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 .

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

O 3H-Glutamine uptake in MeWo cells transfected with si-GCN2 and treated 48 hr later with LDHAi for 6 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


P Gln 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 .

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

P Gln levels in the medium after treatment of MeWo cells with mock- or si-GCN2 for 24 hr and subsequent incubation with LDHA-i for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


Q GC - MS - 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


A GC - MS - 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 .

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

A GC-MS-based quantification of intracellular Asp (upper) and Ser (lower) levels in melanoma cell lines treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


B ( upper ) GC - MS - 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 .

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

B (upper) GC-MS-based 13C6-glucose fractional isotope labeling of Ser and Asp in melanoma cells treated for 72 hr with LDHAi. (lower) GC-MS-based 13C5-glutamine fractional isotope labeling of Asp in melanoma cell lines treated for 72 hr with LDHAi. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - 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 .

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

GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in DMEM (C) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - 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 .

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

GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells cultured in MEM supplemented with Ser (400 μM; D) and treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


GC - MS - 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 .

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

GC-MS-based quantification of intracellular Ser and Asp levels in MeWo cells treated with LDHAi for 1 hr Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


F GC - MS - 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 .

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

F GC-MS-based quantification of intracellular Asp levels in MeWo cells pre-treated with Asp (10 mM) for 6 hr and then treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


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

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

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


H Immunoblotting of ATF4 in MeWo 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-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O

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


A Relative NAD + / NADH 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 .

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

A Relative NAD+/NADH ratio in MeWo cells treated with LDHAi, nicotinamide mononucleotide (NMN, 1 mM), or a combination of the two for 24 hr. Cells were pretreated with nicotinamide mononucleotide for 6 hr and then treated with LDHAi for 24 hr in the combination setting. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


B GC - MS - 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 .

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

B GC-MS-based quantification of total intracellular levels of Ser and Asp and their 13C6-glucose fractional isotope labeling in MeWo cells pretreated with nicotinamide mononucleotide (NMN, 1 mM) for 6 hr and then treated with LDHAi for 24 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


D GC - MS - 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 .

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D GC-MS-based quantification of intracellular Ala levels in MeWo cells treated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


E GC - MS - 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 .

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

E GC-MS-based 13C6-Glc (M+2) and 13C5-Gln (M+4) isotopologue levels of the key TCA cycle metabolites and Asp in MeWo cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


F GC - MS - 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 .

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

F GC-MS-based 13C6-Glc (left) and 13C5-Gln (right) isotopologue levels of Asp in MeWo cells treated with LDHAi for 72 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


G GC - MS - 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 .

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

G GC-MS-based quantification of intracellular Ala (left) and Asp (right) levels in si-GPT2-treated MeWo cells incubated with LDHAi for 1 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups and unpaired t-test for the comparison of two groups. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001


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H Immunoblotting of ATF4 and GPT2 in MeWo cells


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

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

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


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

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


C Athymic nude mice were injected subcutaneously with A375 melanoma 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 .

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

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


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

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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 ) .

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

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 .

O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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

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


A qRT - PCR 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 .

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

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


GC - MS - 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 .

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

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


GC - MS - 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 .

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

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


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

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

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


E , F 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 O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O

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


( A ) Top : SERA6 architecture . Positions of 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 O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

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


( C ) Giemsa - stained thin films 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 O B-SMALL_MOLECULE O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

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


( F ) Representative IFA 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 .

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

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


( G ) Western blots showing MSA180 is a predominantly soluble PV 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 O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O

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


( A ) Replication of DMSO - 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 ) .

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

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


( B ) Stills from simultaneous time - lapse DIC 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 .

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

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


( D ) Time - course comparing fate of SERA6 from DMSO - 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 O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O

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


( A ) Representative IFA 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 .

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

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


( C ) Representative IFA 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 O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

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


( B ) E64 - d , MMV676881 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 .

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

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


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

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


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

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(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 .

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

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


( I ) A tail vein injection model was established using adaptive Huh7 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 .

O O O O B-TISSUE I-TISSUE O O O O O O B-CELL_LINE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

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


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 .

