D , In vitro GST pull - down assays were performed with bacterially expressed and purified GST or GST - tagged AR immobilized onto GSH beads . Immobilized GST protein beads were used to pull down purified his - MBP ( left ) or his - MBP - tagged TRIM33 ( right ) .

D, In vitro GST pull-down assays were performed with bacterially expressed and purified GST or GST-tagged AR immobilized onto GSH beads. Immobilized GST protein beads were used to pull down purified his-MBP (left) or his-MBP-tagged TRIM33 (right).


AR - dependent luciferase assays were performed on LNCaP cells transfected with siTRIM33 or negative control ( NC ) ( A ) , increasing amounts of TRIM33 plasmid ( B ) Western blot analysis shows the protein level of TRIM33 in LNCaP cells after TRIM33 knockdown ( A ) or overexpression ( B ) . Data information : , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 ; * * P < 0 . 01 ; ns = not significant ( two - way ANOVA ) .

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

AR-dependent luciferase assays were performed on LNCaP cells transfected with siTRIM33 or negative control (NC) (A), increasing amounts of TRIM33 plasmid (B) Western blot analysis shows the protein level of TRIM33 in LNCaP cells after TRIM33 knockdown (A) or overexpression (B). Data information: , data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05; **P < 0.01; ns=not significant (two-way ANOVA).


A - D , AR transcript levels were measured by RT - qPCR on LNCaP cells ( A and C ) or VCaP cells ( B and D ) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT . Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies . Data information : In ( A - D , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ( two - way ANOVA ) .

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

A-D, AR transcript levels were measured by RT-qPCR on LNCaP cells (A and C) or VCaP cells (B and D) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT. Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies. Data information: In (A-D, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).


E and F , LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide ( CHX ) . Protein lysates were harvested at the indicated time points for western blot analysis . AR protein levels were quantified and normalized to β - actin and then plotted relative to 0 h . Data information : In F , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ( two - way ANOVA ) .

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

E and F, LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide (CHX). Protein lysates were harvested at the indicated time points for western blot analysis. AR protein levels were quantified and normalized to β-actin and then plotted relative to 0 h. Data information: In F, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).


G , LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h . Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies .

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

G, LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h. Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies.


HA - Ub was expressed in LNCaP cells co - transfected with siTRIM33 ( H ) The cells were treated with MG132 for 4 h before harvesting . Endogenous AR was immunoprecipitated from cell lysates , and interacting proteins were detected by immunoblotting with the indicated antibodies .

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

HA-Ub was expressed in LNCaP cells co-transfected with siTRIM33 (H) The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.


I , HA - Ub was expressed in LNCaP cells co - transfected with the TRIM33 expression construct ( I ) . The cells were treated with MG132 for 4 h before harvesting . Endogenous AR was immunoprecipitated from cell lysates , and interacting proteins were detected by immunoblotting with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HA-Ub was expressed in LNCaP cells co-transfected with the TRIM33 expression construct (I). The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.


LNCaP cells transiently expressing myc - tagged Skp2 with Flag - TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies .

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

LNCaP cells transiently expressing myc-tagged Skp2 with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.


K , LNCaP cells transiently expressing myc - tagged SPOP with Flag - TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies .

O O 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, LNCaP cells transiently expressing myc-tagged SPOP with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.


L , shTRIM33 - or shCtrl - LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies .

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

L, shTRIM33- or shCtrl-LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies.


M , HEK293T cells were co - transfected with flag - TRIM33 and various myc - tagged AR constructs . AR - FL and AR truncated mutants were immunoprecipitated with an anti - myc antibody and then subjected to western blot analysis with the indicated antibodies .

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

M, HEK293T cells were co-transfected with flag-TRIM33 and various myc-tagged AR constructs. AR-FL and AR truncated mutants were immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.


N , HEK293T cells were co - transfected with myc - AR - N and various flag - tagged TRIM33 constructs . AR - N was immunoprecipitated with an anti - myc antibody and then subjected to western blot analysis with the indicated antibodies .

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

N, HEK293T cells were co-transfected with myc-AR-N and various flag-tagged TRIM33 constructs. AR-N was immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.


O , LNCaP cells were transfected with various flag - tagged TRIM33 constructs . AR - associated proteins were immunoprecipitated with an anti - AR antibody and then subjected to western blot analysis with the indicated antibodies .

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

O, LNCaP cells were transfected with various flag-tagged TRIM33 constructs. AR-associated proteins were immunoprecipitated with an anti-AR antibody and then subjected to western blot analysis with the indicated antibodies.


P , LNCaP cells were co - transfected with myc - Skp2 and various flag - TRIM33 constructs . AR and other proteins were detected by western blot analysis with the indicated antibodies .

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

P, LNCaP cells were co-transfected with myc-Skp2 and various flag-TRIM33 constructs. AR and other proteins were detected by western blot analysis with the indicated antibodies.


Q , AR pull - down was performed on protein extracts from MG132 - treated HEK293T cells overexpressing myc - AR , HA - Ub , myc - Skp2 , and various flag - tagged TRIM33 constructs . AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies .

Q, AR pull-down was performed on protein extracts from MG132-treated HEK293T cells overexpressing myc-AR, HA-Ub, myc-Skp2, and various flag-tagged TRIM33 constructs. AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies.


A , TRIM33 mRNA expression levels were analyzed in normal and tumor samples from multiple publicly available patient cohorts . A two - sided Wilcoxon signed - rank test was used for the TCGA Prostate , Ding 2016 , and Jing 2020 datasets , while a two - sided Wilcoxon rank - sum test was used for the other datasets . Measurements shown in box plots are the first and third quartiles and are split by the medians , whiskers extending a 1 . 5 - fold interquartile range beyond the box .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, TRIM33 mRNA expression levels were analyzed in normal and tumor samples from multiple publicly available patient cohorts. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.


B and C , A human PCa progression tissue microarray was used for immunohistochemistry staining . Representative staining images are shown in B . TRIM33 staining by Gleason Scores is summarized in the histogram . Scale bar : 100 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

B and C, A human PCa progression tissue microarray was used for immunohistochemistry staining. Representative staining images are shown in B. TRIM33 staining by Gleason Scores is summarized in the histogram. Scale bar: 100 μm.


A , The AR - TRIM33 coactivated gene signature was compared with two PCa patient - derived AR gene signatures in multiple publicly available patient datasets .

O O O 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, The AR-TRIM33 coactivated gene signature was compared with two PCa patient-derived AR gene signatures in multiple publicly available patient datasets.


B , The expression of the gene signature in normal and tumor samples was examined in the same cohorts used in A . A two - sided Wilcoxon signed - rank test was used for the TCGA Prostate , Ding 2016 , and Jing 2020 datasets , while a two - sided Wilcoxon rank - sum test was used for the other datasets . Measurements shown in box plots are the first and third quartiles and are split by the medians , whiskers extending a 1 . 5 - fold interquartile range beyond the box .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, The expression of the gene signature in normal and tumor samples was examined in the same cohorts used in A. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.


