degradation in indicated yeast strains monitored by - chase experiments for Hrd1 model substrates PrA * - 3xHA Values for each time point are reported as means ± standard deviation ( n = 4 for PrA * - 3xHA For D , data for control strains ( Doa10 ( 94H ) wt Δdoa10 ) are same as in Fig 3D 4A .

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Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the Hrd1 model substrates PrA*-3xHA Values for each time point are reported as means ± standard deviation (n = 4 for PrA*-3xHA For D, data for control strains (Doa10(94H) wt and Δdoa10) are the same as in Fig 3D and 4A.


degradation in indicated yeast strains monitored by - chase experiments for lysine - less CPY * - K0 - HA Values for each time point are reported as means ± standard deviation

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Protein degradation in indicated yeast strains monitored by pulse-chase experiments for the lysine-less CPY*-K0-HA Values for each time point are reported as means ± standard deviation


degradation in indicated yeast strains monitored by - chase experiments for lysine - less CPY * - K0 - HA Values for each time point are reported as means ± standard

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degradation in indicated yeast strains monitored by - chase experiments for FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG - Sbh2

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Protein degradation in indicated yeast strains monitored by pulse-chase experiments for FLAG-Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG-Sbh2


degradation in indicated yeast strains monitored by - chase experiments for FLAG - Sbh2 Values for each time point are reported as means ± standard deviation n = 3 for FLAG -

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C . Confocal of U2OS transiently expressing lifeAct - GFP wild type mutants of lifeAct residues interacting with F - actin , were additionally stained with SiR - actin to confirm actin filaments . scan as indicated with yellow on shows extent of lifeAct ( green ) SiR - actin ( magenta ) co - of actin structures . Scale bar = 5µm .

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C. Confocal images of U2OS cells transiently expressing lifeAct-GFP wild type and mutants of lifeAct residues interacting with F-actin, cells were additionally stained with SiR-actin to confirm the actin filaments. The line scan as indicated with yellow line on the cells shows the extent of lifeAct (green) and SiR-actin (magenta) co-staining of actin structures. Scale bar = 5µm.


D . Binding affinities calculated from titration data of co - sedimentation assays of lifeAct - 14 ( 1 . 2µM ) lifeAct - 17 ( 2 . 2µM ) as indicated . Data points for each were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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D. Binding affinities calculated from titration data of co-sedimentation assays of lifeAct-14 (1.2µM) and lifeAct-17 (2.2µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


C . Representative TIRF of lifeAct binding experiments as indicated . remaining lifeAct are shown in Supplement Figure 4 . Scale bar = 5µm .

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C. Representative TIRF images of lifeAct binding experiments as indicated. The remaining lifeAct concentration images are shown in Supplement Figure 4. Scale bar = 5µm.


D . Mean of lifeAct bound to phalloidin jasplakinolide F - actin ( mean s . e . m ; n = 2 or 3 independent experiments with > 50 actin filament for each set ) .

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D. Mean ratio of lifeAct fluorescent intensity bound to phalloidin and jasplakinolide F-actin (mean and s.e.m; n = 2 or 3 independent experiments with >50 actin filament for each set).


A . Representative Coomassie stained SDS - PAGE gels of UTRN - ABD UTRN - V87D co - sedimentation with F - actin . Pellet ( top ) supernatant ( bottom ) fractions of co - sedimentation reactions of increasing utrophin concentrations , uncropped gel of co - sedimentation reactions are presented in Supplement Figure 6 .

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A. Representative Coomassie stained SDS-PAGE gels of UTRN-ABD and UTRN-V87D co-sedimentation with F-actin. Pellet (top) and supernatant (bottom) fractions of individual co-sedimentation reactions of increasing utrophin concentrations, uncropped gel images of all the co-sedimentation reactions are presented in Supplement Figure 6.


B . Apparent Kd indicated was calculated from titration data of co - sedimentation assays of utrophin wildtype ( 1 . 8µM ) mutants ; H29A ( 2 . 8µM ) , I22D ( 38µM ) , V87D ( > 100µM ) N109A ( 55µM ) as indicated . Data points for each were averaged from 3 independent experiments , error bars represent S . D between independent experiments .

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B. Apparent Kd indicated was calculated from the titration data of co-sedimentation assays of utrophin wildtype (1.8µM) and mutants; H29A (2.8µM), I22D (38µM), V87D (>100µM) and N109A (55µM) as indicated. Data points for each concentration were averaged from 3 independent experiments, error bars represent S.D between independent experiments.


C . Confocal of U20S transfected with GFP tagged UTRN - mini mCherry UTRN - ABD , stained with SIR - Actin shows F - actin structures , mainly stress . Scale bar = 5µm .

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C. Confocal images of U20S cells transfected with GFP tagged UTRN-mini and mCherry UTRN-ABD, stained with SIR-Actin shows F-actin structures, mainly stress fibers. Scale bar = 5µm.


Electron microscopy revealed a number of demyelinated or degenerated axons in PLP - 150Q mice at of 5 months . LAQ ( 5 mg / kg ) treatment at 3 months of for 2 months improved myelination in PLP - 150Q mice . Scale bars : 2μm ( Low magnification ) 0 . 5 μm ( High magnification ) .

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Electron microscopy revealed a number of demyelinated or degenerated axons in PLP-150Q mice at the age of 5 months. LAQ (5 mg/kg) treatment at 3 months of age for 2 months improved myelination in PLP-150Q mice. Scale bars: 2μm (Low magnification) and 0.5 μm (High magnification).


