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 .

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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 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 .

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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 .

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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 .

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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 LDHA in MeWo 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 mock ( NT - siRNA + DMSO ) or si - SLC1A5 - treated for 24 hr 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 ( 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 .

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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 .

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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 .

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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 .

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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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-DISEASE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-DISEASE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-DISEASE O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-DISEASE O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-DISEASE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY O B-GENEPROD O B-DISEASE O O O O O O B-GENEPROD O B-GENEPROD 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 .

B-EXP_ASSAY O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O B-GENEPROD O O O 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 B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-DISEASE O O O B-GENEPROD O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-DISEASE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-DISEASE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-DISEASE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O B-GENEPROD 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-DISEASE O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-DISEASE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 .

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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 .

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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


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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 .

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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 .

O O O B-ORGANISM O O O O B-CELL_LINE B-DISEASE O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


G Relative mTORC1 expression in pan - samples segregated on high ( LDHAhi ) ( n = 2917 ) low LDHA ( LDHAlo ) ( n = 2914 ) expression . p - value of paired Wilcoxon following adjustment multiple hypothesis correction ( FDR ) . Data information : In boxplots , top bottom horizontal lines represent 75th 25th percentile respectively , middle horizontal represents median . of box represents interquartile range top bottom whiskers represent maximum minimum values respectively .

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G Relative mTORC1 expression in pan-tumor samples segregated based on high (LDHAhi) (n=2917) and low LDHA (LDHAlo) (n=2914) expression. The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively.


H Conditional essentiality of indicated genes in LDHAlo LDHAhi lines ( n = 17 , each group ) . p - value of paired Wilcoxon following adjustment multiple hypothesis correction ( FDR ) . p ≤ 0 . 1 is significant . Data information : In boxplots , top bottom horizontal lines represent 75th 25th percentile respectively , middle horizontal represents median . of box represents interquartile range top bottom whiskers represent maximum minimum values respectively .

O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Conditional essentiality of the indicated genes in LDHAlo and LDHAhi cell lines (n=17, each group). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR). p≤0.1 is significant. Data information: In the boxplots, the top and bottom horizontal lines represent the 75th and the 25th percentile respectively, and the middle horizontal line represents the median. The size of the box represents the interquartile range and the top and bottom whiskers represent the maximum and the minimum values respectively.


A qRT - PCR of LDHA mRNA in A375 treated with BRAF inhibitor ( PLX - 4032 , 1 μM ) for 24 hr . Data information : Statistical was performed by unpaired t - for comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of LDHA mRNA levels in A375 cells treated with BRAF inhibitor (PLX-4032, 1 μM) for 24 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.


GC - MS - of ( B ) intracellular 3 - PG in A375 treated with PLX - 4032 ( 1 μM ) for 16 hr . Data information : Statistical was performed by unpaired t - for comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) intracellular 3-PG in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.


GC - MS - of ( C ) lactate in A375 treated with PLX - 4032 ( 1 μM ) for 16 hr . Data information : Statistical was performed by unpaired t - for comparison of two groups . Data are shown as mean ± SD , n = 3 . ns : not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C) lactate levels in A375 cells treated with PLX-4032 (1 μM) for 16 hr. Data information: Statistical analysis was performed by unpaired t-test for the comparison of two groups. Data are shown as mean ± SD, n = 3. ns: not significant, **p ≤ 0.01, ****p ≤ 0.0001.


D Immunoblotting of indicated in A375 treated with PLX - 4032 ( 1 μM ) for 2 , 6 , or 24 hr .

O B-EXP_ASSAY O O O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

D Immunoblotting of the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr.


E , F for expression fold - change of ASNS , GPT2 , PSAT1 , MITF between ( E ) post - treatment ( BRAF inhibitor , BRAF - i ; MEK inhibitor , MEK - i ) pre - treatment , or ( F ) post - treatment ( BRAF - i ) pre - treatment in each of ( y - axis ) . p - value of paired Wilcoxon following adjustment multiple hypothesis correction ( FDR ) are also displayed ; post - treatment expression significantly greater than pre - treatment except in case of MITF , where post - treatment expression is significantly less than pre - treatment .

O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O

E, F Heatmap for expression fold-change of ASNS, GPT2, PSAT1, and MITF between (E) post-treatment (BRAF inhibitor, BRAF-i; MEK inhibitor, MEK-i) and pre-treatment, or (F) post-treatment (BRAF-i) and pre-treatment tumor in each of the patients (y-axis). The p-value of paired Wilcoxon test following the adjustment multiple hypothesis correction (FDR) are also displayed; post-treatment expression significantly greater than pre-treatment except in the case of MITF, where post-treatment expression is significantly less than pre-treatment.