O O O B-GENEPROD O B-DISEASE B-TISSUE B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

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


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 .

O O O B-GENEPROD O B-DISEASE B-TISSUE B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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


( M ) 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 .

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


( C ) Transmission electron microscopy ( 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 .

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


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


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

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(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 .

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(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 .

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


( F - G ) Western blot showing 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 .

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


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

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


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

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


( B - C ) Western blot showing the expression of indicated proteins in adaptive HCC cells with or without ATP5B silencing .

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(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 .

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(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 .

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(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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(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.


( 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 .

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

(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 ) .

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(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 .

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

(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.


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

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-GENEPROD O

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


( 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

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

(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 .

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(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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

Steady-state protein levels of strains as described in (A) cultivated on YP media containing glycerol


Steady - 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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Steady-state protein levels of strains as described in (A) cultivated on YP media containing CM media with galactose as carbon source (D, E).


( A ) Growth analysis of strains expressing the wild type form of Cor1 ( Cor1WT ) or the mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . Growth curves and calculated doubling times from the exponential phase ( insert ) are presented . Cells were cultivated in media containing either glucose or glycerol as carbon source .

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(A) Growth analysis of strains expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Growth curves and calculated doubling times from the exponential phase (insert) are presented. Cells were cultivated in media containing either glucose or glycerol as carbon source.


( B ) Determination of chronological lifespan via propidium iodide ( PI ) staining of cells described above .

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(B) Determination of chronological lifespan via propidium iodide (PI) staining of cells described above.


( D , E ) Analysis of competitive fitness as described in ( C ) . Cor1WT strains harbored a chromosomally - integrated hygromycin selection cassette , whereas Cor1 * * contained a clonNAT selection cassette ( D ) . Subsequently , selection markers were switched as a control ( E ) .

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(D,E) Analysis of competitive fitness as described in (C). Cor1WT strains harbored a chromosomally-integrated hygromycin selection cassette, whereas Cor1** contained a clonNAT selection cassette (D). Subsequently, selection markers were switched as a control (E).


( A , B ) Spectrophotometric measurement of Cyt c oxidase ( CIV ; A ) and NADH Cyt c reductase ( CIII ; B ) activities in solubilized mitochondrial extracts . Mitochondria were isolated from cells expressing the wild type form of Cor1 ( Cor1WT ) or the mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) .

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(A,B) Spectrophotometric measurement of Cyt c oxidase (CIV; A) and NADH Cyt c reductase (CIII; B) activities in solubilized mitochondrial extracts. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**).


( C , D ) Polarographic measurement of KCN - sensitive oxygen consumption , driven by NADH ( C ) or succinate + glycerol - 3 - phosphate ( G3P ; D ) in isolated coupled mitochondria of indicated strains . Measurement was performed in the absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP .

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(C,D) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH (C) or succinate + glycerol-3-phosphate (G3P; D) in isolated coupled mitochondria of indicated strains. Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.


( E ) Respiratory control ratio calculated from polarographic measurements of KCN - sensitive oxygen consumption driven by NADH and succinate + glycerol - 3 - phosphate ( G3P ) in isolated coupled mitochondria of indicated strains .

(E) Respiratory control ratio calculated from polarographic measurements of KCN-sensitive oxygen consumption driven by NADH and succinate + glycerol-3-phosphate (G3P) in isolated coupled mitochondria of indicated strains.


( F , G ) Polarographic measurement of KCN - sensitive oxygen consumption driven by NADH in mitoplasts in the absence ( endog . Cyt c ) or presence ( + exog . Cyt c ) of exogenous oxidized Cyt c . NADH oxidation ( E ) and the calculated ratio + Cyt c / - Cyt c of substrate oxidation ( F ) are visualized .

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(F,G) Polarographic measurement of KCN-sensitive oxygen consumption driven by NADH in mitoplasts in the absence (endog. Cyt c) or presence (+ exog. Cyt c) of exogenous oxidized Cyt c. NADH oxidation (E) and the calculated ratio +Cyt c/- Cyt c of substrate oxidation (F) are visualized.


