Fig 2: Metabolite analysis of SLC25A11 knockdown cancer cells revealed that the MAS is responsible for ATP production in cancer cells. (a, b) Targeted LC-MS/MS metabolite analysis in UACC62 (a) and A549 (b) cells treated with siRNA against SLC25A11 (40 nM) for 24 h. Metabolite levels were measured in triplicate and normalized by BCA assay. (c) Mitochondrial malate levels were measured in SLC25A11-knockdown A549 and UACC62 cells using a mitochondrial fractionation kit and malate assay kit. (d) Mitochondrial NADH levels were measured in SLC25A11-knockdown A549 and UACC62 cells using a mitochondrial fractionation kit and NADH assay kit. Western blot confirming the isolation of mitochondria from cytosol with MDH1 (cytosol marker) and CV-ATP5A (mitochondria marker) antibodies. (Data were presented as mean ± SD. ***p < .001, **p < .01, *p < .05).

Fig 3: Expression and distribution of SLC25A11 in lung cancer and malignant melanoma tissue samples. (a) Tissue microarray was performed to determine the expression of SLC25A11 in normal lung tissues and lung cancer samples. A-1: Normal lung tissue showing no staining (×200; scale bar, 200 µm); A-2: Weak, A-3: Moderate, and A-4: Strong expression of SLC25A11 in lung cancer samples (×400; scale bar, 60 µm). (b) Percent distribution (graph) and SLC25A11 expression patterns in normal and cancer samples (Table) according to histologic subtypes. Statistical significance compared with normal lung tissues was determined by Fisher's exact test. (c) The expression patterns of SLC25A11 in malignant melanoma were determined by IHC. A-1: Weak, A-2: Moderate, A-3: Strong (×400; scale bar, 60 µm). (d) Percent SLC25A11 expression in normal skin and malignant melanoma samples. (Statistical significance was calculated by Fisher's Exact test. ***p < .001, **p < .01, *p < .05).

Fig 4: Genetic loss of SLC25A11 suppressed Kras-mediated lung tumorigenesis. (a) Generation of Slc25a11 knockout mice. A mixture of Cas9 protein and two guide RNAs (gRNA) was injected into the cytoplasm of mouse pronuclei. Mutations in mice were identified by TA cloning and sequencing. A 7 nt deletion in exon 2 of Slc25a11 causes premature translation termination. (b) Representative photomicrographs of tumor nodules detected by haematoxylin and eosin (H&E) and CK-19 staining (scale bar, 5 mm). (c) Quantitative analysis of tumor nodule number and tumor area showed a statistically significant decrease in these parameters in KRASLA2/SLC25A11+/- mice compared with KRASLA2 mice. (Data were analyzed statistically by two-way analysis of variance (ANOVA) tests using GraphPad PRISM 5 ***p < .001, **p < .01, *p < .05).

Fig 5: Knockdown of SLC25A11 inhibited protein translation by inactivating eIF4B. (a) Western blot was performed against phosphorylated mTOR, p70S6K, eIF4B and c-Myc in SLC25A11 knockdown cells and quantification was performed by ImageJ software. (b) Immunofluorescence was performed against eIF4B and c-Myc expression in SLC25A11 knockdown cells and the intensity was analyzed by ZEN software. (scale bar, 20 µm). (c) The indicated cells were treated with SLC25A11 siRNA for 48 h followed by 15 min incubation with puromycin (2 µg/ml). Cell lysates were subjected to immunoblotting using anti-puromycin antibody (SUnSET assay). (d) Possible regulation pathway of SLC25A11. (Data were presented as mean ± SD. ***p < .001, **p < .01, *p < .05).