( A ) Top : SERA6 architecture . Positions of papain - like domain , catalytic triad residues , SUB1 cleavage sites epitopes for antibodies used are indicated . Positions of inserted mTAP or myc3 epitope tags ( between codons Asn886 - Val887 ) introduced in SERA6 - mTAP : loxP are shown . Bottom : time - course of egress by western blot , showing processing of SERA6 - mTAP into multiple species ( representative of 2 independent experiments ) . Schizonts were sampled at indicated times following washout of C2 - mediated arrest . Nomenclature for each species was on apparent molecular mass or predicted composition . Note that predicted mass of p40 species is ~ 27 kDa , but its migration on SDS PAGE may be aberrant due to its acidic nature ; predicted pI of between SUB1 site 1 central papain - like domain ( Asp371 - Lys605 ) is ~ 4 . 4 ( see https : / / web . expasy . org / compute _ pi / ) .

O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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) Top: SERA6 architecture. Positions of the papain-like domain, catalytic triad residues, SUB1 cleavage sites and epitopes for the antibodies used are indicated. Positions of the inserted mTAP or myc3 epitope tags (between codons Asn886-Val887) introduced in SERA6-mTAP:loxP parasites are shown. Bottom: time-course analysis of egress by western blot, showing processing of SERA6-mTAP into multiple protein species (representative of 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2-mediated arrest. Nomenclature for each protein species was based on apparent molecular mass or predicted composition. Note that the predicted mass of the p40 species is ~27 kDa, but its migration on SDS PAGE may be aberrant due to its acidic nature; the predicted pI of sequence between SUB1 site 1 and the central papain-like domain (Asp371-Lys605) is ~4.4 (see https://web.expasy.org/compute_pi/).


( C ) Giemsa - stained thin films western blot of DMSO - RAP - treated SERA6 - WT - myc3 [ SERA6 - mTAP : loxP ] SERA6 - Cys644Ala - myc3 [ SERA6 - mTAP : loxP ] showing that cleavage of host RBC β - spectrin ( red arrowhead ) does not occur unless a functional copy of SERA6 is present , is tightly associated with egress ( reproducible in 2 independent experiments ) . Schizonts were sampled at indicated times following washout of C2 . Scale bar , 5 μm . Also see Fig EV2 .

O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O 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-SMALL_MOLECULE O O O O O O O O O O O O

(C) Giemsa-stained thin films and western blot of DMSO- and RAP-treated SERA6-WT-myc3 [SERA6-mTAP:loxP] and SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites showing that cleavage of host RBC β-spectrin (red arrowhead) does not occur unless a functional copy of SERA6 is present, and is tightly associated with egress (reproducible in 2 independent experiments). Schizonts were sampled at the indicated times following washout of C2. Scale bar, 5 μm. Also see Fig EV2.


( F ) Representative IFA confirming loss of MSA180 - HA3 expression following RAP - treatment ( reproducible in 2 independent experiments ) . Mature C2 - arrested cycle 0 schizonts were co - stained with anti - HA ( red ) anti - SERA5 ( green ) . Merged signals include that of dye 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar , 5 μm .

O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O

(F) Representative IFA images confirming loss of MSA180-HA3 expression following RAP-treatment (reproducible in 2 independent experiments). Mature C2-arrested cycle 0 schizonts were co-stained with anti-HA (red) and anti-SERA5 (green). Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 5 μm.


( G ) Western blots showing MSA180 is a predominantly soluble PV ( representative of 2 independent experiments ) . Mature C2 - arrested MSA180 - HA3 : loxP schizonts were sampled prior to ( Total ) following saponin fractionation to separate RBC cytosol plus soluble PV ( Supernatant ) from other - associated ( Pellet ) . same samples were iprobed with anti - SERA5 anti - MSP1 ( mAb 89 . 1 ) as positive controls for soluble PV merozoite surface respectively . presence of MSA180 p45 - HA3 is likely a post - lysis artefact , perhaps due to SUB1 release during sample manipulation . Note less efficient saponin - mediated release of MSA180 - HA3 compared to SERA5 , indicating limited association of a fraction of MSA180 . See also Fig EV3 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O

(G) Western blots showing MSA180 is a predominantly soluble PV protein (representative of 2 independent experiments). Mature C2-arrested MSA180-HA3:loxP schizonts were sampled prior to (Total) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins (Supernatant) from other parasite proteins and membrane-associated proteins (Pellet). The same samples were iprobed with anti-SERA5 and anti-MSP1 (mAb 89.1) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively. The presence of MSA180 p45-HA3 is likely a post-lysis artefact, perhaps due to SUB1 release during sample manipulation. Note the less efficient saponin-mediated release of MSA180-HA3 compared to SERA5, indicating limited membrane association of a fraction of MSA180. See also Fig EV3.