G - ratios , which were calculated plotted against axon diameter with linear regression , were shown beneath were significantly decreased in PLP - 150Q mice after LAQ treatment ( G = 0 . 6418 ± 0 . 0191 ) , compared with - matched vehicle group ( G = 0 . 7727 ± 0 . 0203 ) . One - way ANOVA with Tukey ' s . * P = 0 . 0166 . At least 182 axons per genotype were examined from 3 mice in each group .

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G-ratios, which were calculated and plotted against axon diameter with linear regression, were shown beneath the micrographs and were significantly decreased in PLP-150Q mice after LAQ treatment (G=0.6418±0.0191), compared with age-matched vehicle group (G=0.7727±0.0203). One-way ANOVA with Tukey's test. *P=0.0166. At least 182 axons per genotype were examined from 3 mice in each group.


Western blotting showing that LAQ ( 5 or 25 mg / kg ) treatment for 2 months up - regulates multiple myelin ( MBP , MOBP MOG ) in corpus callosum in PLP - 150Q mice , which were treated from 3 months of . Ratios of MBP , MOBP or MOG to vinculin obtained from 3 independent experiments were presented on right . One - way ANNOVA followed with Tukey ' s . MBP : * * * P = 0 . 0009 ; MOG : * * * P = 0 . 0003 ; MOBP : * * P = 0 . 0067 . Data are mean ±

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Western blotting showing that LAQ (5 or 25 mg/kg) treatment for 2 months up-regulates multiple myelin proteins (MBP, MOBP and MOG) in the corpus callosum in PLP-150Q mice, which were treated from 3 months of age. Ratios of MBP, MOBP or MOG to vinculin obtained from 3 independent experiments were presented on the right. One-way ANNOVA followed with Tukey's test. MBP: ***P=0.0009; MOG: ***P=0.0003; MOBP: **P=0.0067. Data are mean ±SEM


Quantitative PCR of transcript expression of myelin associated genes ( MBP MOG ) in PLP - 150Q mouse corpus callosum at 5 months of after LAQ ( 5 or 25 mg / kg ) treatment for 2 months . Student ' s t - . MBP : * * * P = 0 . 0007 ; MOG : * * * P = 0 . 0009 . Data are mean ± ( n = 3 ) .

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Quantitative PCR of the transcript expression of myelin associated genes (MBP and MOG) in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 or 25 mg/kg) treatment for 2 months. Student's t-test. MBP: ***P=0.0007; MOG: ***P=0.0009. Data are mean ±SEM (n=3).


MBP promoter was inserted into pGL4 . 1 luciferase report vector was co - expressed with N - terminal MYRF ( nMYRF ) mHTT to assess its transcription activity via luciferase . MYRF markedly enhanced MBP promoter activity . N - terminal mutant HTT significantly inhibited reporter activity , * * * P < 0 . 001 ; which was reversed by LAQ ( 5 μM ) treatment , * * P < 0 . 01 . ratios were obtained from 3 independent experiments . One - way ANNOVA followed with Tukey ' s . Data are mean ± .

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The MBP DNA promoter was inserted into the pGL4.1 luciferase report vector and was co-expressed with N-terminal MYRF (nMYRF) and mHTT to assess its transcription activity via the luciferase assay. MYRF markedly enhanced the MBP promoter activity. N-terminal mutant HTT significantly inhibited the reporter activity, ***P< 0.001; which was reversed by LAQ (5 μM) treatment, **P< 0.01. The ratios were obtained from 3 independent experiments. One-way ANNOVA followed with Tukey's test. Data are mean ±SEM.


Immunoprecipitation of transgenic mHTT from 3 - month - old PLP - 150Q mouse brains revealed that 5 or 25 mg / kg LAQ treatments for 2 months reduced interaction between transgenic mutant HTT MYRF . fMYRF : full - MYRF ; nMYRF : N - terminal MYRF . of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on right . One - way ANNOVA followed with Tukey ' s . * * P = 0 . 009 . Data are mean ± .

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Immunoprecipitation of transgenic mHTT from 3-month-old PLP-150Q mouse brains revealed that 5 or 25 mg/kg LAQ treatments for 2 months reduced the interaction between transgenic mutant HTT and MYRF. fMYRF: full-length MYRF; nMYRF: N-terminal MYRF. The ratio of immunoprecipitated MYRF to input obtained from 3 independent experiments was shown on the right. One-way ANNOVA followed with Tukey's test. **P=0.009. Data are mean ±SEM.


Western blotting of phosphorylated - NF - kB , - Akt , - JNK in PLP - 150Q mouse corpus callosum at 3 months of showing that LAQ ( 5 or 25 mg / kg ) treatment for 2 months could dephosphorylate these . Ratios of phosphorylated to their total obtained from 3 independent experiments were presented on right . One - way ANNOVA with Tukey ' s . NF - kB : * * * P = 0 . 00067 ( left ) 0 . 00086 ( right ) ; JNK : * * * P = 0 . 00013 ( left ) 0 . 00047 ( right ) ; Akt : * * P = 0 . 0014 * * * P = 0 . 00012 . Data are mean ± .

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Western blotting of the phosphorylated- NF-kB, -Akt, -JNK in PLP-150Q mouse corpus callosum at 3 months of age showing that LAQ (5 or 25 mg/kg) treatment for 2 months could dephosphorylate these proteins. Ratios of the phosphorylated proteins to their total proteins obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. NF-kB: ***P=0.00067(left) and 0.00086(right); JNK: ***P=0.00013(left) and 0.00047(right); Akt: **P=0.0014 and ***P=0.00012. Data are mean ±SEM.