( A ) Polarographic measurement of KCN - sensitive oxygen consumption , driven by NADH in isolated coupled mitochondria . Mitochondria were isolated from cells expressing the wild type form of Cor1 ( Cor1WT ) or the mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . Thereby , strains either overexpressed cytochrome c ( Cyt cOE ) or harbored the empty plasmid as a control ( Empty ) . Measurement was performed in the absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP .

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(A) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH in isolated coupled mitochondria. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Thereby, strains either overexpressed cytochrome c (Cyt cOE) or harbored the empty plasmid as a control (Empty). Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.


Analysis of competitive fitness of strains described in ( A ) . Cor1WT strains harbored a chromosomally - integrated clonNAT selection cassette , whereas Cor1 * * contained a hygromycin selection cassette . Strains were cultivated in CM media containing glucose ( B ) as carbon source .

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Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing glucose (B) as carbon source.


Analysis of competitive fitness of strains described in ( A ) . Cor1WT strains harbored a chromosomally - integrated clonNAT selection cassette , whereas Cor1 * * contained a hygromycin selection cassette . Strains were cultivated in CM media containing galactose ( C ) or glycerol ( D ) as carbon source .

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Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing galactose (C) or glycerol (D) as carbon source.


A In vitro differentiation to mature ookinete of wildtype ( WT ) , Δisp1 , Δisp3 , and Δisp1 / 3 parasites . Values are means ± SD ( n = 4 biological replicates ) ; two - tailed t test , * , P < 0 . 05 , * * , P < 0 . 01 , * * * , P < 0 . 001 , ns : not significant .

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A In vitro differentiation to mature ookinete of wildtype (WT), Δisp1, Δisp3, and Δisp1/3 parasites. Values are means ± SD (n=4 biological replicates); two-tailed t test, *, P<0.05, **, P<0.01, ***, P<0.001, ns: not significant.


Giemsa staining of zygotes / ookinetes from in vitro culture . Upper diagrams indicate morphological changes from zygote to ookinete . Black arrow indicates the apical . Scale bar = 5 μm .

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Giemsa staining of zygotes/ookinetes from in vitro culture. Upper diagrams indicate morphological changes from zygote to ookinete. Black arrow indicates the apical. Scale bar = 5 μm.


C Time - course analysis of ookinete differentiation from in vitro culture . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * * , P < 0 . 001 .

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C Time-course analysis of ookinete differentiation from in vitro culture. Values are means ± SEM (n=3 biological replicates), two-tailed t test, ***, P<0.001.


D In vivo ookinete differentiation in midgut of the infected mosquitos . Right panel indicates the parasite smear stained with Giemsa solution . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 . Scale bar = 5 μm .

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D In vivo ookinete differentiation in midgut of the infected mosquitos. Right panel indicates the parasite smear stained with Giemsa solution. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01. Scale bar = 5 μm.


E Number of oocysts in mosquito midgut 7 days post blood feeding . n is the number of mosquitoes in each group . The horizontal line shows the mean value of each group , Mann - Whitney test , * * * * , P < 0 . 0001 . Right panel shows mosquito midguts stained with 0 . 5 % mercurochrome . Scale bar = 50 μm . Three biological replicates performed .

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E Number of oocysts in mosquito midgut 7 days post blood feeding. n is the number of mosquitoes in each group. The horizontal line shows the mean value of each group, Mann-Whitney test, ****, P<0.0001. Right panel shows mosquito midguts stained with 0.5% mercurochrome. Scale bar = 50 μm. Three biological replicates performed.


F Formation of salivary gland sporozoites in mosquitoes 14 days post blood feeding . In each group , at least twenty blood - fed mosquitoes were counted . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 .

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F Formation of salivary gland sporozoites in mosquitoes 14 days post blood feeding. In each group, at least twenty blood-fed mosquitoes were counted. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01.