C , Hierarchical clustering of PCa patients from the TCGA Prostate dataset ( n = 480 ) resulted in two distinct clusters indicated in orange ( high ) and blue ( low ) based on the average expression of our 36 - gene signature .

C, Hierarchical clustering of PCa patients from the TCGA Prostate dataset (n = 480) resulted in two distinct clusters indicated in orange (high) and blue (low) based on the average expression of our 36-gene signature.


E , Kaplan - Meier analysis showing the recurrence - free survival of patients with low and high expression of the 36 - gene signature ( p - value , log - rank test ) .

O O O O O O O O O O O O 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, Kaplan-Meier analysis showing the recurrence-free survival of patients with low and high expression of the 36-gene signature (p-value, log-rank test).


J , Western blot analysis of AR and TRIM3 protein levels in tumor tissues from H .

J, Western blot analysis of AR and TRIM3 protein levels in tumor tissues from H.


( A ) Excision of endogenous Hi in plants with and without HiTG obtained from the same parental plant .

(A) Excision of endogenous Hi in plants with and without HiTG obtained from the same parental plant.


( B ) Patterns of DNA methylation for each cytosine context in an endogenous Hi locus calculated from WGBS data . Vertical dashed lines indicate the termini of Hiun .

O O O O O O 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) Patterns of DNA methylation for each cytosine context in an endogenous Hi locus calculated from WGBS data. Vertical dashed lines indicate the termini of Hiun.


( A - C ) Excision of endogenous Hi in cmt23 ( A ) , drm12 ( B ) , and ddcc ( C ) mutant backgrounds when HiTG is segregated away . Plants with * and * * were used for WGBS , and the results of those with * * were used for the analysis shown in Figures .

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

(A-C) Excision of endogenous Hi in cmt23 (A), drm12 (B), and ddcc (C) mutant backgrounds when HiTG is segregated away. Plants with * and ** were used for WGBS, and the results of those with ** were used for the analysis shown in Figures.


( D ) Excision of endogenous Hi in mutant backgrounds of epigenetic regulators . drm12 : drm1 drm2 double mutant , ddc : drm1 drm2 cmt3 triple mutant , cmt23 : cmt2 cmt3 double mutant , and ddcc : drm1 drm2 cmt2 cmt3 quadruple mutant . Histone acetyltransferase HDA6 / RTS1 is reported as important for silencing of TEs , and TEs activated in hda6 mutant are shared with those activated in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Excision of endogenous Hi in mutant backgrounds of epigenetic regulators. drm12: drm1 drm2 double mutant, ddc: drm1 drm2 cmt3 triple mutant, cmt23: cmt2 cmt3 double mutant, and ddcc: drm1 drm2 cmt2 cmt3 quadruple mutant. Histone acetyltransferase HDA6/RTS1 is reported as important for silencing of TEs, and TEs activated in hda6 mutant are shared with those activated in met1


( A - C ) Patterns of DNA methylation in endogenous Hi ( A ) , AT2TE05755 ( B ) , and AT2TE06955 ( C ) in the drm12 mutant background . ' ( - HiTG ) ' indicates that HiTG was segregated . Vertical dashed lines indicate termini of TEs .

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

(A-C) Patterns of DNA methylation in endogenous Hi (A), AT2TE05755 (B), and AT2TE06955 (C) in the drm12 mutant background. '(-HiTG)' indicates that HiTG was segregated. Vertical dashed lines indicate termini of TEs.


( A ) Patterns of DNA methylation in flanking regions of VANC21 targets when HiTG was segregated in drm12 ( left ) and cmt23 ( right ) mutant background . Grey dashed lines indicate the points of VANC targets .

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(A) Patterns of DNA methylation in flanking regions of VANC21 targets when HiTG was segregated in drm12 (left) and cmt23 (right) mutant background. Grey dashed lines indicate the points of VANC targets.


( C ) Patterns of DNA methylation around VANC21 ( left ) , VANC6 ( middle ) , and VANC1 ( right ) targets .

O 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) Patterns of DNA methylation around VANC21 (left), VANC6 (middle), and VANC1 (right) targets.


( C ) Hi excision in F2 plants obtained from met1 x GK345 . Genotyping results of MET1 and Hi loci are also shown . a - h indicate individuals who are analyzed for their DNA methylation status in ( D ) .

O O O O O O O O O O O O O O O O O O O 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) Hi excision in F2 plants obtained from met1 x GK345. Genotyping results of MET1 and Hi loci are also shown. a-h indicate individuals who are analyzed for their DNA methylation status in (D).


( D ) DNA methylation status in endogenous Hi calculated from the bisulfite sequencing results .

(D) DNA methylation status in endogenous Hi calculated from the bisulfite sequencing results.


( A ) Expression of VANB21 ( At2g23490 ) and VANC21 ( At2g23480 ) measured by RT ‒ qPCR . The values were normalized to the expression levels in the ddm1 mutant . For each genotype , the average values and standard deviations of the three technical replicates are shown . Note that we could not detect the expression of VANC21 in drm12 ( - HiTG ) , although endogenous Hi retained mobility ( Fig . 6C ) . This finding can likely be explained by the expression of VANA21 , a transposase of VANDAL21 , but we failed to compare the expression levels of this gene because of its very low expression level .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of VANB21 (At2g23490) and VANC21 (At2g23480) measured by RT‒qPCR. The values were normalized to the expression levels in the ddm1 mutant. For each genotype, the average values and standard deviations of the three technical replicates are shown. Note that we could not detect the expression of VANC21 in drm12 (-HiTG), although endogenous Hi retained mobility (Fig. 6C). This finding can likely be explained by the expression of VANA21, a transposase of VANDAL21, but we failed to compare the expression levels of this gene because of its very low expression level.


( B ) DNA methylation status of coding and noncoding regions of five VANDAL21 TEs The average values and standard deviations for each region are shown . One - way ANOVA with Tukey ' s multiple comparisons test was used to determine the significant differences ( p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DNA methylation status of coding and noncoding regions of five VANDAL21 TEs The average values and standard deviations for each region are shown. One-way ANOVA with Tukey's multiple comparisons test was used to determine the significant differences (p<0.01).


( C ) Excision of endogenous Hi detected by PCR . For RT - qPCR shown in ( A ) and excision analysis shown in ( C ) , RNA and DNA , respectively , were extracted from the same plants .

O O O O O O O O O O O O O O O O O O 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) Excision of endogenous Hi detected by PCR. For RT-qPCR shown in (A) and excision analysis shown in (C), RNA and DNA, respectively, were extracted from the same plants.


( a ) The Hiw - associated complex was purified by TAP ( see Online Methods ) and analyzed by one - dimensional SDS - PAGE gel followed by Sypro Ruby staining . Mass spectrometry identified the full - length Hiw , β - tubulin , Rae1 and Fsn , indicated by arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The Hiw-associated complex was purified by TAP (see Online Methods) and analyzed by one-dimensional SDS-PAGE gel followed by Sypro Ruby staining. Mass spectrometry identified the full-length Hiw, β-tubulin, Rae1 and Fsn, indicated by arrows.