In vitro phosphorylation of N - terminal MYRF ( nMYRF ) . GST fusion containing nMYRF were incubated overnight with brain lysates from PLP - 23Q or PLP - 150Q mice that were treated with vehicle or 5 mg / kg LAQ ( left panel ) . GST fusion containing wild type N - terminal MYRF , S259A , or S261A were incubated with PLP - 150Q mouse tissue lysates ( right panel ) . beads were then centrifuged analyzed by western blotting with anti - GST ( lower panels ) anti - phosphor - serine ( upper panels ) . Note that LAQ treatment could eliminate mutant HTT ( PLP - 150Q ) - mediated MYRF phosphorylation that S259A substitution prevents MYRF phosphorylation .

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In vitro phosphorylation assay of N-terminal MYRF (nMYRF). GST fusion proteins containing nMYRF were incubated overnight with brain lysates from PLP-23Q or PLP-150Q mice that were treated with vehicle or 5 mg/kg LAQ (left panel). GST fusion proteins containing wild type N-terminal MYRF, S259A, or S261A were incubated with PLP-150Q mouse tissue lysates (right panel). The beads were then centrifuged and analyzed by western blotting with anti-GST (lower panels) and anti-phosphor-serine (upper panels). Note that LAQ treatment could eliminate mutant HTT (PLP-150Q)-mediated MYRF phosphorylation and that S259A substitution prevents MYRF phosphorylation.


Co - immunoprecipitation of nMYRF N - terminal HTT ( 1 - 212 aa ) containing 150Q in HEK293 . Five μM LAQ treatment decreased phosphorylation of immunoprecipitated MYRF amount of co - immunoprecipitated HTT . Λ - PPase served as positive control for dephosphorylation of MYRF . ratios of pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under blots , which were obtained from 3 independent western blotting experiments . One - way ANNOVA with Tukey ' s . * * * P = 6 . 51x10 - 5 . Data are mean ± .

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Co-immunoprecipitation of nMYRF and N-terminal HTT (1-212 aa) containing 150Q in HEK293 cells. Five μM LAQ treatment decreased the phosphorylation of immunoprecipitated MYRF and the amount of co-immunoprecipitated HTT. Λ-PPase served as the positive control for the dephosphorylation of MYRF. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots, which were obtained from 3 independent western blotting experiments. One-way ANNOVA with Tukey's test. ***P=6.51x10-5. Data are mean ±SEM.


Co - transfection of nMYRF with N - terminal mutant HTT in HEK293 immunoprecipitation of nMYRF with or without LAQ ( 5 μM ) treatment . Coomassie blue confirms presence of immunoprecipitated N - terminal MYRF bands ( arrow ) .

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Co-transfection of nMYRF with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of nMYRF with or without LAQ (5 μM) treatment. Coomassie blue staining confirms the presence of immunoprecipitated N-terminal MYRF bands (arrow).


Transcriptional activity of wide type N - terminal MYRF , S259A , or S261A with MBP promoter reporter in HEK293 was detected using a luciferase . Compared to wide type MYRF or S261A , S259A ( non - phosphorylated ) remained transcriptional activity that was not inhibited by N - terminal mutant HTT . ratios were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s . * * * P < 0 . 001 . Data are mean ± .

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Transcriptional activity of wide type N-terminal MYRF, S259A, or S261A with the MBP promoter reporter in HEK293 cells was detected using a luciferase assay. Compared to wide type MYRF or S261A, S259A (non-phosphorylated) remained transcriptional activity that was not inhibited by N-terminal mutant HTT. The ratios were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.


Co - transfection of wide type N - terminal MYRF , S259A , or S261A with N - terminal mutant HTT in HEK293 immunoprecipitation of MYRF . Compared to wide type MYRF S261A , less S259A ( non - phosphorylated ) was precipitated with mutant HTT . ratios of pho - serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under blots were obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s . * * * P = 2 . 30x10 - 5 . Data are mean ± .

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Co-transfection of wide type N-terminal MYRF, S259A, or S261A with N-terminal mutant HTT in HEK293 cells and immunoprecipitation of MYRF. Compared to wide type MYRF and S261A, less S259A (non-phosphorylated) was precipitated with mutant HTT. The ratios of the pho-serine MYRF or precipitated HTT to immunoprecipitated MYRF are shown under the blots and were obtained from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P=2.30x10-5 . Data are mean ±SEM.


Western blotting of corpus callosum in PLP - 23Q or PLP - 150Q mice with anti - pS259 an antibody to total MYRF . Note that LAQ ( 5 mg / kg ) treatment for 2 months decreased phosphorylation in both full ( fMYRF ) N - terminal MYRF ( nMYRF ) in PLP - 150Q mice .

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Western blotting analysis of the corpus callosum in PLP-23Q or PLP-150Q mice with anti-pS259 and an antibody to total MYRF. Note that LAQ (5 mg/kg) treatment for 2 months decreased the phosphorylation in both full length (fMYRF) and N-terminal MYRF (nMYRF) in PLP-150Q mice.


Ratios of phosphor - S259 - MYRF ( pMYRF ) total MYRF to GAPDH . ratios were obtained from 3 independent western blotting experiments . Student ' s t - ; * * * P = 0 . 0007 . Data are mean ± .

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Ratios of phosphor-S259-MYRF (pMYRF) and total MYRF to GAPDH. The ratios were obtained from 3 independent western blotting experiments. Student's t-test; ***P=0.0007. Data are mean ±SEM.


