Fig 2: Lipoate synthesis is dispensable under low oxygen tensions.A, volcano plots depicting log2 fold changes and corresponding FDR for all proteins in FDX1 and LIAS KO samples compared with controls in normoxia (21% O2) and hypoxia (1% O2). FDX1 and LIAS are shown in blue, gray horizontal line denotes FDR = 0.01. B, immunoblots for FDX1, LIAS, lipoylated PDH and KGDH, E2 subunit proteins of PDH (DLAT) and KGDH (DLST) enzyme complexes, and control proteins ACTIN and TOM20 on lysates of HepG2 cells edited with control (CTRL), FDX1, or LIAS guides and grown in 21% or 1% O2. Double asterisk indicates band of interest. C, immunoblots for GDF15, ASNS, GLRX2, and control proteins ACTIN and TOM20 on lysates of HepG2 cells edited with control (CTRL), FDX1, or LIAS guides and grown in 21% or 1% O2. D, three-day proliferation assay of HepG2 cells edited with control (CTRL), FDX1, or LIAS guides and grown in 21% or 1% O2. E, KGDH activity assayed by a KGDH enzyme activity kit using HepG2 cells edited with control, FDX1, or LIAS guides and grown in 21% or 1% O2. F, bar plot displaying fold change in oxygen consumption rate (OCR) as assessed via permeabilized cell seahorse assays run at 21% and 1% O2 on HepG2 cells edited with control (CTRL), FDX1, or LIAS guides. Oxygen consumption rates following injection of a-KG were normalized to baseline readings per well. Dashed line indicates normalized baseline. G, YSI measured glucose consumption over a 3-day period in HepG2 cells edited with control (CTRL), FDX1, or LIAS guides and grown in 21% or 1% O2, normalized to final cell count on day 3. H, YSI measured lactate production over a 3-day period in HepG2 cells edited with control (CTRL), FDX1, or LIAS guides and grown in 21% or 1% O2, normalized to final cell count on day 3. All bar plots show mean ± SD of three independent experiments. ns = p > 0.05, *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001. Two-way ANOVA with Bonferroni’s post-test. a-KG, a-ketoglutarate; FDR, false discovery rate; KGDH, a-ketoglutarate dehydrogenase; LIAS, lipoyl synthase; PDH, pyruvate dehydrogenase.

Fig 3: Expression and clinical significance of CPRMs in CRC. A. The boxplot shows the mRNA expression levels of 16 CPRMs between the normal and tumor CRC samples in GTEX and TCGA-COREAD cohorts. T-test: * P < 0.05, * * P < 0.01, * ** P < 0.001. B. The radar charts show the mRNA expression levels of 16 CPRMs in different CRC cell lines in CCLE datasets. C. The waterfall diagram shows the mutation frequency of 16 CPRMs in 538 patients with colorectal cancer in the TCGA-COREAD cohorts. D. The vertical lollipop chart shows the frequencies of CNV gain, loss, and non-CNV among CPRMs in the TCGA-COREAD cohorts. E. The doughnut chart shows the locations of CNV alterations in CPRMs on 23 chromosomes. F. The forest map shows the results of Univariate regression analysis on the average survival rate of 16 CPRMs in the TCGA-COREAD cohorts. G. The Kaplan-Meier curves show disease-specific survival, overall survival, and progression-free interval of CDKN2A and DLAT in the TCGA-COREAD cohorts using the log-rank test. H. The boxplots show the relationship between mRNA expression levels of CPRMs and the chemotherapy, targeted therapy or immunotherapy resistance in CRC individuals. T-test: * P < 0.05, * * P < 0.01, * ** P < 0.001. I. The thermogram shows the correlation between mRNA expression levels of 16 CPRMs and TME infiltrating cells or common molecular biology pathways in CRC individuals. Spearman correlation analysis: * P < 0.05, * * P < 0.01, * ** P < 0.001.