( b ) The Rae1 - associated complex purified from fly brains by TAP was analyzed by one - dimensional SDS - PAGE gel followed by Coomassie blue G - 250 staining . Mass spectrometry identified Hiw , Nup98 and Rae1 , indicated by arrows .

O O O O O O O O O O O O O O O O O O O O O O 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) The Rae1-associated complex purified from fly brains by TAP was analyzed by one-dimensional SDS-PAGE gel followed by Coomassie blue G-250 staining. Mass spectrometry identified Hiw, Nup98 and Rae1, indicated by arrows.


( c ) Larval brain lysate of wild - type ( WT ) , hiw null mutant ( hiwΔN ) and Fsn mutant ( Fsnf06595 / Df ( 2R ) 7872 ) flies was subject to co - immunoprecipitation with antibody to Hiw ( Hiw2b ) . Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1 . The membrane was also blotted with antibody to β - tubulin to ensure equal amount of total proteins in the input samples . Full - length blots are presented in Supplementary Figure 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Larval brain lysate of wild-type (WT), hiw null mutant (hiwΔN) and Fsn mutant (Fsnf06595/Df(2R)7872) flies was subject to co-immunoprecipitation with antibody to Hiw (Hiw2b). Both the input and the immunoprecipitated complexes were analyzed by western blots with antibodies to Hiw or Rae1. The membrane was also blotted with antibody to β-tubulin to ensure equal amount of total proteins in the input samples. Full-length blots are presented in Supplementary Figure 8.


( a ) The gene structure of Rae1 . The black - arrowed boxes indicate exons . A transposable element , P { GawB } NP3499 , located ∼ 400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1 . PCR analysis indicates that ∼ 900 bp and a ∼ 1 . 5 - k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12 , respectively . ( b ) Western blot analysis on Rae1 protein in the brain lysate of wild - type , Df ( 2R ) 5764 / + , Rae1EX28 / + , or late second instar Rae1EX28 , Rae1EX28 / Df ( 2R ) 5764 ( / Df ) and Rae1EXB12 / Df ( 2R ) 5764 larvae . β - tubulin was used as the loading control . Full - length blots are presented in Supplementary Figure 9 . ( c ) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains . Rae1 protein levels were first normalized to β - tubulin protein levels and are presented as percentage of wild - type level . * P 0 . 05 , * * P 0 . 001 . Error bars denote s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The gene structure of Rae1. The black-arrowed boxes indicate exons. A transposable element, P{GawB}NP3499, located ∼400 bp upstream of the first exon of Rae1 was used to generate imprecise excision mutants of Rae1. PCR analysis indicates that ∼900 bp and a ∼1.5-k bp DNA fragment in the Rae1 promoter region are missing in Rae1EX28 and Rae1EXB12, respectively. (b) Western blot analysis on Rae1 protein in the brain lysate of wild-type, Df(2R)5764/+, Rae1EX28/+, or late second instar Rae1EX28, Rae1EX28/Df(2R)5764(/Df) and Rae1EXB12 /Df(2R)5764 larvae. β-tubulin was used as the loading control. Full-length blots are presented in Supplementary Figure 9. (c) Quantification of Rae1 protein levels in the Rae1 heterozygous and homozygous mutant brains. Rae1 protein levels were first normalized to β-tubulin protein levels and are presented as percentage of wild-type level. *P 0.05, **P 0.001. Error bars denote s.e.m.


( a ) Representative confocal images of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) and FasII ( red ) , in late 2nd / early 3rd instar wild - type , Rae1EX28 , Rae1EX28 presynaptic rescue ( Rae1EX28 ; elav - Gal4 / UAS - NTAP - Rae1 ) and Rae1EX28 wallenda suppression ( Rae1EX28 ; wnd3 ) larvae . Scale bar represents 10 μm . ( b , c ) Quantification of bouton number ( b ) and size ( c ) in wild - type , Rae1EX28 / Df , Rae1EX28 , Rae1EX28 rescue , Rae1EX28 ; wnd3 suppression and hiwΔN larval NMJs ( segment A3 muscle 6 / 7 ; n = 20 , 21 , 18 , 20 , 20 and 11 , respectively , for bouton number ; n = 271 , 465 , 548 , 726 , 486 and 826 , respectively , for bouton size ) . There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28 / Df ( P > 0 . 1 for both comparisons ) . * P 0 . 001 . Error bars denote s . e . m .

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

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in late 2nd/early 3rd instar wild-type, Rae1EX28, Rae1EX28 presynaptic rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) and Rae1EX28 wallenda suppression (Rae1EX28; wnd3) larvae. Scale bar represents 10 μm. (b,c) Quantification of bouton number (b) and size (c) in wild-type, Rae1EX28/Df, Rae1EX28, Rae1EX28 rescue, Rae1EX28; wnd3 suppression and hiwΔN larval NMJs (segment A3 muscle 6/7; n = 20, 21, 18, 20, 20 and 11, respectively, for bouton number; n = 271, 465, 548, 726, 486 and 826, respectively, for bouton size). There was no significant difference in either bouton number or bouton size between Rae1EX28 and Rae1EX28/Df (P > 0.1 for both comparisons). *P 0.001. Error bars denote s.e.m.


( a ) Representative confocal images of muscle 4 synapses stained for both DVGLUT ( green ) and FasII ( red ) in wild - type , Rae1EX28 / + , hiwND51 , hiwND51 ; Rae1EX28 / + , hiwΔN and hiwΔN ; Rae1EX28 / + larvae . Scale bar represents 10 μm . ( b ) Quantification of bouton number at muscle 4 NMJs in wild - type , Rae1EX28 / + , hiwND51 , hiwND51 ; Rae1EX28 / + , hiwΔN and hiwΔN ; Rae1EX28 / + larvae ( n = 27 , 23 , 42 , 46 , 23 and 25 cells , respectively ) . There was a significant increase in the number of boutons formed in hiwND51 ; Rae1EX28 / + compared with hiwND51 3rd instar larval NMJs ( * P 0 . 001 ) . The number of boutons in the hiwΔN ; Rae1EX28 / + double mutant was not significantly different from that in hiwΔN ( * * P > 0 . 5 ) , demonstrating a lack of enhancement of the hiwΔN phenotype by Rae1EX28 / + . Error bars denote s . e . m .

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

(a) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae. Scale bar represents 10 μm. (b) Quantification of bouton number at muscle 4 NMJs in wild-type, Rae1EX28/+, hiwND51, hiwND51; Rae1EX28/+, hiwΔN and hiwΔN; Rae1EX28/+ larvae (n = 27, 23, 42, 46, 23 and 25 cells, respectively). There was a significant increase in the number of boutons formed in hiwND51; Rae1EX28/+ compared with hiwND51 3rd instar larval NMJs (*P 0.001). The number of boutons in the hiwΔN; Rae1EX28/+ double mutant was not significantly different from that in hiwΔN (**P > 0.5), demonstrating a lack of enhancement of the hiwΔN phenotype by Rae1EX28/+. Error bars denote s.e.m.