( A ) Replication of DMSO - RAP - treated MSA180 - HA3 : loxP over three erythrocytic cycles . Parasitaemia values are averages from replicates in different blood sources . similar growth rate of DMSO - treated parental B11 clone shows that genetic modifications to generate MSA180 - HA3 : loxP did not affect viability . Error bars , ± S . D ( B11 : n = 3 ; DMSO - RAP - treated MSA180 - HA3 : loxP : n = 6 each ) .

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O

(A) Replication of DMSO- and RAP-treated MSA180-HA3:loxP parasites over three erythrocytic cycles. Parasitaemia values are averages from replicates in different blood sources. The similar growth rate of DMSO-treated parasites and the parental B11 clone shows that the genetic modifications to generate MSA180-HA3:loxP parasites did not affect parasite viability. Error bars, ± S.D (B11: n=3; DMSO- and RAP-treated MSA180-HA3:loxP: n=6 each).


( B ) Stills from simultaneous time - lapse DIC microscopic examination of DMSO - RAP - treated MSA180 - HA3 : loxP cycle 0 schizonts following removal of C2 - arrest ( representative of 2 independent experiments ) . Note defect in RBCM rupture merozoite release in RAP - treated despite normal PVM rupture ( indicated by a sudden increased visibility motility of merozoites ) RBCM poration ( green ring indicating access binding of phalloidin to host RBC cytoskeleton ) . Scale bars , 5 μm .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O

(B) Stills from simultaneous time-lapse DIC and fluorescence microscopic examination of DMSO- and RAP-treated MSA180-HA3:loxP cycle 0 schizonts following removal of C2-arrest (representative of 2 independent experiments). Note the defect in RBCM rupture and merozoite release in RAP-treated parasites despite normal PVM rupture (indicated by a sudden increased visibility and motility of merozoites) and RBCM poration (green ring indicating access and binding of phalloidin to the host RBC cytoskeleton). Scale bars, 5 μm.


( D ) Time - course comparing fate of SERA6 from DMSO - RAP - treated MSA180 - HA3 : loxP ( representative of 2 independent experiments ) . Schizonts were sampled at indicated times following removal of C2 - arrest , or arrested with E64 - d ( 50 μM ) . RAP - treated SERA6 - Cys644Ala - myc3 [ SERA6 - mTAP : loxP ] were included in blot to allow comparison with processing of catalytically - dead SERA6 ( SERA6 - Cys644Ala - myc3 ) . Note accumulation of SERA6 p65 poor conversion to SERA6 p40 in RAP - treated ( ΔMSA180 ) .

O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O

(D) Time-course comparing fate of SERA6 from DMSO- and RAP-treated MSA180-HA3:loxP parasites (representative of 2 independent experiments). Schizonts were sampled at the indicated times following removal of C2-arrest, or arrested with E64-d (50 μM). RAP-treated SERA6-Cys644Ala-myc3 [SERA6-mTAP:loxP] parasites were included in the blot to allow comparison with processing of catalytically-dead SERA6 (SERA6-Cys644Ala-myc3). Note the accumulation of SERA6 p65 and poor conversion to SERA6 p40 in the RAP-treated (ΔMSA180) parasites.


( A ) Representative IFA demonstrating dynamic changes in localisation of SERA6 - mTAP during egress ( reproducible in 4 independent experiments ) . SERA6 - mTAP : loxP schizonts were sampled at indicated times following removal of C2 - arrest , fixed co - stained with DAPI ( blue ) , anti - HA ( red ) anti - ankyrin ( green ; ankyrin is a major component of RBC cytoskeleton ) . Scale bar , 5 μm .

O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O

(A) Representative IFA images demonstrating the dynamic changes in localisation of SERA6-mTAP during egress (reproducible in 4 independent experiments). SERA6-mTAP:loxP schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green; ankyrin is a major component of the RBC cytoskeleton). Scale bar, 5 μm.