Anti - Ser259 - MYRF immunohistochemical of corpus callosum ( CC , indicated between two dotted lines ) of PLP - 23Q PLP - 150Q mice showing decreased expression of Ser259 - MYRF by LAQ ( 5 mg / kg ) treatment for 2 months . at 10X ( upper ) 40X ( lower ) are shown . Scale bars : 40 μm .

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Anti-Ser259-MYRF immunohistochemical staining of the corpus callosum (CC, indicated between two dotted lines) of PLP-23Q and PLP-150Q mice showing decreased expression of Ser259-MYRF by LAQ (5 mg/kg) treatment for 2 months. Micrographs at 10X (upper) and 40X (lower) are shown. Scale bars: 40 μm.


Quantitative statistics of stained by anti - Ser259 MYRF in each filed was presented . Twenty random fields ( 40X ) of each section were examined to anti - Ser259 positive , n = 3 mice in each group . One - way ANNOVA with Tukey ' s . * * * P = 0 . 0003 ; * * P = 0 . 0039 . Data are mean ± .

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Quantitative statistics of the cells stained by anti-Ser259 MYRF in each filed was presented. Twenty random fields (40X) of each section were examined to count anti-Ser259 positive cells, n=3 mice in each group. One-way ANNOVA with Tukey's test. ***P=0.0003; **P=0.0039. Data are mean ±SEM.


Western blotting of MBP Ser259 - MYRF expression in postmortem brain cortex tissues in 4 HD 4 non - HD control individuals . Ratios of MBP to GAPDH , or Ser259 ( pMYRF ) to total MYRF are presented on right . One - way ANNOVA with Tukey ' s . MBP : * * * P = 0 . 0004 ; pMYRF : * * P = 0 . 0051 . Data are mean ± .

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Western blotting analysis of MBP and Ser259-MYRF expression in the postmortem brain cortex tissues in 4 HD patients and 4 non-HD control individuals. Ratios of MBP to GAPDH, or Ser259 (pMYRF) to total MYRF are presented on the right. One-way ANNOVA with Tukey's test. MBP: ***P=0.0004; pMYRF: **P=0.0051. Data are mean ±SEM.


Western blotting of expression of Ser259 ( pMYRF ) total MYRF ( full - : fMYRF N - terminal : nMYRF ) in 13 - months - old HD - KI WT mouse cortex . Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath blots . One - way ANNOVA with Tukey ' s . * * * P = 0 . 00083 . Data are mean ± .

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Western blotting analysis of the expression of Ser259 (pMYRF) and total MYRF (full-length: fMYRF and N-terminal: nMYRF) in 13-months-old HD-KI and WT mouse cortex. Ratios of pMYRF to total MYRF obtained from 3 independent experiments were presented beneath the blots. One-way ANNOVA with Tukey's test. ***P=0.00083. Data are mean ±SEM.


Heatmaps of gene expression in PLP - 150Q mouse corpus callosum at 5 months after LAQ ( 5 mg / kg ) treatment for 2 months revealed 27 significantly upregulated 103 significantly downregulated genes .

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Heatmaps of gene expression in PLP-150Q mouse corpus callosum at 5 months after LAQ (5 mg/kg) treatment for 2 months revealed 27 significantly upregulated and 103 significantly downregulated genes.


networks of up - regulated gene were heat mapped typically , on clusters of " REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS " , " POSITIVE _ REGULATE _ OF _ PHOSPHORUS _ METABOLIC _ PROCESS "

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The individual networks of up-regulated gene were heat mapped typically, on the clusters of "REGULATE_OF_PHOSPHORUS_METABOLIC_PROCESS", and "POSITIVE_REGULATE_OF_PHOSPHORUS_METABOLIC _PROCESS"


Western blotting showing down - regulation of PRKG2 in PLP - 150Q mouse corpus callosum at 3 months of by 5 mg / kg LAQ . Ratios of PRKG2 or phosphorylated VASP ( p - VASP ) to GAPDH obtained from 3 independent experiments were presented on right . One - way ANNOVA with Tukey ' s . PRKG2 : * * P = 0 . 0076 ( left ) * * * P = 0 . 0015 ( right ) ; p - VASP : * * * P = 3 . 86x10 - 5 ( left ) 1 . 01x10 - 5 ( right ) . Data are mean ± .

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Western blotting showing down-regulation of PRKG2 in PLP-150Q mouse corpus callosum at 3 months of age by 5 mg/kg LAQ. Ratios of PRKG2 or phosphorylated VASP (p-VASP) to GAPDH obtained from 3 independent experiments were presented on the right. One-way ANNOVA with Tukey's test. PRKG2: **P=0.0076(left) and ***P=0.0015(right); p-VASP: ***P=3.86x10-5(left) and 1.01x10-5(right). Data are mean ±SEM.


Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP - 150Q mouse corpus callosum at 5 months of after LAQ ( 5 mg / kg ) treatment for 2 months . Only PRKG2 was decreased by LAQ . n = 3 mice in each group . Student ' s t - ; * * P = 0 . 0073 ; * * * P = 0 . 00041 . Data are mean ± .

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 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

Quantitative PCR of PRKG2 or PRKG1 gene expression in PLP-150Q mouse corpus callosum at 5 months of age after LAQ (5 mg/kg) treatment for 2 months. Only PRKG2 was decreased by LAQ. n=3 mice in each group. Student's t-test; **P=0.0073; ***P=0.00041. Data are mean ±SEM.


different PRKG2 promoter regions were inserted into pGL4 . 1 luciferase report vector . promoter reporter vector of PRKG2 ( - 472 / + 131 ) was determined for transcription activity in cultured N2a , obtained from 3 independent experiments . One - way ANNOVA with Tukey ' s ; * * * P < 0 . 001 . Data are mean ± .

