Fig. 6.

Mechanistic study of CPT1A regulating the proliferation and apoptosis of CRC cells. (A) Gene expression of CPT1A and its association with patient lymph node metastasis (left panel) and Kaplan-Meier curve survival prognosis analysis (right patients) of colorectal cancer patients (based on TCGA PAN CANCER ATLAS data), N₀ indicates no lymph node metastasis (n = 339), N₁ indicates 1–3 lymph node metastases (n = 141), N₂ indicates 4 or more lymph node metastases (n = 110); (B) Western blot showing CPT1A protein levels in different colorectal cancer cells; (C) Correlation analysis of CPT1A protein levels and 3D tumor spheroid area; and (D) sensitivity to DHP-B (left panel) or ETO (,right panel) in different cell lines. The confidence interval (blue scale) was calculated using Pearson's correlation with confidence interval under the “ggscatter” function in the “ggplot2” package of Rstudio. Data are mean from five independent experiments; (E) Isotope tracing experiment with [U–¹³C]-palmitic acid (or [U–¹³C]-myristic acid) in SW 620 cells with CPT1A knockdown, showing the ratio of labeled (m+1∼m + n) to unlabeled (m+0) long-chain acylcarnitines; and the relative abundance of unlabeled and U-[¹³C]-labeled long-chain acylcarnitines; (F) Nile red staining combined with fluorescence confocal microscopy to observe the accumulation of intracellular fatty acids in SW620 cells before and after CPT1A knockdown (left panel), statistical results are the ratio of fluorescence intensity in the unloading phase to that in the loading phase (right panel, n = 5). (G–H) Oxygen consumption rates (OCR) of SW620 CPT1A knockdown cells assayed with 180 μM BSA-conjugated palmitate (G) under basal conditions (H, left panel) and in response to FCCP (H, right panel). Results for maximal OCR are expressed as a percent increase in OCR relative to BSA-treated cells. Graphs show mean ± SEM, n = 5. (I) Flow cytometry detection of mitochondrial permeability transition pore detection results before and after SW620 CPT1A knockdown, using the percentage of relative fluorescence intensity of Calcein AM detected (compared to CoCl₂ group or DMSO group) 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 or mentioned otherwise; 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.)