( C ) Representative IFA demonstrating localisation of MSA180 - HA3 during egress . MSA180 - HA3 [ B11 ] schizonts were sampled at indicated times following removal of C2 - arrest , fixed co - stained with DAPI ( blue ) , anti - HA ( red ) anti - ankyrin ( green ) . Scale bar , 5 μm . See also Fig EV5 .

O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(C) Representative IFA images demonstrating localisation of MSA180-HA3 during egress. MSA180-HA3 [B11] schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green). Scale bar, 5 μm. See also Fig EV5.


( B ) E64 - d , MMV676881 Compound 31 inhibit RBCM rupture merozoite release despite normal PVM rupture , indicated by loss of a continuous ring of EXP2 - mNeon signal ( green ) RBCM poration ( red ring indicating access binding of phalloidin to host RBC cytoskeleton ) ( reproducible in 2 independent experiments ) . P . falciparum EXP2 - mNeon schizonts were allowed to reach point of egress in presence of C2 ( 1 µM ) , E64 - d ( 10 µM ) , MMV676881 ( 2 µM ) , Compound 31 ( 2 µM ) or DMSO ( 0 . 1 % v / v ; control ) then examined by DIC microscopy . Scale bar , 5 μm .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(B) E64-d, MMV676881 and Compound 31 inhibit RBCM rupture and merozoite release despite normal PVM rupture, indicated by loss of a continuous ring of EXP2-mNeon signal (green) and RBCM poration (red ring indicating access and binding of phalloidin to the host RBC cytoskeleton) (reproducible in 2 independent experiments). P. falciparum EXP2-mNeon schizonts were allowed to reach the point of egress in the presence of C2 (1 µM), E64-d (10 µM), MMV676881 (2 µM), Compound 31 (2 µM) or DMSO (0.1% v/v; control) then examined by DIC and fluorescence microscopy. Scale bar, 5 μm.


( C ) Western blot comparing dose - response of E64 - d , MMV676881 Compound 31 - mediated inhibition of maturation of SERA6 - mTAP from SERA6 - mTAP : loxP ( reproducible in 3 independent experiments ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(C) Western blot comparing dose-response of E64-d, MMV676881 and Compound 31-mediated inhibition of maturation of SERA6-mTAP from SERA6-mTAP:loxP parasites (reproducible in 3 independent experiments).


( C ) Immunoblots of TOMM34 expression in parental metformin adaptive HCC .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O B-DISEASE O

(C) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells.


( G ) ( Left ) , migration ( Middle ) invasion ( Right ) of metformin adaptive Huh7 with ( shTOM ) or without ( shNC ) knockdown of TOMM34 are shown ( 5 × 104 for Transwell ) . Scale bars , ( Left ) 25 μm , ( Middle ) ( Right ) 100 μm . ( n = 3 biological replicates , Two - way ANOVA ) . Data information : Data are presented as means ± S . D .

O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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) The morphology (Left), migration (Middle) and invasion (Right) of metformin adaptive Huh7 cells with (shTOM) or without (shNC) knockdown of TOMM34 are shown (5×104 cells for Transwell assay). Scale bars, (Left) 25 μm, (Middle) and (Right) 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.


( I ) A tail vein injection model was established using adaptive Huh7 to assess migratory ability . Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 6 mice for each group , Student ' s t - ) . Data information : Data are presented as means ± S . D .

O O O O B-TISSUE I-TISSUE O O O O O O B-CELL_LINE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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) A tail vein injection model was established using adaptive Huh7 cells to assess cell migratory ability. Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=6 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


effects of TOMM34 on HCC lung metastasis in tail vein injection mouse model ( shTOM : shTOMM34 , TOMM34 knockdown ; Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 5 mice for each group , Student ' s t - ) . Data information : Data are presented as means ± S . D .

O O B-GENEPROD O B-DISEASE B-TISSUE B-EXP_ASSAY O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model (shTOM: shTOMM34, TOMM34 knockdown cells; Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


effects of TOMM34 on HCC lung metastasis in tail vein injection mouse model OE - TOM : OE - TOMM34 , TOMM34 overexpressed ) . Scale bars , ( Left ) 1000 μm , ( Right ) 25 μm . ( n = 5 mice for each group , Student ' s t - ) . Data information : Data are presented as means ± S . D .