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The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (-472/+131) was determined for transcription activity in cultured N2a cells, obtained from 3 independent experiments. One-way ANNOVA with Tukey's test; ***P<0.001. Data are mean ±SEM.


treatment of LAQ ( 5 or 10 μM ) caused a marked inhibition on transcription activity of PRKG2 promoter detected by luciferase from 3 independent experiments . One - way ANNOVA with Tukey ' s . * * * P < 0 . 001 . Data are mean ± .

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The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from 3 independent experiments. One-way ANNOVA with Tukey's test. ***P<0.001. Data are mean ±SEM.


qPCR of transcripts of Cyp1a1 Ugt1a6a , which were mediated by AhR , in PLP - 150Q mouse brain ( corpus callosum ) . Note that Cyp1a1 Ugt1a6a were significantly increased by LAQ ( 5 mg / kg ) . n = 3 mice in each group . Student ' s t - ; Cyp1a1 : * * * P = 0 . 00042 ; Ugt1a6a ; * * * P = 0 . 00057 . Data are mean ± .

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The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP-150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n=3 mice in each group. Student's t-test; Cyp1a1: ***P=0.00042; Ugt1a6a; ***P=0.00057. Data are mean ±SEM.


PRKG2 promoter activity in N2a transfected with AhR siRNA or its scrambled siRNA control then treated with 5 μM LAQ . values of promoter activity via luciferase report were obtained from three independent experiments . One - way ANNOVA with Tukey ' s . * * * P < 0 . 001 . Data are mean ± .

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The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One-way ANNOVA with Tukey's test. ***P< 0.001. Data are mean ±SEM.


semi - PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti - AhR in ChIP . N2a were treated with 10 μM LAQ or 10 nM 2 , 3 , 7 , 8 - TCDD or DMSO for 12 hr . of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti - AhR in ChIP . N2a were treated with 10 μM LAQ or 10 nM 2 , 3 , 7 , 8 - TCDD or DMSO for 12 hr . results were obtained from three independent experiments . One - way ANNOVA with Tukey ' s . LAQ : * * * P = 0 . 00065 ; 2 , 3 , 7 , 8 - TCDD : * * * P = 0 . 00014 . Data are mean ±

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

The semi-PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti-AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8-TCDD or DMSO for 12 hr. The results were obtained from three independent experiments. One-way ANNOVA with Tukey's test. LAQ: ***P=0.00065; 2,3,7,8-TCDD: ***P=0.00014. Data are mean ±SEM


Western blotting showing that phosphorylation of transfected N - terminal MYRF ( pMYRF ) was promoted by treatment with PKG activator of 8 - Br - cGMP ( 10 or 100 μM ) for 1 hr or co - transfected 150Q - HTT for 24 hr , which was blocked by treatment of PKG inhibitors of RKRARKE ( 50 μM ) or KT5823 ( 5 μM ) for 12 hr . trans - nMYRF : transfected N - terminal MYRF .

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Western blotting showing that the phosphorylation of transfected N-terminal MYRF (pMYRF) was promoted by treatment with the PKG activator of 8-Br-cGMP (10 or 100 μM) for 1 hr or co-transfected 150Q-HTT for 24 hr, which was blocked by treatment of the PKG inhibitors of RKRARKE (50 μM) or KT5823 (5 μM) for 12 hr. trans-nMYRF: transfected N-terminal MYRF.


Western blotting of N2a that was transfected with AAV - PRKG2 for 48 hr . blots were probed with anti - Ser259 ( pMYRF ) antibody . Note that overexpression of PRKG2 increased phosphorylation of N - terminal MYRF ( nMYRF ) in a dose - dependent manner .

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Western blotting of N2a cell line that was transfected with AAV-PRKG2 for 48 hr. The blots were probed with anti-Ser259 (pMYRF) antibody. Note that overexpression of PRKG2 increased phosphorylation of N-terminal MYRF (nMYRF) in a dose-dependent manner.


Stereotaxic injection of AAV - GFP into corpus callosum ( CC , indicated between two dotted lines ) in mouse brain . Ctx : cortex , Str : striatum . Scale bar : 100 μm . . Immunohistochemical with anti - Ser259 ( pMYRF ) showing that AAV - PRKG2 , but not AAV - GFP , promotes MYRF phosphorylation in corpus callosum . Scale bar : 40 μm .

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Stereotaxic injection of AAV-GFP virus into the corpus callosum (CC, indicated between two dotted lines) in the mouse brain. Ctx: cortex, Str: striatum. Scale bar: 100 μm..   Immunohistochemical staining with anti-Ser259 (pMYRF) showing that AAV-PRKG2, but not AAV-GFP, promotes MYRF phosphorylation in the corpus callosum. Scale bar: 40 μm.


Quantitative of pMYRF - or total MYRF - positive in each filed ( 40X ) . Twenty random fields in each section were examined , n = 3 mice in each group . One - way ANNOVA with Tukey ' s ; * * * P = 8 . 79x10 - 5 . Data are mean ± .

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Quantitative analysis of the pMYRF- or total MYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=8.79x10-5. Data are mean ±SEM.


Double immunofluorescent with antibodies to PRKG2 Ser259 showing that AAV - PRKG2 , but not AAV - GFP ( green ) , promotes MYRF phosphorylation ( red ) in injected corpus callosum . Scale bar : 40 μm .