G Western blot of ISP1 and ISP3 in zygote of the Δisp1 / 3 parasites episomally complemented with 3V5 - tagged isp1 or 6HA - tagged isp3 genes from either P . yoelii or P . falciparum . P28 as loading control . The DTS ( isp1 : : 3V5 ; isp3 : : 6HA ) is a doubly - tagged

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G Western blot of ISP1 and ISP3 in zygote of the Δisp1/3 parasites episomally complemented with 3V5-tagged isp1 or 6HA-tagged isp3 genes from either P. yoelii or P. falciparum. P28 as loading control. The DTS (isp1::3V5;isp3::6HA) is a doubly-tagged parasite


H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites . Nuclei are stained with Hoechst 33342 . Scale bar = 5 μm .

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H IFA analysis of tagged ISP1 and ISP3 in zygotes of the complemented parasites. Nuclei are stained with Hoechst 33342. Scale bar = 5 μm.


I In vitro ookinete differentiation of the complemented parasites . Values are means ± SEM ( n = 3 biological replicates ) , two - tailed t test , * * , P < 0 . 01 , * * * , P < 0 . 001 .

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I In vitro ookinete differentiation of the complemented parasites. Values are means ± SEM (n=3 biological replicates), two-tailed t test, **, P<0.01, ***, P<0.001.


IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1 : : 6HA and isp3 : : 6HA parasites , respectively . Scale bar = 5 μm .

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IFA of ISP1 and ISP3 expression from zygote to ookinete of the isp1::6HA and isp3::6HA parasites, respectively. Scale bar = 5 μm.


B Co - localization of ISP1 and ISP3 from zygote to ookinete in the doubly - tagged strain ( DTS , isp1 : : 3V5 ; isp3 : : 6HA ) . Scale bar = 5 μm .

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B Co-localization of ISP1 and ISP3 from zygote to ookinete in the doubly-tagged strain (DTS, isp1::3V5;isp3::6HA). Scale bar = 5 μm.


C Western blot of ISP1 and ISP3 expression in zygotes of isp1 : : 6HA and isp3 : : 6HA parasites . The numbers are the relative intensities of band in blot . Right panel : the quantification of band intensity . Values are means ± SEM ( n = 4 biological replicates ) , two - tailed t test , * * * * , P < 0 . 0001 .

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C Western blot of ISP1 and ISP3 expression in zygotes of isp1::6HA and isp3::6HA parasites. The numbers are the relative intensities of band in blot. Right panel: the quantification of band intensity. Values are means ± SEM (n=4 biological replicates), two-tailed t test, ****, P<0.0001.


D Western blot of ISP1 and ISP3 expression in zygotes of DTS , DTS / Δisp1 , and DTS / Δisp3 parasites . The numbers are the relative intensities of band in blot . Three replicates performed .

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D Western blot of ISP1 and ISP3 expression in zygotes of DTS, DTS/Δisp1, and DTS/Δisp3 parasites. The numbers are the relative intensities of band in blot. Three replicates performed.


E RT - qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT , Δisp1 , Δisp3 , and Δisp1 / 3 parasites . The expression is normalized to the 18s rRNA . Values are means ± SEM ( n = 4 biological replicates ) .

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E RT-qPCR analysis of isp1 and isp3 transcripts in zygotes of the WT, Δisp1, Δisp3, and Δisp1/3 parasites. The expression is normalized to the 18s rRNA. Values are means ± SEM (n=4 biological replicates).


F IFA analysis of ISP1 and ISP3 expression dynamic of DTS / Δisp1 and DTS / Δisp3 parasites . Scale bar = 5 μm .

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F IFA analysis of ISP1 and ISP3 expression dynamic of DTS/Δisp1 and DTS/Δisp3 parasites. Scale bar = 5 μm.


B Fluorescent microscopy of ISP and its N - terminal 20 residues ( N20 ) fused with a BFP : : 3V5 peptide episomally expressed in zygotes . Scale bar = 5 μm .

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B Fluorescent microscopy of ISP and its N-terminal 20 residues (N20) fused with a BFP::3V5 peptide episomally expressed in zygotes. Scale bar = 5 μm.


