Fig. 3.

DHP-B affects fatty acid oxidation and mitochondrial permeability in colorectal cancer cells. (A) GSEA enrichment analysis of the RNA-seq data of SW620 cells treated with DHP-B, showing the significantly enriched pathways and NES values (P < 0.001); (B) GO enrichment analysis of the RNA-seq data of SW620 cells treated with DHP-B, showing the significantly enriched terms and -Log₁₀P values; (C) Kaplan-Meier survival curves of the transcriptomic expression level of fatty acid oxidation pathway (GOBP_FATTY_ACID_BETA_OXIDATION) and the survival of clinical colorectal cancer patients (TCGA, Firehouse Legacy). The high-expression group (orange) and low-expression group (blue) were divided based on the median value of expressions. The difference between the survival curves was statistically analyzed by log-rank test. (D) Nile red staining and fluorescence confocal microscopy of intracellular fatty acids in SW620 cells in different treatment groups (CTR: DMSO, DHP-B: 5 μM, ETO: 100 μM). The loading phase is the stage of incubation with palmitic acid for 24 h, and the unloading phase is the nutrient restriction stage of culturing with low glucose DMEM medium for 48 h. The ratio of fluorescence intensity in the unloading phase to that in the loading phase is shown; (E) Schematic diagram of the isotope tracing experiment with [U–¹³C]-palmitic acid (or [U–¹³C]-myristic acid); (F) U-[¹³C]-isotopic labeling of long-chain acylcarnitines in SW620 cells. The ratio of labeled (m+1∼m + n) to unlabeled (m+0) long-chain acylcarnitines in SW620 cells treated with different drugs; and the relative abundance of unlabeled (¹²C) and labeled (¹³C) long-chain acylcarnitines are shown. Data are median ± IQR from ten random fields per sample. (G) Oxygen consumption rates (OCR) of SW620 cells treated with different drugs assayed with 180 μM BSA-conjugated palmitate (PA, left panel) under basal conditions (middle panel) and in response to FCCP (right panel). Results for maximal OCR are expressed as a percent increase in OCR relative to BSA-treated cells (BSA). Data are mean ± SEM from five independent experiments; (H) Oil red staining and quantification of positive area (quantified by Image J 1.5.3 software) in frozen tumor sections from different treatment groups, ten random fields per sample were counted and averaged; (I) Flow cytometry analysis and quantification of mitochondrial permeability transition pore opening in different drug treatment groups, Iono is an abbreviation for Ionomycin, Co²⁺ is an abbreviation for CoCl₂, and the relative fluorescence intensity of Calcein AM detected (compared to CTR) percentage to evaluate the permeability of mitochondria after drug treatment. All of the studies above were examined using at least five biological replicates, and the results were expressed as mean ± SD（if different, it has been mentioned before）; NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, upaired t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)