag, the bound HA-ULK1 was detected using anti-HA.


F . Co - immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells .

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F. Co-immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells.


G . Co - immunoprecipitation of Flag - STK38L with ULK1 , or TRIM27 in HEK293T cells with or without starvation treatment . Flag - STK38L was immunoprecipitated using anti - Flag , the immunoprecipitates and lysates were analyzed with anti - ULK1 , anti - TRIM27 .

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G. Co-immunoprecipitation of Flag-STK38L with ULK1, or TRIM27 in HEK293T cells with or without starvation treatment. Flag-STK38L was immunoprecipitated using anti-Flag, the immunoprecipitates and lysates were analyzed with anti-ULK1, anti-TRIM27.


H . Co - immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout . The cells were treated with starvation before lysis .

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H. Co-immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout. The cells were treated with starvation before lysis.


I . Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc - STK38L , or Flag - TRIM27 , or both . The lysates were analyzed using anti - ULK1 , anti - Flag , anti - Myc , or anti - actin .

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I. Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc-STK38L, or Flag-TRIM27, or both. The lysates were analyzed using anti-ULK1, anti-Flag, anti-Myc, or anti-actin.


J . Ubiquitination analysis of HA - ULK1 in HEK293T cells co - expressing Flag - TRIM27 and His - Ub , or together with Myc - STK38L .

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J. Ubiquitination analysis of HA-ULK1 in HEK293T cells co-expressing Flag-TRIM27 and His-Ub, or together with Myc-STK38L.


K . Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout , the cells were treated with starvation for the indicated time .

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K. Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time.


L . Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout , the cells were treated with starvation for the indicated time . Bafilomycin A1 ( BAF A1 ) was used to inhibit the fusion between autophagosomes and lysosomes .

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L. Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time. Bafilomycin A1 (BAF A1) was used to inhibit the fusion between autophagosomes and lysosomes.


A . Western blot analysis of ULK1 levels in HeLa cells stably expressing Flag - TRIM27 , the cells were transfected with either Myc - vector , Myc - STK38L , or Myc - STK38L - K119A . Then the cells were treated with starvation before lysis .

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A. Western blot analysis of ULK1 levels in HeLa cells stably expressing Flag-TRIM27, the cells were transfected with either Myc-vector, Myc-STK38L, or Myc-STK38L-K119A. Then the cells were treated with starvation before lysis.


B . Phosphorylation of HA - ULK1 in HeLa STK38L knockout cells with or without expression of Flag - STK38L . HA - ULK1 was immunoprecipitated and then analyzed by western blot using anti - phosphoserine or anti - phosphothreonine , and the cell lysates were analyzed using anti - HA and anti - Myc .

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B. Phosphorylation of HA-ULK1 in HeLa STK38L knockout cells with or without expression of Flag-STK38L. HA-ULK1 was immunoprecipitated and then analyzed by western blot using anti-phosphoserine or anti-phosphothreonine, and the cell lysates were analyzed using anti-HA and anti-Myc.


C . Co - immunoprecipitation of Flag - TRIM27 with HA - ULK1 or HA - ULK1 - S495A in HEK293T cells . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated using anti - HA , the immunoprecipitates and lysates were analyzed with anti - HA or anti - Flag .

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C. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 or HA-ULK1-S495A in HEK293T cells. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with anti-HA or anti-Flag.


D . Ubiquitination analysis of HA - ULK1 WT or HA - ULK1 S495A in HEK293T cells co - expressing Flag - TRIM27 and His - Ub .

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D. Ubiquitination analysis of HA-ULK1 WT or HA-ULK1 S495A in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.


E . Phosphorylation of HA - ULK1 in HEK293T cells co - expressing Myc - STK38L with or without CIP treatment . HA - ULK1 was immunoprecipitated with anti - HA and analyzed by western blot using a specific antibody against p - ULK1 - S495 ( Ser495 ) .

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E. Phosphorylation of HA-ULK1 in HEK293T cells co-expressing Myc-STK38L with or without CIP treatment. HA-ULK1 was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).


F . Phosphorylation of HA - ULK1 or HA - ULK1 S495A in HEK293T cells co - expressing Myc - STK38L . HA - ULK1 or HA - ULK1 S495A was immunoprecipitated with anti - HA and analyzed by western blot using a specific antibody against p - ULK1 - S495 ( Ser495 ) .

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F. Phosphorylation of HA-ULK1 or HA-ULK1 S495A in HEK293T cells co-expressing Myc-STK38L. HA-ULK1 or HA-ULK1 S495A was immunoprecipitated with anti-HA and analyzed by western blot using a specific antibody against p-ULK1-S495 (Ser495).