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Double immunofluorescent staining with antibodies to PRKG2 and Ser259 showing that AAV-PRKG2, but not AAV-GFP (green), promotes MYRF phosphorylation (red) in the injected corpus callosum. Scale bar: 40 μm.


Western blotting of mouse corpus callosum tissues using anti - Ser259 ( pMYRF ) , phosphor - PRKG2 ( pPRKG2 ) or phosphor - VASP ( pVASP ) . Note that overexpression of mouse PRKG2 leads to phosphorylation of MYRF VASP . fMYRF : full - MYRF , nMYRF : N - terminal MYRF .

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Western blotting analysis of the mouse corpus callosum tissues using anti-Ser259 (pMYRF), phosphor-PRKG2 (pPRKG2) or phosphor-VASP (pVASP). Note that overexpression of mouse PRKG2 leads to the phosphorylation of MYRF and VASP. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.


Construction of AAV - U6 - gRNA - CMV - RFP vector expressing PRKG2 gRNA under U6 promoter RFP under CMV promoter , respectively . AAV - gRNA - PRKG2 was injected into corpus callosum ( CC , indicated between two dotted lines ) in PLP - 150Q / Cas9 mouse at 2 months of injected tissues were isolated 4 after injection ( lower panel ) . Ctx : cortex , Str : striatum , LV : lateral ventricle . Scale bar : 100

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Construction of AAV-U6-gRNA-CMV-RFP vector expressing PRKG2 gRNA under the U6 promoter and RFP protein under the CMV promoter, respectively. AAV-gRNA-PRKG2 was injected into the corpus callosum (CC, indicated between two dotted lines) in PLP-150Q/Cas9 mouse at 2 months of age and the injected tissues were isolated 4 weeks after injection (lower panel). Ctx: cortex, Str: striatum, LV: lateral ventricle. Scale bar: 100 μm


Western blotting of PLP - 150Q / Cas9 mouse corpus callosum tissues using antibody to Ser259 ( pMYRF ) , phosphor - PRKG2 or MBP . Note that reduction of mouse PRKG2 by CRISPR / Cas9 leads to decreased MYRF phosphorylation increased expression of MBP . ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under blots . Student ' s t - ; pMYRF : * * * P = 0 . 00026 ; MBP ; * * P = 0 . 0036 . Data are presented as mean ± . fMYRF : full - MYRF , nMYRF : N - terminal MYRF .

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Western blotting analysis of the PLP-150Q/Cas9 mouse corpus callosum tissues using the antibody to Ser259 (pMYRF), phosphor-PRKG2 or MBP. Note that reduction of mouse PRKG2 by CRISPR/Cas9 leads to the decreased MYRF phosphorylation and the increased expression of MBP. The ratios of pMYRF to MYRF or MBP to GAPDH obtained from 3 independent experiments were presented under the blots. Student's t-test; pMYRF: ***P=0.00026; MBP; **P=0.0036. Data are presented as mean ±SEM. fMYRF: full-length MYRF, nMYRF: N-terminal MYRF.


Immunohistochemical with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in corpus callosum of PLP - 150Q mouse . Scale bar : 40 μm .

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Immunohistochemical staining with antibody to Ser259 showed that knocking down PRKG2 reduced MYRF phosphorylation in the corpus callosum of PLP-150Q mouse. Scale bar: 40 μm.


Quantitative of pMYRF - positive in each filed ( 40X ) . Twenty random fields in each section were examined , n = 3 mice in each group . One - way ANNOVA with Tukey ' s ; * * * P = 0 . 00022 . Data are mean ± .

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Quantitative analysis of the pMYRF-positive cells in each filed (40X). Twenty random fields in each section were examined, n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=0.00022. Data are mean ±SEM.


Immunofluorescent with antibodies to Ser259 or MBP showed AAV - PRKG2 - gRNA ( red ) injection increased MBP expression ( green ) as compared with AAV - gRNA - control . Scale bar : 40 μm .

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Immunofluorescent staining with antibodies to Ser259 or MBP showed the AAV- PRKG2-gRNA (red) injection increased MBP expression (green) as compared with AAV-gRNA-control. Scale bar: 40 μm.


Quantitative of MBP - positive in corpus callosum injected with control - gRNA or PRKG2 - gRNA . Twenty random fields ( 40X ) in each section were examined . n = 3 mice in each group . One - way ANNOVA with Tukey ' s ; * * * P = 4 . 43x10 - 5 . Data are mean ± .

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Quantitative analysis of the MBP-positive cells in the corpus callosum injected with control-gRNA or PRKG2-gRNA. Twenty random fields (40X) in each section were examined. n=3 mice in each group. One-way ANNOVA with Tukey's test; ***P=4.43x10-5. Data are mean ±SEM.


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

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

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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 A375 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 A375 stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical was performed by two - way Anova for time - dependent proliferation changes by one - way Anova for comparison of more than two groups . Data are shown as 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 A375 stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical was performed by two - way Anova for time - dependent proliferation changes by one - way Anova for comparison of more than two groups . Data are shown as 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 ) 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 A375 stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data are shown as 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 ) glutamine ( Gln , C ) in medium after treatment of melanoma lines with LDHAi for 72 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups , N . D . , not determined . Data are shown as mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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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 after transfection of melanoma with non - targeting ( NT ) or LDHA - specific siRNA for 96 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups N . D . , not determined . Data are shown as 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 - of intracellular Gln in Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups N . D . , not determined . Data are shown as 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

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 of expression of indicated SLC family transporters in MeWo ( left ) A375 ( right ) melanoma after treatment with LDHAi for 48 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups N . D . , not determined . Data are shown as 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 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

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 treated with LDHAi for 96 hr .