( a ) Schematic presentation of NTAP - tagged ( NT - ) and HM - tagged ( HM - ) hiw transgenes encoding full - length , mutated or truncated Hiw proteins . Hiw functional domains are marked with colored boxes . The positions of amino - acid substitution in NT - HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT - Hiw - RCC1 and NT - Hiw - PHR are indicated . The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants , to cause dominant negative overgrowth phenotype when expressed in wild - type background and to bind to Rae1 . ( b ) Western blot analysis of fly brain lysate using antibody to TAP ( PAP , Sigma ) or Myc revealed the expression of wild - type and mutant hiw transgenes in predicted size .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic presentation of NTAP-tagged (NT-) and HM-tagged (HM-) hiw transgenes encoding full-length, mutated or truncated Hiw proteins. Hiw functional domains are marked with colored boxes. The positions of amino-acid substitution in NT-HiwΔRING and the added amino acid residue necessary to maintain the reading frame in NT-Hiw-RCC1 and NT-Hiw-PHR are indicated. The right column summarizes the ability of the given hiw transgene to rescue the synaptic overgrowth phenotype in hiw mutants, to cause dominant negative overgrowth phenotype when expressed in wild-type background and to bind to Rae1. (b) Western blot analysis of fly brain lysate using antibody to TAP (PAP, Sigma) or Myc revealed the expression of wild-type and mutant hiw transgenes in predicted size.


( c ) Representative confocal images of muscle 4 synapses stained for DVGLUT ( green ) and FasII ( red ) in wild - type ( + / hiwND8 ; + / + ; GS - elav - Gal4 / + ) , NT - Hiw overexpression ( + / hiwND8 ; UAS - NT - hiw / + ; GS - elav - Gal4 / + ) , NT - Hiw - NT overexpression ( + / hiwND8 ; UAS - NT - hiw - NT / + ; GS - elav - Gal4 / + ) , NT - Hiw - CT1000 overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - CT1000 ) , NT - Hiw - PHR overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - PHR ) , HM - Hiw - HindIII overexpression ( + / hiwND8 ; UAS - HW - hiw - HindIII / + ; GS - elav - Gal4 / + ) , NT - Hiw - RCC1 overexpression ( NT - hiw - RCC1 / hiwND8 ; + / + ; GS - elav - Gal4 / + ) , or NT - Hiw - CT overexpression ( + / hiwND8 ; + / + ; GS - elav - Gal4 / UAS - NT - hiw - CT ) 3rd larval NMJs . Overexpression is indicated by an up arrow . Scale bar represents 10 μm . ( d ) Quantification of the number of boutons at muscle 4 NMJs in wild - type larvae or larvae overexpressing wild - type or mutant hiw transgenes ( n = 23 , 24 , 26 , 27 , 26 , 21 , 25 and 27 , respectively ; * P 0 . 001 ) . Error bars denote s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of muscle 4 synapses stained for DVGLUT (green) and FasII (red) in wild-type (+/hiwND8; +/+; GS-elav-Gal4/+), NT-Hiw overexpression (+/hiwND8; UAS-NT-hiw/+; GS-elav-Gal4/+), NT-Hiw-NT overexpression (+/hiwND8; UAS-NT-hiw-NT/+; GS-elav-Gal4/+), NT-Hiw-CT1000 overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT1000), NT-Hiw-PHR overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-PHR), HM-Hiw-HindIII overexpression (+/hiwND8; UAS-HW-hiw-HindIII/+; GS-elav-Gal4/+), NT-Hiw-RCC1 overexpression (NT-hiw-RCC1/hiwND8; +/+; GS-elav-Gal4/+), or NT-Hiw-CT overexpression (+/hiwND8; +/+; GS-elav-Gal4/UAS-NT-hiw-CT) 3rd larval NMJs. Overexpression is indicated by an up arrow. Scale bar represents 10 μm. (d) Quantification of the number of boutons at muscle 4 NMJs in wild-type larvae or larvae overexpressing wild-type or mutant hiw transgenes (n = 23, 24, 26, 27, 26, 21, 25 and 27, respectively; *P 0.001). Error bars denote s.e.m.


( a ) Indicated NTAP - tagged hiw transgenes ( described in Fig . 5 ) and a TAP only transgene were expressed in neurons under the control of the BG380 - Gal4 driver . Larval brain lysates from each sample were subject to IgG pulldown . Both the inputs and the IgG pulldown complexes were analyzed by western blot using antibody to Rae1 . Rae1 was present in all of the pulldown complexes except for those from TAP only , NT - Hiw - CT1000 and NT - Hiw - NT . Full - length blots are presented in Supplementary Figure 10 . ( b ) Quantification of the interaction between various Hiw transgenic proteins and Rae1 . Rae1 intensities in TAP pulldown blots were normalized to both the intensities and molecular weights of the corresponding TAP pulldown proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Indicated NTAP-tagged hiw transgenes (described in Fig. 5) and a TAP only transgene were expressed in neurons under the control of the BG380-Gal4 driver. Larval brain lysates from each sample were subject to IgG pulldown. Both the inputs and the IgG pulldown complexes were analyzed by western blot using antibody to Rae1. Rae1 was present in all of the pulldown complexes except for those from TAP only, NT-Hiw-CT1000 and NT-Hiw-NT. Full-length blots are presented in Supplementary Figure 10. (b) Quantification of the interaction between various Hiw transgenic proteins and Rae1. Rae1 intensities in TAP pulldown blots were normalized to both the intensities and molecular weights of the corresponding TAP pulldown proteins.


( c ) Representative confocal images of muscle 4 synapses stained for both DVGLUT ( green ) and FasII ( red ) in wild - type ( BG380 / Y ; + / + ; + / + ) , NT - Hiw - CT and GAP - GFP ( BG380 / Y ; UAS - GAP - GFP / + ; UAS - NT - hiw - CT / + ) , and NT - Hiw - CT and Rae1 ( BG380 / Y ; + / + ; UAS - NT - hiw - CT / UAS - GFP - Rae1 ) larvae . Scale bar represents 10 μm . ( d ) Quantification of number of boutons at muscle 4 NMJs in wild - type , NT - Hiw - CT overexpression ( BG380 / Y ; + / + ; UAS - NT - hiw - CT / + ) , NT - Hiw - CT and GAP - GFP , and NT - Hiw - CT and Rae1 ( n = 24 , 27 , 32 and 26 , respectively ) larvae . Error bars denote s . e . m . ; * P 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative confocal images of muscle 4 synapses stained for both DVGLUT (green) and FasII (red) in wild-type (BG380/Y;+/+; +/+), NT-Hiw-CT and GAP-GFP (BG380/Y; UAS-GAP-GFP/+; UAS-NT-hiw-CT/+), and NT-Hiw-CT and Rae1 (BG380/Y; +/+; UAS-NT-hiw-CT/UAS-GFP-Rae1) larvae. Scale bar represents 10 μm. (d) Quantification of number of boutons at muscle 4 NMJs in wild-type, NT-Hiw-CT overexpression (BG380/Y; +/+; UAS-NT-hiw-CT/+), NT-Hiw-CT and GAP-GFP, and NT-Hiw-CT and Rae1 (n = 24, 27, 32 and 26, respectively) larvae. Error bars denote s.e.m.; *P 0.001.


