Figure 1A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK - 2837808A ( 10 μM ) for the indicated duration under hypoxic ( 1 % O2 ) and normoxic conditions . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . In ( A ) statistical comparison for the treatments that showed a significant change . B ( left ) Immunoblotting of LDHA in melanoma cell lines transfected with non - targeting siRNA ( NT - siRNA ) or LDHA - specific siRNA for 72 hr . ( right ) Proliferation of melanoma cell lines transfected with NT - siRNA or LDHA - specific siRNA for the indicated timepoints . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . In ( B ) , statistical comparison for the treatments that showed a significant change . Immunoblotting of LDHA of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . proliferation under normoxic ( D ) conditions of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . proliferation under hypoxic conditions of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data information : Statistical analysis was performed by two - way Anova for time - dependent proliferation changes and by one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . statistical significance is denoted by blue ( for si / sh - LDHA # 1 , hypoxia ) and black ( for sish - LDHA # 2 , hypoxia ) stars respectively . G Anchorage - independent growth of MeWo and A375 cells stably transduced with empty vector ( EV ) or LDHA - specific shRNAs . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Proliferation of melanoma cell lines treated with LDHA inhibitor GSK-2837808A (10 μM) for the indicated duration under hypoxic (1% O2) and normoxic conditions. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (A) statistical comparison for the treatments that showed a significant change.B (left) Immunoblotting of LDHA in melanoma cell lines transfected with non-targeting siRNA (NT-siRNA) or LDHA-specific siRNA for 72 hr. (right) Proliferation of melanoma cell lines transfected with NT-siRNA or LDHA-specific siRNA for the indicated timepoints. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. In (B), statistical comparison for the treatments that showed a significant change.Immunoblotting of LDHA of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs.proliferation under normoxic (D) conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.proliferation under hypoxic conditions of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data information: Statistical analysis was performed by two-way Anova for time-dependent proliferation changes and by one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. statistical significance is denoted by blue (for si/sh-LDHA#1, hypoxia) and black (for sish-LDHA#2, hypoxia) stars respectively.G Anchorage-independent growth of MeWo and A375 cells stably transduced with empty vector (EV) or LDHA-specific shRNAs. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Figure 2B , C Glucose ( B ) and glutamine ( Gln , C ) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . D Medium Gln levels after transfection of melanoma cells with non - targeting ( NT ) or LDHA - specific siRNA for 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . E GC - MS - based quantification of intracellular Gln levels in cells Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . F qRT - PCR analysis of expression of the indicated SLC family transporters in MeWo ( left ) and A375 ( right ) melanoma cells after treatment with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . G Immunoblotting of SLC1A5 in melanoma cells treated with LDHAi for 96 hr . H Immunoblotting of LDHA and SLC1A5 in melanoma cell lines transfected with NT - siRNA or indicated si - LDHA for 96 hr . I Immunoblotting of SLC1A5 and LDHA in MeWo cells stably transduced with empty vector ( EV ) or four different LDHA - specific shRNAs . J Immunoblotting of SLC1A5 in MeWo cells mock ( NT - siRNA + DMSO ) or si - SLC1A5 - treated for 24 hr and then treated with LDHAi for 48 hr . K Medium Gln levels ( left ) , intracellular Gln levels ( right ) of MeWo cells Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . L Proliferation of melanoma cell lines for the indicated timepoints following treatment with LDHA - i , si - SLC1A5 or both . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . two - way Anova was used for the proliferation . N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . M ( left ) Long - term viability of melanoma cells treated as indicated for 8 days . ( right ) corresponding quantification showing change in viability . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . N Immunoblotting of SLC1A5 and cleaved caspase - 3 in melanoma cells subjected to mock ( NT - siRNA + DMSO ) or si - SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Unpaired t - test was used for the comparison of two groups , N . D . , not determined . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B, C Glucose (B) and glutamine (Gln, C) levels in the medium after treatment of melanoma cell lines with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.D Medium Gln levels after transfection of melanoma cells with non-targeting (NT) or LDHA-specific siRNA for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.E GC-MS-based quantification of intracellular Gln levels in cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.F qRT-PCR analysis of expression of the indicated SLC family transporters in MeWo (left) and A375 (right) melanoma cells after treatment with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.G Immunoblotting of SLC1A5 in melanoma cells treated with LDHAi for 96 hr.H Immunoblotting of LDHA and SLC1A5 in melanoma cell lines transfected with NT-siRNA or indicated si-LDHA for 96 hr.I Immunoblotting of SLC1A5 and LDHA in MeWo cells stably transduced with empty vector (EV) or four different LDHA-specific shRNAs.J Immunoblotting of SLC1A5 in MeWo cells mock (NT-siRNA + DMSO) or si-SLC1A5-treated for 24 hr and then treated with LDHAi for 48 hr.K Medium Gln levels (left), intracellular Gln levels (right) of MeWo cells Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.L Proliferation of melanoma cell lines for the indicated timepoints following treatment with LDHA-i, si-SLC1A5 or both. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. two-way Anova was used for the proliferation. N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.M (left) Long-term viability of melanoma cells treated as indicated for 8 days. (right) corresponding quantification showing change in viability. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.N Immunoblotting of SLC1A5 and cleaved caspase-3 in melanoma cells subjected to mock (NT-siRNA + DMSO) or si-SLC1A5 treatment for 24 hr followed by exposure to LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups, N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Figure 3GC - 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 . 0001B 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 . 0001C ( 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 . 0001Immunoblotting 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 . 0001Immunoblotting 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 . 0001Immunoblotting 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 . 0001H 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 . 0001I ( 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 . 0001J Immunoblotting of cleaved caspase - 3 and p - ( S780 ) - Rb in melanoma cells treated as indicated for 72 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3GC-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 ≤ 0.0001B 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 ≤ 0.0001C (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 ≤ 0.0001Immunoblotting 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 ≤ 0.0001Immunoblotting 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 ≤ 0.0001Immunoblotting 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 ≤ 0.0001H 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 ≤ 0.0001I (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 ≤ 0.0001J 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


