Fig 1.
Study design and repeatability analyses.
(A) Workflow plot of the study design. (B) Cell viability was measured by trypan blue exclusion assay at different time points after hypoxia treatment. Images (left) and quantitative data (right) are presented, n.s. stands for not significant. (C) PCA plot of HL1 cells in response to hypoxia treatment for different time. (D) Distribution of identified peptide length. (E) The distribution of mass error indicates a high accuracy of modified peptide data obtained from MS. (F) Spearman’s correlation analysis showed the correlation of differentially expressed proteins (log2 ratio) between biological replicates.
Fig 2.
DEP analyses and gene ontology classification.
(A) Differentially expressed protein (DEP) annotation. (B) Histogram of gene ontology classification. The results are summarized in three main categories: biological process, cellular component and molecular function. The right y-axis indicates the number of genes in a category. The left y-axis indicates the percentage of a specific category of genes in that main category.
Fig 3.
Visualization of significantly enriched GO terms in HL1 in response to hypoxia.
A) DEPs were enriched according to biological processes (A), cellular components (B), and molecular functions (C). DEPs involved in oxidoreductase activity are presented (D).
Fig 4.
A treemap overview of significant GO biological processes in hypoxia treated HL1 cells.
The diagram shows that HL1 cells exposed to prolonged hypoxic conditions (72 h vs. 48 h) had a significant effect on proteins associated with mitochondria, the mitochondrial inner membrane, and the oxidoreductase complex. The relative sizes of the treemap boxes are based on the |log10(p value)| of the respective GO term, related terms are visualized with the same color, the color represents the significant p value of this kind of term after -log10.
Fig 5.
KEGG enrichment and DEP representation for significant pathways.
A) KEGG enrichment-based clustering for DEPs between hypoxia treatment for 48 h and 72 h. DEPs involved in fatty acid metabolism (B), the TCA cycle (C), and oxidative phosphorylation (D) are presented.
Fig 6.
Hypoxia maintains cell growth by activating the degradation of fatty acids.
HL1 cells were treated under hypoxia for 72 h. The whole cell lysates were subjected to WB analysis (A), fatty acids measurement (B) and immunofluorescent analysis (C). The protein levels of CD36 on the cell membrane or in the total cell were quantified on the base of immunofluorescent analysis (D), A.U. stands for arbitrary unit. The membrane/total ratio of CD36 were calculated (E). HL1 cells were culture in medium supplemented with oleic acid treated with hypoxia for 72 h, and followed by measuring the concentration of fatty acids (F). HL1 cells, which were infected with lentivirus encoding shRNA targeting CPT1A or ECI 1. Cells were cultured in medium supplemented with oleic acid treated with hypoxia for 72 h. The whole cell lysates were subjected to WB analysis (G) or RTCA analysis (H).