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The effects of TOMM34 on HCC lung metastasis in the tail vein injection mouse model OE-TOM: OE-TOMM34, TOMM34 overexpressed cells). Scale bars, (Left) 1000 μm, (Right) 25 μm. (n=5 mice for each group, Student's t-test). Data information: Data are presented as means ± S.D.


( L ) Western blot showing effects of TOMM34 on expression of EMT markers in HCC ( shTOM : shTOMM34 , TOMM34 knockdown ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed ) .

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(L) Western blot analysis showing the effects of TOMM34 on the expression of EMT markers in HCC cells (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( M ) expression of TOMM34 in clinical HCC samples with or without metastasis . Scale bars , ( Left ) 100 μm , ( Right ) 25 μm . ( n = 33 in metastasis present group , n = 53 in metastasis absent group , Student ' s t - ) . Data information : Data are presented as means ± S . D .

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(M) The protein expression of TOMM34 in clinical HCC samples with or without metastasis. Scale bars, (Left) 100 μm, (Right) 25 μm. (n = 33 in metastasis present group, n = 53 in metastasis absent group, Student's t-test). Data information: Data are presented as means ± S.D.


( C ) Transmission electron microscopy ( ) of Huh7 shNC shTOM ( shTOMM34 ) . Yellow arrowheads indicate mitochondria , bule arrowheads indicate nucleus , red arrowheads indicate endoplasmic reticulum . Scale bars , ( Left ) 20 μm , ( Middle ) 4 μm , ( Right ) 1 μm . ( n = 5 biological replicates , One - way ANOVA ) . Data information : Data are presented as means ± S . D .

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(C) Transmission electron microscopy (TEM) of Huh7 shNC and shTOM (shTOMM34) cells. Yellow arrowheads indicate mitochondria, bule arrowheads indicate nucleus, red arrowheads indicate endoplasmic reticulum. Scale bars, (Left) 20 μm, (Middle) 4 μm, (Right) 1 μm. (n=5 biological replicates, One-way ANOVA). Data information: Data are presented as means ± S.D.


( G ) Western blot showing expression of main mitochondrial respiratory chain subunit ( shTOM : shTOMM34 , TOMM34 knockdown ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed ) .

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(G) Western blot analysis showing the protein expression of the main mitochondrial respiratory chain complex subunit (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).


( B ) Co - IP assays indicate interaction between exogenous TOMM34 ATP5B in HEK293T .

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(B) Co-IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells.


( C ) Co - IP assays indicate interaction between endogenous TOMM34 ATP5B in Huh7 HCC .

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(C) Co-IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells.


( E ) mRNA expression of ATP5B in indicated were determined by qPCR ( n = 3 technical replicates , Two - way ANOVA ) or immunoblotting ( shTOM : shTOMM34 , TOMM34 knockdown ; OE - TOM : OE - TOMM34 , TOMM34 - overexpressed ) . Data information : Data are presented as means ± S . D .

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(E) The mRNA and protein expression of ATP5B in indicated cells were determined by qPCR (n=3 technical replicates, Two-way ANOVA) or immunoblotting (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34-overexpressed cells). Data information: Data are presented as means ± S.D.


( F - G ) Western blot showing level of ATP5B in Huh7 ( F ) or PLC / PRF / 5 ( G ) treated with or without 10 mg / mL CHX for 12 hours or 25 μM MG132 for 8 hours . Relative grey value of ATP5B was calculated using Image J ( shTOM : shTOMM34 , TOMM34 knockdown ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed ; CHX , cyclohexane ) . ( n = 3 technical replicates , Student ' s t - ) . Data information : Data are presented as means ± S . D .

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(F-G) Western blot showing protein level of ATP5B in Huh7 (F) or PLC/PRF/5 (G) cells treated with or without 10 mg/mL CHX for 12 hours or 25 μM MG132 for 8 hours. Relative grey value of ATP5B was calculated using Image J (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells; CHX, cyclohexane). (n=3 technical replicates, Student's t-test). Data information: Data are presented as means ± S.D.


( H ) Tissue immunofluorescence showing co - localization of TOMM34 ATP5B at invasive front of clinical HCC tissues . Scale bars , 20 μm .

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(H) Tissue immunofluorescence assay showing the co-localization of TOMM34 and ATP5B at the invasive front of clinical HCC tissues. Scale bars, 20 μm.


( I ) A positive correlation between TOMM34 ATP5B in human HCC samples was shown by representative immunohistochemical Scale bars , 50 μm . ( n = 72 ) .

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(I) A positive correlation between TOMM34 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars, 50 μm. (n=72).