G . In vitro kinase assay using GST - STK38L , or GST - STK38L K119A ( a kinase - inactive mutant ) and HA - ULK1 as substrate . GST - STK38L or GST - STK38L K119A was pulled down using glutathione Sepharose beads . HA - ULK1 was immunoprecipitated from HEK293T using anti - HA and treated with lambda phosphatase , the reaction product was analyzed by western blot using p - ULK1 - S495 ( Ser495 ) .

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G. In vitro kinase assay using GST-STK38L, or GST-STK38L K119A (a kinase-inactive mutant) and HA-ULK1 as substrate. GST-STK38L or GST-STK38L K119A was pulled down using glutathione Sepharose beads. HA-ULK1 was immunoprecipitated from HEK293T using anti-HA and treated with lambda phosphatase, the reaction product was analyzed by western blot using p-ULK1-S495 (Ser495).


H . Ubiquitination analysis of Myc - STK38L in HEK293T cells co - expressing Flag - TRIM27 and His - Ub .

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H. Ubiquitination analysis of Myc-STK38L in HEK293T cells co-expressing Flag-TRIM27 and His-Ub.


I . Ubiquitination analysis of Flag - STK38L in HEK293T cells co - expressing His - Ub with or without TRIM27 knockdown , cells were cultured in fed or starved conditions as indicated .

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I. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing His-Ub with or without TRIM27 knockdown, cells were cultured in fed or starved conditions as indicated.


J . Ubiquitination analysis of Flag - STK38L in HEK293T cells co - expressing Myc - TRIM27 with either His - Ub ( wild - type ) or His - Ub K6R .

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J. Ubiquitination analysis of Flag-STK38L in HEK293T cells co-expressing Myc-TRIM27 with either His-Ub (wild-type) or His-Ub K6R.


K . Ubiquitination analysis of Myc - STK38L WT , Myc - STK38L - K432R + K434R or Myc - STK38L - K181R + K215R + K224R + K432R + K434R ( Myc - STK38L - 5KR ) mutant in HEK293T cells co - expressing HA - TRIM27 and His - Ub .

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K. Ubiquitination analysis of Myc-STK38L WT, Myc-STK38L-K432R+K434R or Myc-STK38L-K181R+K215R+K224R+K432R+K434R (Myc-STK38L-5KR) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.


L . Ubiquitination analysis of Myc - STK38L K1 - 38R mutant ( All lysines were mutated to arginines ) , or Myc - STK38L - R181 / 215 / 224 / 432 / 434K ( All lysines were mutated to arginines except the indicated five lysine residues ) mutant in HEK293T cells co - expressing HA - TRIM27 and His - Ub .

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L. Ubiquitination analysis of Myc-STK38L K1-38R mutant (All lysines were mutated to arginines), or Myc-STK38L-R181/215/224/432/434K (All lysines were mutated to arginines except the indicated five lysine residues) mutant in HEK293T cells co-expressing HA-TRIM27 and His-Ub.


M . Phosphorylation of HA - ULK1 S495 ( Ser495 ) in HEK293T cells co - expressing Myc - STK38L WT , or Myc - STK38L - 5KR mutant in K . The cells were cultured in fed or starved conditions as indicated and HA - ULK1 was immunoprecipitated using anti - HA , the immunoprecipitates and lysates were analyzed with a specific antibody against p - ULK1 - S495 ( Ser495 ) , anti - Myc , anti - HA , or anti - actin .

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M. Phosphorylation of HA-ULK1 S495(Ser495) in HEK293T cells co-expressing Myc-STK38L WT, or Myc-STK38L-5KR mutant in K. The cells were cultured in fed or starved conditions as indicated and HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-Myc, anti-HA, or anti-actin.


N . Phosphorylation of HA - ULK1 in HeLa cells with or without TRIM27 - knockout , cells were cultured in fed or starved conditions as indicated , HA - ULK1 was immunoprecipitated using anti - HA , the immunoprecipitates and lysates were analyzed with a specific antibody against p - ULK1 - S495 ( Ser495 ) , anti - HA , or anti - actin .

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N. Phosphorylation of HA-ULK1 in HeLa cells with or without TRIM27-knockout, cells were cultured in fed or starved conditions as indicated, HA-ULK1 was immunoprecipitated using anti-HA, the immunoprecipitates and lysates were analyzed with a specific antibody against p-ULK1-S495 (Ser495), anti-HA, or anti-actin.