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

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


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

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

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 LDHA in MeWo stably transduced with empty vector ( EV ) or four different LDHA - specific shRNAs .

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

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 mock ( NT - siRNA + DMSO ) or si - SLC1A5 - treated for 24 hr then treated with LDHAi for 48 hr .

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

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 ( left ) , intracellular Gln ( right ) of MeWo Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups , N . D . , not determined . Data are shown as 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

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 lines for indicated timepoints following treatment with LDHA - i , si - SLC1A5 or both . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . two - way Anova was used for proliferation . N . D . , not determined . Data are shown as 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 treated as indicated for 8 days . ( right ) corresponding showing change in viability . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups , N . D . , not determined . Data are shown as 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

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 caspase - 3 in melanoma subjected to mock ( NT - siRNA + DMSO ) or si - SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups , N . D . , not determined . Data are shown as 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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - of intracellular essential acid in MeWo treated for 72 hr with LDHAi Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 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

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 - of intracellular essential acid in MeWo mock or si - SLC1A5 - transfected for 24 hr then treated with LDHAi for 48 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 transfected with non - targeting ( NT ) or SLC7A5 - specific siRNAs for 48 hr . ( right ) GC - MS - of intracellular essential acid in MeWo mock or si - SLC7A5 - transfected for 24 hr then treated with LDHAi for 48 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

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

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

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

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

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

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

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 treated as indicated for 10 days . ( bottom ) corresponding showing change in viability . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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

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

O O O B-MEASURED_VAR I-MEASURED_VAR I-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

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

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

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

O O B-MEASURED_VAR 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

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

O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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 SLC1A5 in melanoma lines treated with LDHAi for 2 , 24 , or 48 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups .

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

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

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

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

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 - of intracellular essential acid in MeWo treated share Mock LDHA - i controls . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 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

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 lines mock - or si - ATF4 - transfected incubated with LDHAi as indicated for up to 96 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Unpaired t - was used for comparison of two groups , two - way Anova was used for proliferation . Data are shown as 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 B-CONTROLLED_VAR 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

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

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

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 - of intracellular asparagine ( Asn ) alanine ( Ala ) in MeWo 1205Lu treated with LDHAi for 72 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 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

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 - of intracellular Asn Ala in MeWo mock - transfected or transfected with si - ATF4 ( K ) for 24 hr then treated with LDHAi for 48 hr . shared Mock LDHA - i controls . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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

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 - of intracellular Asn Ala in MeWo mock - transfected or si - SLC1A5 ( L ) for 24 hr then treated with LDHAi for 48 hr . shared Mock LDHA - i controls . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR 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

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

O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SLC1A5 in MeWo mock - or si - GCN2 - treated for 24 hr then incubated with LDHAi for 48 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups .

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


O 3H - Glutamine uptake in MeWo transfected with si - GCN2 treated 48 hr later with LDHAi for 6 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O 3H-Glutamine uptake in MeWo cells transfected with si-GCN2 and treated 48 hr later with LDHAi for 6 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


P Gln in medium after treatment of MeWo with mock - or si - GCN2 for 24 hr subsequent incubation with LDHA - i for 48 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . Data are shown as 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 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

P Gln levels in the medium after treatment of MeWo cells with mock- or si-GCN2 for 24 hr and subsequent incubation with LDHA-i 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


Q GC - MS - of intracellular Gln essential acid in MeWo Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups . proliferation . Data are shown as 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

Q GC-MS-based quantification of intracellular Gln and essential amino acid levels in MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. 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


A GC - MS - of intracellular Asp ( upper ) Ser ( lower ) in melanoma lines treated with LDHAi for 72 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

A GC-MS-based quantification of intracellular Asp (upper) and Ser (lower) levels in melanoma cell lines 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 and unpaired t-test for the comparison of 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 ( upper ) GC - MS - 13C6 - glucose fractional isotope labeling of Ser Asp in melanoma treated for 72 hr with LDHAi . ( lower ) GC - MS - 13C5 - glutamine fractional isotope labeling of Asp in melanoma lines treated for 72 hr with LDHAi . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (upper) GC-MS-based 13C6-glucose fractional isotope labeling of Ser and Asp in melanoma cells treated for 72 hr with LDHAi. (lower) GC-MS-based 13C5-glutamine fractional isotope labeling of Asp in melanoma cell lines 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 and unpaired t-test for the comparison of 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 - of intracellular Ser Asp in MeWo cultured in DMEM ( C ) treated with LDHAi for 24 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in DMEM (C) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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 - of intracellular Ser Asp in MeWo cultured in MEM supplemented with Ser ( 400 μM ; D ) treated with LDHAi for 24 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in MEM supplemented with Ser (400 μM; D) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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 - of intracellular Ser Asp in MeWo treated with LDHAi for 1 hr Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells treated with LDHAi for 1 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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


F GC - MS - of intracellular Asp in MeWo pre - treated with Asp ( 10 mM ) for 6 hr then treated with LDHAi for 1 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

F GC-MS-based quantification of intracellular Asp levels in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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


G Immunoblotting of ATF4 in MeWo pre - treated with Asp ( 10 mM ) for 6 hr then treated with GSK - 2837808A ( 10 μM ) for 1 , 2 , or 24 hr .

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

G Immunoblotting of ATF4 in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with GSK-2837808A (10 μM) for 1, 2, or 24 hr.