( a ) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene . Quantification of the number of boutons in wild - type , Rae1EX28 , Rae1EX28 ; Hiw rescue ( Res ) ( Rae1EX28 ; elav - Gal4 / UAS - GFP - hiw ) , hiwΔN and hiwΔN ; Rae1 rescue ( hiwΔN ; elav - Gal4 / UAS - NTAP - Rae1 ) larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 7 and 11 , respectively ) . ( b ) Representative confocal images of wild - type ( BG380 - Gal4 / + ; + / + ; UAS - GFP - hiw / + ) or Rae1 mutant ( BG380 - Gal4 / + ; Rae1EX28 ; UAS - GFP - hiw / + ) ventral ganglions with UAS - GFP - hiw trangene expressed in neurons . The larvae were stained with antibodies to horseradish peroxidase ( HRP , red ) and GFP ( green ) . Scale bar represents 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Neuronal expression of neither Hiw nor Rae1 rescues the synaptic terminal overgrowth phenotype caused by the loss of function of the other gene. Quantification of the number of boutons in wild-type, Rae1EX28, Rae1EX28; Hiw rescue (Res) (Rae1EX28; elav-Gal4/UAS-GFP-hiw), hiwΔN and hiwΔN; Rae1 rescue (hiwΔN; elav-Gal4/UAS-NTAP-Rae1) larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 7 and 11, respectively). (b) Representative confocal images of wild-type (BG380-Gal4/+; +/+; UAS-GFP-hiw/+) or Rae1 mutant (BG380-Gal4/+; Rae1EX28; UAS-GFP-hiw/+) ventral ganglions with UAS-GFP-hiw trangene expressed in neurons. The larvae were stained with antibodies to horseradish peroxidase (HRP, red) and GFP (green). Scale bar represents 10 μm.


( c ) Western blots of total proteins extracted from wild - type , hiwΔN , Rae1EX28 and Rae1EX28 rescue ( Rae1EX28 ; elav - Gal4 / UAS - NTAP - Rae1 ) larval brains probed with indicated antibodies . Full - length blots are presented in Supplementary Figure 11 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blots of total proteins extracted from wild-type, hiwΔN, Rae1EX28 and Rae1EX28 rescue (Rae1EX28; elav-Gal4/UAS-NTAP-Rae1) larval brains probed with indicated antibodies. Full-length blots are presented in Supplementary Figure 11.


( d ) Western blot analysis on total proteins extracted from hiwΔN , wild - type ( C155 - Gal4 / + ) and Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) larval brains . Full - length blots are presented in Supplementary Figure 12 . ( e ) Quantification of Hiw protein levels in the wild - type and the Rae1 expression larval brains ( * P 0 . 001 , based on seven independent experiments ) . Arrows indicate a 38 - kDa endogenous Rae1 protein and the arrowheads indicate the 58 - kDa transgenic NTAP - Rae1 protein . Error bars denote s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.


( a ) Representative confocal images of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) and FasII ( red ) , in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + and Rae1EX28 ; atg18 / + wandering larvae . Scale bar represents 10 μm . ( b ) Quantification of the number of boutons in wild - type , atg1 / + , atg2 / + , atg18 / + , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + and Rae1EX28 ; atg18 / + larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 12 , 12 , 16 , 12 and 21 , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ wandering larvae. Scale bar represents 10 μm. (b) Quantification of the number of boutons in wild-type, atg1/+, atg2/+, atg18/+, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 12, 12, 16, 12 and 21, respectively).


( c ) Western blot analysis on total proteins extracted from wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + and Rae1EX28 ; atg18 / + larval brains . The neural form of β - catenin ( 82 kDa , see Online Methods ) was used as an internal control of neuronal proteins . Full - length blots are presented in Supplementary Figure 13 . ( d ) Quantification of Hiw protein levels in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + and Rae1EX28 ; atg18 / + larval brains ( based on six 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 O O O O O O O O O O O O O O O O O O O O O O

(c) Western blot analysis on total proteins extracted from wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains. The neural form of β-catenin (82 kDa, see Online Methods) was used as an internal control of neuronal proteins. Full-length blots are presented in Supplementary Figure 13. (d) Quantification of Hiw protein levels in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains (based on six independent experiments).


( e ) Representative confocal images of muscle 4synapses stained for both DVGLUT ( green ) and FasII ( red ) in wandering larvae of wild - type ( C155 - Gal4 / + ) , Atg1Gap - GFP ( C155 - Gal4 / + ; UAS - Gap - GFP / + ; UAS - Atg16B / + ) , and Atg1NTAP - Rae1 ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ; UAS - Atg1 / + ) . Scale bar represents 10 μm . ( f ) Quantification of the number of boutons at muscle 4synapses in wild - type , Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) , Atg1Gap - GFP and Atg1NTAP - Rae1 wandering larvae ( segment A2 - 4 , n = 28 , 27 , 33 and 34 , respectively ) . Error bars denote s . e . m . * P 0 . 001 , * * P 0 . 05 .

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

(e) Representative confocal images of muscle 4synapses stained for both DVGLUT (green) and FasII (red) in wandering larvae of wild-type (C155-Gal4/+), Atg1Gap-GFP (C155-Gal4/+; UAS-Gap-GFP/+; UAS-Atg16B/+), and Atg1NTAP-Rae1 (C155-Gal4/+; UAS-NTAP-Rae1/+; UAS-Atg1/+). Scale bar represents 10 μm. (f) Quantification of the number of boutons at muscle 4synapses in wild-type, Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+), Atg1Gap-GFP and Atg1NTAP-Rae1 wandering larvae (segment A2-4, n = 28, 27, 33 and 34, respectively). Error bars denote s.e.m. *P 0.001, **P 0.05.


PCP - MS strategy to identify proteins interacting with specific proteasome subtypes . U937 cells were cross - linked with formaldehyde and lysed , and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device . Protein complexes were then separated on a 15 - 40 % glycerol gradient . Each fraction of the gradient was analyzed by nano - LC - MS / MS . Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein , after internal standard calibration using a mix of 8 isotopically labeled peptides . The PCP analysis was performed as described in the Materials and Methods section .

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

PCP-MS strategy to identify proteins interacting with specific proteasome subtypes. U937 cells were cross-linked with formaldehyde and lysed, and proteins were concentrated and ultrafiltrated on a 100 kDa cutoff device. Protein complexes were then separated on a 15-40% glycerol gradient. Each fraction of the gradient was analyzed by nano-LC-MS/MS. Protein quantification was performed using the mean XIC of the three most intense validated peptides for each protein, after internal standard calibration using a mix of 8 isotopically labeled peptides. The PCP analysis was performed as described in the Materials and Methods section.