A . Comparison of the relative mRNA levels of TRIM27 in normal breast tissues ( Normal , n = 113 ) and indicated breast tumors ( Basal ， n = 143 ； Her2 ， n = 67 ； LumA ， n = 427 ； LumB ， n = 190 ) in the TCGA database . The number of biological replicates are shown as indicated . P < 0 . 0001 ( one - way ANOVA ) .

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A. Comparison of the relative mRNA levels of TRIM27 in normal breast tissues (Normal, n=113) and indicated breast tumors (Basal，n=143；Her2，n=67；LumA，n=427；LumB，n=190) in the TCGA database. The number of biological replicates are shown as indicated. P < 0.0001 (one-way ANOVA).


B . Analysis of the correlation between ULK1 and TRIM27 expression in the CPTAC proteomics database . P = 0 . 0027 ( Pearson ' s correlation method ) .

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B. Analysis of the correlation between ULK1 and TRIM27 expression in the CPTAC proteomics database. P = 0.0027 (Pearson's correlation method).


Western blot analysis of ULK1 and TRIM27 levels from tumor tissues ( T ) , Pericarcinomatous tissues ( P ) , and their matched surrounding normal tissues ( N ) . GAPDH was used as a loading control ( C ) .

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Western blot analysis of ULK1 and TRIM27 levels from tumor tissues (T), Pericarcinomatous tissues (P), and their matched surrounding normal tissues (N). GAPDH was used as a loading control (C).


F . Representative tumors in MMTV - PyMT mice of the indicated Trim27 genotypes . Scale bar , 5mm .

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F. Representative tumors in MMTV-PyMT mice of the indicated Trim27 genotypes. Scale bar, 5mm.


G . Western blot analysis of indicated proteins in Trim27 - / - PyMT or Trim27 + / - PyMT mice in primary breast tumors . Actin was used as a loading control .

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G. Western blot analysis of indicated proteins in Trim27-/- PyMT or Trim27+/- PyMT mice in primary breast tumors. Actin was used as a loading control.


H . Kaplan - Meier tumor - free survival curves of Trim27 + / - PyMT ( median = 75 d , number = 20 ) , or Trim27 - / - PyMT mice ( median = 95 d , number = 16 ) . P < 0 . 0001 ( Mantel - Cox test ) .

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H. Kaplan-Meier tumor-free survival curves of Trim27+/- PyMT (median = 75 d, number = 20), or Trim27 -/- PyMT mice (median = 95 d, number = 16). P < 0.0001 (Mantel-Cox test).


Pulmonary surface nodules in Trim27 + / - PyMT mice and Trim27 - / - PyMT mice ( I Scale bar , 1mm .

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Pulmonary surface nodules in Trim27+/- PyMT mice and Trim27-/- PyMT mice (I Scale bar, 1mm.


Pulmonary surface nodules and hematoxylin and eosin ( H & E ) staining in Trim27 + / - PyMT mice and Trim27 - / - PyMT mice J ) . Scale bar , 1mm . ( K ) . Bar chart showing quantification for the number of metastatic nodules per lung lope ( Trim27 + / - , n = 6 mice ; Trim27 - / - n = 6 mice ) , when compared at the same final timepoint . Each symbol represents a mouse ; Data are presented as mean ± s . d . from three independent experiments ; * * * P < 0 . 001 ( unpaired student ' s t - test ) .

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Pulmonary surface nodules and hematoxylin and eosin (H&E) staining in Trim27+/- PyMT mice and Trim27-/- PyMT mice J). Scale bar, 1mm. (K). Bar chart showing quantification for the number of metastatic nodules per lung lope (Trim27+/-, n=6 mice; Trim27-/- n=6 mice), when compared at the same final timepoint. Each symbol represents a mouse; Data are presented as mean ± s.d. from three independent experiments; ***P<0.001 (unpaired student's t-test).


Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter - targeted strain at ring stage . Levels of dCas9 and histone H3 in the cytoplasmic ( Cyt ) , nuclear ( excluding chromatin , Nuc ) , and chromatin ( Ch ) fractions are revealed with anti - HA and anti - H3 antibodies , respectively . dCas9 is enriched in the immunoprecipitated fraction ( IP ) compared to the unbound supernatant ( SN ) and input ( i . e . chromatin fraction ) of the IP . Molecular weights are shown to the right .

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

Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter-targeted strain at ring stage. Levels of dCas9 and histone H3 in the cytoplasmic (Cyt), nuclear (excluding chromatin, Nuc), and chromatin (Ch) fractions are revealed with anti-HA and anti-H3 antibodies, respectively. dCas9 is enriched in the immunoprecipitated fraction (IP) compared to the unbound supernatant (SN) and input (i.e. chromatin fraction) of the IP. Molecular weights are shown to the right.