C Solubility assay detecting membrane association of ISP1 _ FL and ISP1 _ N20 using different detergents . Cytosolic soluble proteins are in hypotonic buffer ( Hypo ) , peripheral membrane proteins in carbonate buffer ( Carb ) ， integral membrane proteins in Triton X - 100 buffer ( Trx ) , and insoluble proteins in pellet ( P ) . BFP : : 3V5 is in the soluble fraction , while ISP1 _ FL and ISP1 _ N20 are in the membrane - associated fractions . ER protein BiP is a loading control .

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C Solubility assay detecting membrane association of ISP1_FL and ISP1_N20 using different detergents. Cytosolic soluble proteins are in hypotonic buffer (Hypo), peripheral membrane proteins in carbonate buffer (Carb)，integral membrane proteins in Triton X-100 buffer (Trx), and insoluble proteins in pellet (P). BFP::3V5 is in the soluble fraction, while ISP1_FL and ISP1_N20 are in the membrane-associated fractions. ER protein BiP is a loading control.


D Acyl - RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain . Proteins from F ( flow - through ) , W ( wash ) , and E ( elution ) are analyzed . P28 as a loading control .

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D Acyl-RAC method detecting palmitoylation of ISP1 and ISP3 in zygotes of the DTS strain. Proteins from F (flow-through), W (wash), and E (elution) are analyzed. P28 as a loading control.


E Substitutions of N - terminal cysteine or glycine residues to alanine ( G2A or C7A / C8A ) ablates palmitoylation of ISP1 . ISP1 was fused with BFP : : 3V5 and episomally expressed in zygotes . Right panel shows quantification of palmitoylated protein band from two independent repeats , values are means ± SD .

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E Substitutions of N-terminal cysteine or glycine residues to alanine (G2A or C7A/C8A) ablates palmitoylation of ISP1. ISP1 was fused with BFP::3V5 and episomally expressed in zygotes. Right panel shows quantification of palmitoylated protein band from two independent repeats, values are means ± SD.


F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation . Scale bar = 5 μm .

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F G2A and C7AC8A mutations cause cytoplasmic distribution of ISP1 during zygote to ookinete differentiation. Scale bar = 5 μm.


G Membrane association of ISP1 with either G2A or C7AC8A substitutions .

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G Membrane association of ISP1 with either G2A or C7AC8A substitutions.


H Expression and palmitoylation of ISP1 in ∆ isp1 / 3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant .

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H Expression and palmitoylation of ISP1 in ∆isp1/3 parasites complemented with the 3V5 tagged WT ISP1 or C7AC8A mutant.


I IFA of ISP1 at zygote of the complemented parasites . Scale bar = 5 μm .

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I IFA of ISP1 at zygote of the complemented parasites. Scale bar = 5 μm.


In vitro differentiation to mature ookinete of the complemented parasites . Values are means ± SEM ( n = 4 replicates ) , two - tailed t test , * * * , P < 0 . 001 , ns : not significant .

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In vitro differentiation to mature ookinete of the complemented parasites. Values are means ± SEM (n=4 replicates), two-tailed t test, ***, P<0.001, ns: not significant.


A Two - colored IFA of ISP1 / DHHC2 and ISP3 / DHHC2 at zygotes of the triple tagged strain isp1 : : 3V5 / isp3 : : 6HA / dhhc2 : : 4Myc ( TTS ) . Right graph shows overlay of fluorescence peaks along cell periphery . Scale bar = 5 μm .

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A Two-colored IFA of ISP1/DHHC2 and ISP3/DHHC2 at zygotes of the triple tagged strain isp1::3V5/isp3::6HA/dhhc2::4Myc (TTS). Right graph shows overlay of fluorescence peaks along cell periphery. Scale bar = 5 μm.


B Western blot of DHHC2 , ISP1 , and P28 ( plasma membrane protein ) of the TTS zygotes treated with PBS , trypsin ( Try ) , or heat - inactivated ( HI ) trypsin . Upper panel shows the PPM and IMC structures .

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B Western blot of DHHC2, ISP1, and P28 (plasma membrane protein) of the TTS zygotes treated with PBS, trypsin (Try), or heat-inactivated (HI) trypsin. Upper panel shows the PPM and IMC structures.