PCP analysis of the 19S regulatory complex . Protein abundance profiles of 16 proteins of the 19S RP ( Rpt1 - 6 , Rpn1 - 3 , Rpn5 , 7 - 9 , 11 - 13 , gray lanes ) and of their median abundance ( black lane ) ( left panel ) . PCP analysis is performed by plotting the χ2 values ( representing the Euclidian distance between the abundance profile of each protein and the reference profile ) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 ( middle left panel ) . The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values . Different zooms of the graph are represented ( middle right and right panels ) . Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits ( right panel ) .

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

PCP analysis of the 19S regulatory complex. Protein abundance profiles of 16 proteins of the 19S RP (Rpt1-6, Rpn1-3, Rpn5, 7-9, 11-13, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values (representing the Euclidian distance between the abundance profile of each protein and the reference profile) of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 19S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and blue dots represent 19S subunits (right panel).


PCP analysis of proteasome 20S complex . Protein abundance profiles of 17 proteins of the 20S CP ( α1 - α7 , β1 - β7 , β1i , β2i , β5i , gray lanes ) and of their median abundance ( black lane ) ( left panel ) . PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 ( middle left panel ) . The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values . Different zooms of the graph are represented ( middle right and right panels ) . Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits .

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

PCP analysis of proteasome 20S complex. Protein abundance profiles of 17 proteins of the 20S CP (α1-α7, β1-β7, β1i, β2i, β5i, gray lanes) and of their median abundance (black lane) (left panel). PCP analysis is performed by plotting the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 (middle left panel). The median profile of the 20S complex subunits was used as the reference profile for the calculation of the χ2 values. Different zooms of the graph are represented (middle right and right panels). Light gray dots represent the proteins quantified in all the fractions of the density gradient and red dots represent 20S subunits.


Protein correlation profiling ( PCP ) analysis using the median profile of the PA28αβ regulator as the reference profileProfiles of the PA28α , PA28β , and the β2i proteins ( blue , red and green lines , respectively ) .

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Protein correlation profiling (PCP) analysis using the median profile of the PA28αβ regulator as the reference profileProfiles of the PA28α, PA28β, and the β2i proteins (blue, red and green lines, respectively).


Plot of the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1 . A zoom of the graph in ( B ) is represented and χ2 coordinates for PA28α , PA28β , and β2i proteins are highlighted as blue , red , and green dots , respectively . Light gray dots represent the χ2 coordinates of the proteins quantified in all the fractions of the gradient . The median profile of the PA28α and PA28β subunits was used as the reference profile for the calculation of the χ2 values .

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

Plot of the χ2 values of the experimental replicate 2 as a function of the χ2 values of the experimental replicate 1.A zoom of the graph in (B) is represented and χ2 coordinates for PA28α, PA28β, and β2i proteins are highlighted as blue, red, and green dots, respectively. Light gray dots represent the χ2 coordinates of the proteins quantified in all the fractions of the gradient. The median profile of the PA28α and PA28β subunits was used as the reference profile for the calculation of the χ2 values.


A Proteasome complexes were immunopurified from nine formaldehyde - crosslinked human cell lines and analyzed by nano - LC - MS / MS . Protein abundance indexes ( PAIs ) were used to represent the abundance of proteins in purified proteasome samples . The correlation between two different proteins was quantified using coefficients of determination ( R2 ) .

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

A Proteasome complexes were immunopurified from nine formaldehyde-crosslinked human cell lines and analyzed by nano-LC-MS/MS. Protein abundance indexes (PAIs) were used to represent the abundance of proteins in purified proteasome samples. The correlation between two different proteins was quantified using coefficients of determination (R2).


B - E Correlations of abundances of α7 and α6 ( B ) , Rpn3 and Rpn1 ( C ) , PA28β and PA28α ( D ) , and Rpn3 and PA28β ( 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

B-E Correlations of abundances of α7 and α6 (B), Rpn3 and Rpn1 (C), PA28β and PA28α (D), and Rpn3 and PA28β (E).


Heat - map representing the correlations ( expressed as the R2 ) between the abundances of 73 known proteasome - interacting proteins ( PIPs ) and the abundances of 8 reference proteins or protein complexes , PA28γ , β2i ( representing the iP20S ) , PA28αβ , ncP20S ( median of α1 - α7 , β3 , β4 , β6 , and β7 profiles ) , 19S ( median of Rpt1 - 6 , Rpn1 - 3 , 5 - 14 profiles ) , PI31 , β5 ( representing the sP20S ) , and PA200 . For protein complexes , the median PAI of their subunits in each of the 24 AP - MS experiments was used : α1 - α7 , β3 , β4 , β6 , and β7 subunits for the ncP20S , Rpt1 - 6 , Rpn1 - 3 , 5 - 14 for the 19S RP , and PA28α and PA28β subunits for the PA28αβ RP . The R2 values were hierarchically clustered . Three distinct clusters of composition detailed hereafter could be obtained . Cluster 1 ( from top to bottom ) : Rpt3 , Rpn13 , α2 , Rpn7 , USP14 , hHR23B , α1 , β6 , β3 , α4 , α7 , Rpn6 , Rpn3 , Rpt4 , Rpn10 , Rpn5 , Rpt5 , Rpn1 , Rpn11 , Rpt1 , Rpn9 , Rpn2 , Rpn8 , α3 , α6 , β4 , Rpt6 , PDC6 , Rpn12 , APEH , Ubiquilin - 1 , α5 , β7 , CCT7 , CCT4 , CCT2 , CCT3 , CCT5 , CCT6A , DNAJA1 , HSP90AB1 , HSP90AA1 , PNP , Rpt2 . Cluster 2 ( from top to bottom ) : 14 - 3 - 3ζ / δ , CAND1 , GSR , UBE3C , β2 , ATP5A1 , ATP5B , β1 , PA200 , β5 , FBXO7 , UCHL5 , TXNL1 , ECM29 , PI31 . Cluster 3 ( from top to bottom ) : PA28β , PITH1 , β2i , PA28α , PA28γ , β5i , β1i .

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

Heat-map representing the correlations (expressed as the R2) between the abundances of 73 known proteasome-interacting proteins (PIPs) and the abundances of 8 reference proteins or protein complexes, PA28γ, β2i (representing the iP20S), PA28αβ, ncP20S (median of α1-α7, β3, β4, β6, and β7 profiles), 19S (median of Rpt1-6, Rpn1-3, 5-14 profiles), PI31, β5 (representing the sP20S), and PA200. For protein complexes, the median PAI of their subunits in each of the 24 AP-MS experiments was used: α1-α7, β3, β4, β6, and β7 subunits for the ncP20S, Rpt1-6, Rpn1-3, 5-14 for the 19S RP, and PA28α and PA28β subunits for the PA28αβ RP. The R2 values were hierarchically clustered. Three distinct clusters of composition detailed hereafter could be obtained. Cluster 1 (from top to bottom): Rpt3, Rpn13, α2, Rpn7, USP14, hHR23B, α1, β6, β3, α4, α7, Rpn6, Rpn3, Rpt4, Rpn10, Rpn5, Rpt5, Rpn1, Rpn11, Rpt1, Rpn9, Rpn2, Rpn8, α3, α6, β4, Rpt6, PDC6, Rpn12, APEH, Ubiquilin-1, α5, β7, CCT7, CCT4, CCT2, CCT3, CCT5, CCT6A, DNAJA1, HSP90AB1, HSP90AA1, PNP, Rpt2. Cluster 2 (from top to bottom): 14-3-3ζ/δ, CAND1, GSR, UBE3C, β2, ATP5A1, ATP5B, β1, PA200, β5, FBXO7, UCHL5, TXNL1, ECM29, PI31. Cluster 3 (from top to bottom): PA28β, PITH1, β2i, PA28α, PA28γ, β5i, β1i.