Figure 4Immunoblotting of ATF4 in melanoma cell lines ( A ) treated with the LDHA inhibitor LDHAi for 96 hr Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Immunoblotting of ATF4 in melanoma cell lines ( B ) transfected with non - targeting ( NT ) or LDHA - specific siRNAs for 96 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . C Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector ( EV ) or indicated sh - LDHAs . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . D Immunoblotting of ATF4 , c - MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2 , 24 , or 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . E Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock - or si - ATF4 - treated for 24 hr and then incubated with LDHAi for 48 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . F qRT - PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si - ATF4 for 24 hr and then incubated with LDHAi for 24 hr . Data information : Statistical analysis was performed by ordinary one - way Anova for the comparison of more than two groups . Data are shown as the mean ± SD , n = 3 . ns : not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001GC - 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 . 0001H 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 . 0001qRT - 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 . 0001GC - 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 . 0001GC - 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 . 0001GC - 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 . 0001M ( 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 . 0001N 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 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 . 0001P 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 . 0001Q 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immunoblotting of ATF4 in melanoma cell lines (A) treated with the LDHA inhibitor LDHAi for 96 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.Immunoblotting of ATF4 in melanoma cell lines (B) transfected with non-targeting (NT) or LDHA-specific siRNAs for 96 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.C Immunoblotting of ATF4 in MeWo cells stably transduced with empty vector (EV) or indicated sh-LDHAs. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.D Immunoblotting of ATF4, c-MYC and SLC1A5 in melanoma cell lines treated with LDHAi for 2, 24, or 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.E Immunoblotting of ATF4 and SLC1A5 in melanoma cell lines mock- or si-ATF4-treated for 24 hr and then incubated with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups.F qRT-PCR analysis of SLC1A5 transcript levels in melanoma cell lines transfected with si-ATF4 for 24 hr and then incubated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001GC-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 ≤ 0.0001H 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 ≤ 0.0001qRT-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 ≤ 0.0001GC-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 ≤ 0.0001GC-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 ≤ 0.0001GC-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 ≤ 0.0001M (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 ≤ 0.0001N 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 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 ≤ 0.0001P 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 ≤ 0.0001Q 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


Figure 5A 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 . 0001B ( 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 . 0001GC - 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 . 0001GC - 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 . 0001GC - 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 . 0001F 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 . 0001G 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A 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 ≤ 0.0001B (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 ≤ 0.0001GC-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 ≤ 0.0001GC-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 ≤ 0.0001GC-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 ≤ 0.0001F 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 ≤ 0.0001G 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 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).