H Immunoblotting of ATF4 in MeWo pre - treated with Ser ( 400 μM ) , Asp ( 10 mM ) , or Ser ( 400 μM ) + Asp ( 10 mM ) for 6 hr then treated for 1 hr with LDHAi ( 10 μM ) .

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

H Immunoblotting of ATF4 in MeWo cells pre-treated with Ser (400 μM), Asp (10 mM), or Ser (400 μM) + Asp (10 mM) for 6 hr and then treated for 1 hr with LDHAi (10 μM).


A Relative NAD + / NADH in MeWo treated with LDHAi , nicotinamide mononucleotide ( NMN , 1 mM ) , or a combination of two for 24 hr . were pretreated with nicotinamide mononucleotide for 6 hr then treated with LDHAi for 24 hr in combination setting . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-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

A Relative NAD+/NADH ratio in MeWo cells treated with LDHAi, nicotinamide mononucleotide (NMN, 1 mM), or a combination of the two for 24 hr. Cells were pretreated with nicotinamide mononucleotide for 6 hr and then treated with LDHAi for 24 hr in the combination setting. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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 - of total intracellular of Ser Asp their 13C6 - glucose fractional isotope labeling in MeWo pretreated with nicotinamide mononucleotide ( NMN , 1 mM ) for 6 hr then treated with LDHAi for 24 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

B GC-MS-based quantification of total intracellular levels of Ser and Asp and their 13C6-glucose fractional isotope labeling in MeWo cells pretreated with nicotinamide mononucleotide (NMN, 1 mM) for 6 hr and then treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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


D GC - MS - of intracellular Ala in MeWo treated with LDHAi for 1 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

D GC-MS-based quantification of intracellular Ala levels in MeWo cells treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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


E GC - MS - 13C6 - Glc ( M + 2 ) 13C5 - Gln ( M + 4 ) isotopologue of key TCA cycle metabolites Asp in MeWo treated with LDHAi for 72 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 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

E GC-MS-based 13C6-Glc (M+2) and 13C5-Gln (M+4) isotopologue levels of the key TCA cycle metabolites and Asp in MeWo 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 and unpaired t-test for the comparison of 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


F GC - MS - 13C6 - Glc ( left ) 13C5 - Gln ( right ) isotopologue of Asp in MeWo treated with LDHAi for 72 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

F GC-MS-based 13C6-Glc (left) and 13C5-Gln (right) isotopologue levels of Asp in MeWo 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 and unpaired t-test for the comparison of 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


G GC - MS - of intracellular Ala ( left ) Asp ( right ) in si - GPT2 - treated MeWo incubated with LDHAi for 1 hr . Data information : Statistical was performed by ordinary one - way Anova for comparison of more than two groups unpaired t - for comparison of two groups . Data are shown as 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G GC-MS-based quantification of intracellular Ala (left) and Asp (right) levels in si-GPT2-treated MeWo cells incubated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of 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 Immunoblotting of ATF4 and GPT2 in MeWo cells


A ( left ) Immunoblotting of LDHA ATF4 in A375 expressing inducible ( Tet - On ) sh - LDHA expression vector treated with doxycycline ( Dox ) for 48 hr . ( right ) qRT - PCR ( n = 3 ) of ATF4 target genes in A375 treated with doxycycline ( 0 . 5 μg / ml ) for 24 hr . Statistical was performed by unpaired t - . Data are shown as mean ± , ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

A (left) Immunoblotting of LDHA and ATF4 in A375 cells expressing inducible (Tet-On) sh-LDHA expression vector treated with doxycycline (Dox) for 48 hr. (right) qRT-PCR (n=3) analysis of the ATF4 target genes in A375 cells treated with doxycycline (0.5 μg/ml) for 24 hr. Statistical analysis was performed by unpaired t-test. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


B Proliferation of inducible ( Tet - On ) sh - LDHA expression vector harboring A375 ( n = 3 ) treated with Dox ( 0 . 5

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B Proliferation of inducible (Tet-On) sh-LDHA expression vector harboring A375 cells (n=3) treated with Dox (0.5 μg


C Athymic nude mice were injected subcutaneously with A375 melanoma expressing inducible ( Tet - On ) sh - LDHA expression vector . Starting day 5 , were treated on alternate days with 1 g / L doxycycline , 4 mg / kg rapamycin , or with a combination of two ( n = 8 in each group ) . growth was determined by measuring volume . Statistical was performed using Welch ' s t - ( two - ) . Data are shown as mean ± ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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C Athymic nude mice were injected subcutaneously with A375 melanoma cells expressing inducible (Tet-On) sh-LDHA expression vector. Starting day 5, animals were treated on alternate days with 1 g/L doxycycline, 4 mg/kg rapamycin, or with a combination of the two (n=8 in each group). The tumor growth was determined by measuring the tumor volume. Statistical analysis was performed using Welch's t-test (two-tailed). Data are shown as the mean ± SEM ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


D Immunoblotting of resected tumors ( 4 from each group ) for indicated .

D Immunoblotting of resected tumors (4 from each group) for the indicated proteins.


Representative of H & E caspase - 3 from each treatment group ( E ) .

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Representative images of H&E and cleaved caspase-3 staining from each treatment group (E).


of caspase - 3 ( F ) ( n = 4 , each 4 fields ) . Scale bars , 50 μm . Statistical was performed using two - way Anova . Data are shown as mean ± , ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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Quantification of cleaved caspase-3 (F) (n = 4, each 4 fields). Scale bars, 50 μm. Statistical analysis was performed using two-way Anova. Data are shown as the mean ± SEM , ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.