The two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits . Western blots against the immuno - ( β1i , β2i , β5i ) and standard ( β1 , β2 , β5 ) catalytic subunits of the 20S proteasome . Calnexin is used as a loading control . Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non - adjacent in the gel . Importantly , the bands were assembled from the same blot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 two HEK EBNA cell lines express only standard proteasome or immunoproteasome subunits. Western blots against the immuno- (β1i, β2i, β5i) and standard (β1, β2, β5) catalytic subunits of the 20S proteasome. Calnexin is used as a loading control. Black lines delineate the boundary between vertically sliced images that juxtapose lanes that were non-adjacent in the gel. Importantly, the bands were assembled from the same blot.


Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome . The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions ( n = 4 ) .

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Relative normalized abundance indexes of proteasome regulators in HEK EBNA cells containing only immunoproteasome compared to HEK EBNA cells containing only standard proteasome. The normalized abundance indexes for each regulator were set to 1 for standard proteasome conditions (n = 4).


Kinetics of IFN - γ treatment on HeLa cells . HeLa cells were stimulated for 0 , 24 , 48 , or 72 h with IFN - γ . Western blots were performed on total cell lysates with antibodies against the β2i , α2 , and α5 subunits . IRF - 1 was used to control IFN - γ treatment efficiency , and GAPDH was used as a loading control .

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Kinetics of IFN-γ treatment on HeLa cells. HeLa cells were stimulated for 0, 24, 48, or 72 h with IFN-γ. Western blots were performed on total cell lysates with antibodies against the β2i, α2, and α5 subunits. IRF-1 was used to control IFN-γ treatment efficiency, and GAPDH was used as a loading control.


For each time point of the IFN - γ treatment , proteasome complexes were purified and analyzed by LC - MS / MS . Proteasome complexes dynamics was measured by label - free quantitative proteomics . The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI ( n = 3 ) .

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

For each time point of the IFN-γ treatment, proteasome complexes were purified and analyzed by LC-MS/MS. Proteasome complexes dynamics was measured by label-free quantitative proteomics. The normalized abundance index of each protein or protein complex obtained at each time point was compared to the one obtained at the 0 h time point to obtain a regulator relative normalized PAI (n = 3).


A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK - 2837808A ( 10 μM ) for the indicated duration under hypoxic ( 1 % O2 ) and normoxic conditions . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . In ( A ) statistical comparison for the treatments that showed a significant change .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Proliferation of melanoma cell lines treated with LDHA inhibitor GSK-2837808A (10 μM) for the indicated duration under hypoxic (1% O2) and normoxic conditions. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (A) statistical comparison for the treatments that showed a significant change.


B ( left ) Immunoblotting of LDHA in melanoma cell lines transfected with non - targeting siRNA ( NT - siRNA ) or LDHA - specific siRNA for 72 hr . ( right ) Proliferation of melanoma cell lines transfected with NT - siRNA or LDHA - specific siRNA for the indicated timepoints . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . In ( B ) , statistical comparison for the treatments that showed a significant change .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (left) Immunoblotting of LDHA in melanoma cell lines transfected with non-targeting siRNA (NT-siRNA) or LDHA-specific siRNA for 72 hr. (right) Proliferation of melanoma cell lines transfected with NT-siRNA or LDHA-specific siRNA for the indicated timepoints. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (B), statistical comparison for the treatments that showed a significant change.


Immunoblotting of LDHA of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs .

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Immunoblotting of LDHA of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs.


proliferation under normoxic ( D ) conditions of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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proliferation under normoxic (D) conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


proliferation under hypoxic conditions of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . statistical significance is denoted by blue ( for si / sh - LDHA # 1 , hypoxia ) and black ( for sish - LDHA # 2 , hypoxia ) stars respectively .

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proliferation under hypoxic conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. statistical significance is denoted by blue (for si/sh-LDHA#1, hypoxia) and black (for sish-LDHA#2, hypoxia) stars respectively.


G Anchorage - independent growth of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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G Anchorage-independent growth of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


B , C Glucose ( B ) and glutamine ( Gln , C ) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

O O O B-MEASURED_VAR O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Glucose (B) and glutamine (Gln, C) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


D Medium Gln levels after transfection of melanoma cells with non - targeting ( NT ) or LDHA - specific siRNA for 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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D Medium Gln levels after transfection of melanoma cells with non-targeting (NT) or LDHA-specific siRNA for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


E GC - MS - based quantification of intracellular Gln levels in cells Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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E GC-MS-based quantification of intracellular Gln levels in cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


F qRT - PCR analysis of expression of the indicated SLC family transporters in MeWo ( left ) and A375 ( right ) melanoma cells after treatment with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

F qRT-PCR analysis of expression of the indicated SLC family transporters in MeWo (left) and A375 (right) melanoma cells after treatment with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


G Immunoblotting of SLC1A5 in melanoma cells treated with LDHAi for 96 hr .

G Immunoblotting of SLC1A5 in melanoma cells treated with LDHAi for 96 hr.


H Immunoblotting of LDHA and SLC1A5 in melanoma cell lines transfected with NT - siRNA or indicated si - LDHA for 96 hr .

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H Immunoblotting of LDHA and SLC1A5 in melanoma cell lines transfected with NT-siRNA or indicated si-LDHA for 96 hr.


I Immunoblotting of SLC1A5 and LDHA in MeWo cells stably transduced with empty vector ( EV ) or four different LDHA - specific shRNAs .

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I Immunoblotting of SLC1A5 and LDHA in MeWo cells stably transduced with empty vector (EV) or four different LDHA-specific shRNAs.


J Immunoblotting of SLC1A5 in MeWo cells mock ( NT - siRNA + DMSO ) or si - SLC1A5 - treated for 24 hr and then treated with LDHAi for 48 hr .

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J Immunoblotting of SLC1A5 in MeWo cells mock (NT-siRNA + DMSO) or si-SLC1A5-treated for 24 hr and then treated with LDHAi for 48 hr.