Figure 6A 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 . 0001B 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 . 0001D 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 . 0001E 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 . 0001F 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 . 0001G 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 . 0001H Immunoblotting of ATF4 and GPT2 in MeWo

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O

Figure 6A 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 ≤ 0.0001B 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 ≤ 0.0001D 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 ≤ 0.0001E 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 ≤ 0.0001F 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 ≤ 0.0001G 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 ≤ 0.0001H Immunoblotting of ATF4 and GPT2 in MeWo cells


Figure 7A ( 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 cells ( n = 3 ) treated with Dox ( 0 . 5 μgC 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 the indicated proteins . Representative images of H & E and cleaved caspase - 3 staining from each treatment group ( E ) . 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 - 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A (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 cells (n=3) treated with Dox (0.5 μgC 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 the indicated proteins.Representative images of H&E and cleaved caspase-3 staining from each treatment group (E).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-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 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.


Figure 8A 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 - 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 - 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 the indicated proteins in A375 cells treated with PLX - 4032 ( 1 μM ) for 2 , 6 , or 24 hr . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A 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-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-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 the indicated proteins in A375 cells treated with PLX-4032 (1 μM) for 2, 6, or 24 hr.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.


Figure 1 ( 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 / ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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/).


Figure 2 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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.


Figure 4 ( 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 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 - 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 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- 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.


Figure 5 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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 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.


Figure 6 ( 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 and Compound 31 - mediated inhibition of maturation of SERA6 - mTAP from SERA6 - mTAP : loxP parasites ( reproducible in 3 independent experiments ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 and Compound 31-mediated inhibition of maturation of SERA6-mTAP from SERA6-mTAP:loxP parasites (reproducible in 3 independent experiments).


Figure 2 ( C ) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells . ( 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 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 . 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 . 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 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) Immunoblots analysis of TOMM34 expression in parental and metformin adaptive HCC cells.(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 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.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.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 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) 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.


Figure 3 ( 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 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 ) .

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Figure 3(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 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).


Figure 4 ( B ) Co - IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells . ( C ) Co - IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells . ( 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 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 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 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars , 50 μm . ( n = 72 ) .

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Figure 4(B) Co-IP assays indicate the interaction between exogenous TOMM34 and ATP5B in HEK293T cells.(C) Co-IP assays indicate the interaction between endogenous TOMM34 and ATP5B in Huh7 HCC cells.(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 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 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 and ATP5B levels in human HCC samples was shown by representative immunohistochemical images Scale bars, 50 μm. (n=72).


Figure 5 ( B - C ) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing . ( D - E ) Transwell assays showing the migration and invasion of adaptive HCC cells with or without the silence of ATP5B ( 5 × 104 adaptive Huh7 cells , 1 × 105 adaptive PLC / PRF / 5 cells ) , Scale bars , 100 μm . ( n = 3 biological replicates , Two - way ANOVA ) . Data information : Data are presented as means ± S . D . ( G ) Western blot showing the expression of EMT markers in cells with ( shTOM ) or without ( shNC ) knockdown of TOMM34 or overexpression of ATP5B . ( H ) Transwell assays showing the migration and invasion of indicated cells ( 5 × 104 cells ; shTOM , shTOMM34 , TOMM34 knockdown cells ) . Scale bars , 100 μm . ( n = 4 biological replicates , two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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Figure 5(B-C) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing.(D-E) Transwell assays showing the migration and invasion of adaptive HCC cells with or without the silence of ATP5B (5×104 adaptive Huh7 cells, 1×105 adaptive PLC/PRF/5 cells), Scale bars, 100 μm. (n=3 biological replicates, Two-way ANOVA). Data information: Data are presented as means ± S.D.(G) Western blot showing the expression of EMT markers in cells with (shTOM) or without (shNC) knockdown of TOMM34 or overexpression of ATP5B.(H) Transwell assays showing the migration and invasion of indicated cells (5×104 cells; shTOM, shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=4 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.