C Knockdown of dhhc2 expression . Upper panel shows the promoter swap strategy applied in TTS strain , generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter . Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes . Three replicates performed .

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C Knockdown of dhhc2 expression. Upper panel shows the promoter swap strategy applied in TTS strain, generating the dhhc2kd mutant with endogenous dhhc2 promoter replaced with clag promoter. Western blot confirming decreased expression of DHHC2 in dhhc2kd zygotes. Three replicates performed.


D Two - colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes . Scale bar = 5 μm . Bottom panel , quantifications of DHHC2 fluorescence . Values are means ± SD ( n : the number of cells analyzed ) . Mann - Whitney test , * * * * , P < 0 . 0001 .

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D Two-colored IFA of DHHC2 and P28 proteins in the TTS and dhhc2kd zygotes. Scale bar = 5 μm. Bottom panel, quantifications of DHHC2 fluorescence. Values are means ± SD (n: the number of cells analyzed). Mann-Whitney test, ****, P<0.0001.


E Western blot of DHHC2 , ISP1 and ISP3 in the TTS and dhhc2kd zygotes . Two replicates performed .

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E Western blot of DHHC2, ISP1 and ISP3 in the TTS and dhhc2kd zygotes. Two replicates performed.


F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl - RAC method . NH : NH2OH . Right panel indicates quantification of protein band intensity from three replicates , values are means ± SD .

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F Palmitoylation of ISP1 and ISP3 in the TTS and dhhc2kd zygotes using Acyl-RAC method. NH: NH2OH. Right panel indicates quantification of protein band intensity from three replicates, values are means ± SD.


G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd . Scale bar = 5 μm .

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G IFA of ISP1 and ISP3 in zygotes of TTS and dhhc2kd. Scale bar = 5 μm.


H Giemsa staining of zygote / ookinete from in vitro cultured TTS and dhhc2kd parasites . Scale bar = 5 μm .

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H Giemsa staining of zygote/ookinete from in vitro cultured TTS and dhhc2kd parasites. Scale bar = 5 μm.


I Time - course analysis of in vitro ookinete differentiation . Values are means ± SD from three independent tests .

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I Time-course analysis of in vitro ookinete differentiation. Values are means ± SD from three independent tests.


J IFA of DHHC2 expression in the dhhc2 : : 6HA and dhhc2 : : 6HA ; ∆ isp1 / 3 zygotes . Scale bar = 5 μm .

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J IFA of DHHC2 expression in the dhhc2::6HA and dhhc2::6HA;∆isp1/3 zygotes. Scale bar = 5 μm.


K Western blot of DHHC2 in zygotes of dhhc2 : : 6HA and dhhc2 : : 6HA ; ∆ isp1 / 3 .

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K Western blot of DHHC2 in zygotes of dhhc2::6HA and dhhc2::6HA;∆isp1/3.


L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P . yoelii , PfDHHC2 of P . falciparum , or PyDHHC2 with PAT catalytic deficient mutation C128A . Scale bar = 5 μm .

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L IFA of DHHC2 and ISP1 in zygotes of dhhc2kd parasites complemented with 4Myc tagged WT PyDHHC2 of P. yoelii, PfDHHC2 of P. falciparum, or PyDHHC2 with PAT catalytic deficient mutation C128A. Scale bar = 5 μm.


M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated .

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M Western blot of DHHC2 in the complemented dhhc2kd parasites as indicated.


N In vitro differentiation to mature ookinetes of the complemented parasites . Values are means ± SD from three independent tests . two - tailed t test , ns : not significant .

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N In vitro differentiation to mature ookinetes of the complemented parasites. Values are means ±SD from three independent tests. two-tailed t test, ns: not significant.


Western blot of episomally expressed DHHC2 : : 6HA or variants containing truncated segment ( C1 : 210 - 232 , C2 : 233 - 262 , C3 : 263 - 284 residues ) in WT zygotes .