K Medium Gln levels ( left ) , intracellular Gln levels ( right ) of MeWo cells Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Medium Gln levels (left), intracellular Gln levels (right) of MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


L Proliferation of melanoma cell lines for the indicated timepoints following treatment with LDHA - i , si - SLC1A5 or both . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . two - way Anova was used for the proliferation . N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

L Proliferation of melanoma cell lines for the indicated timepoints following treatment with LDHA-i, si-SLC1A5 or both. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. two-way Anova was used for the proliferation. N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


M ( left ) Long - term viability of melanoma cells treated as indicated for 8 days . ( right ) corresponding quantification showing change in viability . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

M (left) Long-term viability of melanoma cells treated as indicated for 8 days. (right) corresponding quantification showing change in viability. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


N Immunoblotting of SLC1A5 and cleaved caspase - 3 in melanoma cells subjected to mock ( NT - siRNA + DMSO ) or si - SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

N Immunoblotting of SLC1A5 and cleaved caspase-3 in melanoma cells subjected to mock (NT-siRNA + DMSO) or si-SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


GC - MS - based quantification of intracellular essential amino acid levels in MeWo cells treated for 72 hr with LDHAi Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells treated for 72 hr with LDHAi Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


B GC - MS - based quantification of intracellular essential amino acid levels in MeWo cells mock or si - SLC1A5 - transfected for 24 hr and then treated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells mock or si-SLC1A5-transfected for 24 hr and then treated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


C ( left ) Immunoblotting of SLC7A5 in MeWo cells transfected with non - targeting ( NT ) or SLC7A5 - specific siRNAs for 48 hr . ( right ) GC - MS - based quantification of intracellular essential amino acid levels in MeWo cells mock or si - SLC7A5 - transfected for 24 hr and then treated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (left) Immunoblotting of SLC7A5 in MeWo cells transfected with non-targeting (NT) or SLC7A5-specific siRNAs for 48 hr. (right) GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells mock or si-SLC7A5-transfected for 24 hr and then treated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


Immunoblotting of total and phosphorylated S6 , ULK1 , and eIF4EBP1 in melanoma cells ( D ) treated with LDHAi for 96 hr Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (D) treated with LDHAi for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


Immunoblotting of total and phosphorylated S6 , ULK1 , and eIF4EBP1 in melanoma cells ( E ) transfected with non - targeting ( NT ) or LDHA - specific siRNAs for 96 hr Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (E) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


Immunoblotting of total and phosphorylated S6 , ULK1 , and eIF4EBP1 in melanoma cells ( F ) si - SLC1A5 - transfected for 24 hr followed by treatment with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

Immunoblotting of total and phosphorylated S6, ULK1, and eIF4EBP1 in melanoma cells (F) si-SLC1A5-transfected for 24 hr followed by treatment with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


H Proliferation of melanoma cells treated with LDHAi , m - TORC1 inhibitor ( Rapamycin , 100 nM ) , or a combination of the two . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , and two - way Anova was used for the proliferation . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

H Proliferation of melanoma cells treated with LDHAi, m-TORC1 inhibitor (Rapamycin, 100 nM), or a combination of the two. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


I ( top ) Long - term viability of melanoma cells treated as indicated for 10 days . ( bottom ) corresponding quantification showing change in viability . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (top) Long-term viability of melanoma cells treated as indicated for 10 days. (bottom) corresponding quantification showing change in viability. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


J Immunoblotting of cleaved caspase - 3 and p - ( S780 ) - Rb in melanoma cells treated as indicated for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

J Immunoblotting of cleaved caspase-3 and p-(S780)-Rb in melanoma cells treated as indicated for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


Immunoblotting of ATF4 in melanoma cell lines ( A ) treated with the LDHA inhibitor LDHAi for 96 hr Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups .

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

Immunoblotting of ATF4 in melanoma cell lines (A) treated with the LDHA inhibitor LDHAi for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.


Immunoblotting of ATF4 in melanoma cell lines ( B ) transfected with non - targeting ( NT ) or LDHA - specific siRNAs for 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups .

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

Immunoblotting of ATF4 in melanoma cell lines (B) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.


C Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector ( EV ) or indicated sh - LDHAs . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups .

O O O O O O O O O O O O O O O O O O O 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 Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector (EV) or indicated sh-LDHAs. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.


D Immunoblotting of ATF4 , c - MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2 , 24 , or 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups .

O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblotting of ATF4, c-MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2, 24, or 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.


E Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock - or si - ATF4 - treated for 24 hr and then incubated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock- or si-ATF4-treated for 24 hr and then incubated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.


F qRT - PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si - ATF4 for 24 hr and then incubated with LDHAi for 24 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si-ATF4 for 24 hr and then incubated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - based quantification of intracellular essential amino acid levels in MeWo cells treated and share Mock and LDHA - i controls . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

GC-MS-based quantification of intracellular essential amino acid levels in MeWo cells treated and share Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


H Proliferation of melanoma cell lines mock - or si - ATF4 - transfected and incubated with LDHAi as indicated for up to 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , and two - way Anova was used for the proliferation . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Proliferation of melanoma cell lines mock- or si-ATF4-transfected and incubated with LDHAi as indicated for up to 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


qRT - PCR analysis of asparagine synthetase ( ASNS ) and glutamine - pyruvate transaminase 2 ( GPT2 ) mRNA levels in the indicated cell lines after 24 hr treatment with LDHAi . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

qRT-PCR analysis of asparagine synthetase (ASNS) and glutamine-pyruvate transaminase 2 (GPT2) mRNA levels in the indicated cell lines after 24 hr treatment with LDHAi. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - based quantification of intracellular asparagine ( Asn ) and alanine ( Ala ) levels in MeWo and 1205Lu cells treated with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

GC-MS-based quantification of intracellular asparagine (Asn) and alanine (Ala) levels in MeWo and 1205Lu cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - based quantification of intracellular Asn and Ala levels in MeWo cells mock - transfected or transfected with si - ATF4 ( K ) for 24 hr and then treated with LDHAi for 48 hr . and shared Mock and LDHA - i controls . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or transfected with si-ATF4 (K) for 24 hr and then treated with LDHAi for 48 hr. and shared Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - based quantification of intracellular Asn and Ala levels in MeWo cells mock - transfected or si - SLC1A5 ( L ) for 24 hr and then treated with LDHAi for 48 hr . and shared Mock and LDHA - i controls . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

GC-MS-based quantification of intracellular Asn and Ala levels in MeWo cells mock-transfected or si-SLC1A5 (L) for 24 hr and then treated with LDHAi for 48 hr. and shared Mock and LDHA-i controls. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


M ( left ) Immunoblotting of ASNS in MeWo cells mock - or si - ASNS - transfected for 24 hr and then incubated with LDHAi for 48 hr . ( right ) Proliferation of MeWo cells mock - or si - ASNS - transfected for 24 hr and then incubated with LDHAi for the indicated times . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , and two - way Anova was used for the proliferation . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

M (left) Immunoblotting of ASNS in MeWo cells mock- or si-ASNS-transfected for 24 hr and then incubated with LDHAi for 48 hr. (right) Proliferation of MeWo cells mock- or si-ASNS-transfected for 24 hr and then incubated with LDHAi for the indicated times. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, and two-way Anova was used for the proliferation. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


N Immunoblotting of GCN2 , ATF4 and SLC1A5 in MeWo cells mock - or si - GCN2 - treated for 24 hr and then incubated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups .

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

N Immunoblotting of GCN2, ATF4 and SLC1A5 in MeWo cells mock- or si-GCN2-treated for 24 hr and then incubated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.