Figure 6 ( A - B ) Co - IP assay was performed to detect the interaction between TOMM34 and ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC / PRF / 5 cells . ( C - D ) Immunoblotting of EMT markers of HCC cells treated with or without 200 nM Gboxin for 24 hours ( shTOM : shTOMM34 , TOMM34 knockdown cells ; OE - TOM : OE - TOMM34 , TOMM34 overexpressed cells ) . ( E - F ) Transwell assay showing the migration and invasion of HCC cells treated with or without 200 nM Gboxin for 36 hours ( 5 × 104 cells ; shTOM : shTOMM34 , TOMM34 knockdown cells ) . Scale bars , 100 μm . ( n = 3 biological replicates , two - way ANOVA ) . Data information : Data are presented as means ± S . D .

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Figure 6(A-B) Co-IP assay was performed to detect the interaction between TOMM34 and ATP5B treated with or without Gboxin for 24 hours in parental or adaptive PLC/PRF/5 cells.(C-D) Immunoblotting of EMT markers of HCC cells treated with or without 200 nM Gboxin for 24 hours (shTOM: shTOMM34, TOMM34 knockdown cells; OE-TOM: OE-TOMM34, TOMM34 overexpressed cells).(E-F) Transwell assay showing the migration and invasion of HCC cells treated with or without 200 nM Gboxin for 36 hours (5×104 cells; shTOM: shTOMM34, TOMM34 knockdown cells). Scale bars, 100 μm. (n=3 biological replicates, two-way ANOVA). Data information: Data are presented as means ± S.D.


Figure 1 ( D ) Blue - native gel electrophoresis of S . cerevisiae strains with indicated mutations in Cor1 .

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Figure 1(D) Blue-native gel electrophoresis of S. cerevisiae strains with indicated mutations in Cor1.


Figure 2 ( A ) Blue - native gel electrophoresis and immunoblots of cells expressing the wild type form of Cor1 ( Cor1WT ) , as well as the mutants Cor1N63A , N187A , D192A ( Cor1 * ) and Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) in wild type ( WT ) background as well as in cells lacking CRD1 ( crd1Δ ) . Blots were probed with antibodies against Cor1 to visualize Complex III ( CIII ) , as well as Cox1 , to monitor Complex IV ( CIV ) . CV : Complex V ; Cor1Cor2 : multimer consisting of Cor1 and Cor2 ( B ) Reduced - minus - oxidized difference spectra of strains described in ( A ) . Heme peaks are indicated for Cor1WT in WT background . Steady - state protein levels of strains as described in ( A ) cultivated on YP media containing glycerolSteady - state protein levels of strains as described in ( A ) cultivated on YP media containing CM media with galactose as carbon source ( D , E ) .

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Figure 2(A) Blue-native gel electrophoresis and immunoblots of cells expressing the wild type form of Cor1 (Cor1WT), as well as the mutants Cor1N63A, N187A, D192A (Cor1*) and Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**) in wild type (WT) background as well as in cells lacking CRD1 (crd1Δ). Blots were probed with antibodies against Cor1 to visualize Complex III (CIII), as well as Cox1, to monitor Complex IV (CIV). CV: Complex V; Cor1Cor2: multimer consisting of Cor1 and Cor2(B) Reduced-minus-oxidized difference spectra of strains described in (A). Heme peaks are indicated for Cor1WT in WT background.Steady-state protein levels of strains as described in (A) cultivated on YP media containing glycerolSteady-state protein levels of strains as described in (A) cultivated on YP media containing CM media with galactose as carbon source (D, E).