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Western blot of episomally expressed DHHC2::6HA or variants containing truncated segment (C1:210-232, C2:233-262, C3:263-284 residues) in WT zygotes.


C IFA of episomally expressed DHHC2 : : 6HA and three truncation mutants in zygotes and retorts . Scale bar = 5 μm .

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C IFA of episomally expressed DHHC2::6HA and three truncation mutants in zygotes and retorts. Scale bar = 5 μm.


D Acyl - RAC method detecting palmitoylation of endogenous DHHC2 in the dhhc2 : : 6HA zygotes .

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D Acyl-RAC method detecting palmitoylation of endogenous DHHC2 in the dhhc2::6HA zygotes.


E Palmitoylation of DHHC2 : : 6HA and C4A mutant episomally expressed in zygotes . C4A : substitutions of C - terminal four cysteine to alanine . Right panel indicates the quantification of protein band intensity from two replicates , values are means ± SD .

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E Palmitoylation of DHHC2::6HA and C4A mutant episomally expressed in zygotes. C4A: substitutions of C-terminal four cysteine to alanine. Right panel indicates the quantification of protein band intensity from two replicates, values are means ± SD.


F Cytoplasmic distribution of C4A mutant during zygote to ookinete differentiation . Scale bar = 5 μm .

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F Cytoplasmic distribution of C4A mutant during zygote to ookinete differentiation. Scale bar = 5 μm.


G IFA of DHHC2 at the dhhc2 : : 6HA zygotes treated with 100 μΜ 2 - BP . Scale bar = 5 μm .

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G IFA of DHHC2 at the dhhc2::6HA zygotes treated with 100 μΜ 2-BP. Scale bar = 5 μm.


H In vitro ookinete differentiation of the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant . Values are means ± SD from three independent tests .

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H In vitro ookinete differentiation of the dhhc2kd parasites complemented with either WT DHHC2 or C4A mutant. Values are means ± SD from three independent tests.


I Palmitoylation of the episomally expressed DHHC2 : : 6HA with mutation in respective individual cysteine ( C255A , C258A , C260A , and C262A ) . Representative of three independent repeats .

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I Palmitoylation of the episomally expressed DHHC2::6HA with mutation in respective individual cysteine (C255A, C258A, C260A, and C262A). Representative of three independent repeats.


J Palmitoylation of HA - tagged and human codon optimized DHHC2 ectopically expressed in human HEK293T cells . Both C4A and C128A mutations ablate DHHC2 palmitoylation .

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J Palmitoylation of HA-tagged and human codon optimized DHHC2 ectopically expressed in human HEK293T cells. Both C4A and C128A mutations ablate DHHC2 palmitoylation.


K DHHC2 _ C128A - HA mutant protein could be palmitoylated by the co - transfected DHHC2WT - Myc , but not DHHC2 _ C128A - Myc in HEK293T cells . Representative of two independent repeats .

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K DHHC2_C128A-HA mutant protein could be palmitoylated by the co-transfected DHHC2WT-Myc, but not DHHC2_C128A-Myc in HEK293T cells. Representative of two independent repeats.


L Co - immunoprecipitation of the DHHC2 _ WT - HA and DHHC2 _ WT - Myc co - expressed in HEK293T cells .

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L Co-immunoprecipitation of the DHHC2_WT-HA and DHHC2_WT-Myc co-expressed in HEK293T cells.


M C - terminus 75 residues peptide ( Cter75 ) of DHHC2 could be palmitoylated as an independent substrate by co - transfected DHHC2WT - HA in HEK293T cells . Cter75 is fused with a BFP : : Myc for easy detection via western blot . Cter75 * peptide contains C4A mutation .

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M C-terminus 75 residues peptide (Cter75) of DHHC2 could be palmitoylated as an independent substrate by co-transfected DHHC2WT-HA in HEK293T cells. Cter75 is fused with a BFP::Myc for easy detection via western blot. Cter75* peptide contains C4A mutation.


N Palmitoylation of tagged DHHC2 ectopically expressed in bacteria E . coli .

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N Palmitoylation of tagged DHHC2 ectopically expressed in bacteria E.coli.