Aberrant CPT1A mRNA level in CRC.

A. The expression of CPT1A in six GEO microarrays. B. Real-time PCR for CPT1A in 24-paired CRC and adjacent non-tumour tissues. C. Western blot for CPT1A in sixteen-paired CRC and adjacent non-tumour tissues. D. Lower CPT1A mRNA level in COAD than the normal counterparts from TCGA in ualcan database. E. Lower CPT1A mRNA level in READ than the normal counterparts from TCGA in ualcan database. F. IHC assay for CPT1A in two patients. ***, P < 0.001, ** P < 0.01, * P < 0.05.

Correlation of CPT1A with overall survival and neoadjuvant therapy response in rectal cancer patients.

A. IHC assay for CPT1A in two groups of patients, upper with low CPT1A expression and lower with high CPT1A expression (IHC score ≥ 6). B. The Overall survival (OS) was estimated by the Kaplan-Meier method in rectal cancer patients with low (n = 33) or high CPT1A expression (n = 43). C. The OS was estimated by the Kaplan-Meier method in rectal cancer patients in TCGA database with low (n = 46) or high CPT1A expression (n = 46). D. IHC assay for CPT1A in two groups of patients, upper with TRG-1 and lower with TRG-2 (TRG means tumour regression grade, AJCC standard, 0, complete response: No remaining viable cancer cells; 1, moderate response: Only small clusters or single cancer cells remaining; 2, minimal response: Residual cancer remaining, but with predominant fibrosis). E. Dot plot showing the IHC score and TRG score of patients. F. Correlation of CPT1A with TRG score, size of dot represents the number. ***, P < 0.001, ** P < 0.01, * P < 0.05.

Radio-sensitivity of stable knock-out or overexpression of CPT1A.

A. The protein level of CPT1A in HCT 116-NC, HCT 116-KO1, HCT 116-KO2, HCT 116-KO3, SW480-RFP, and SW480-OE cell lines. B. The mRNA level of CPT1A in HCT 116-NC, HCT 116-KO (KO2), SW480-RFP, and SW480-OE cell lines. C. Colony-forming assay of HCT 116-NC and HCT 116-KO cell lines. D. The map of multi-target, single-hit model. E. Colony-forming assay of SW480-RFP and SW480-OE cell lines. F. The map of multi-target, single-hit model. G. Comet assay of HCT 116-NC, HCT 116-KO, SW480-RFP, and SW480-OE. H. Protein expression of γ-H2A.X in HCT 116-NC and HCT 116-KO cell lines. I. Protein expression of γ-H2A.X in SW480-RFP and SW480-OE cell lines. ***, P < 0.001, ** P < 0.01, * P < 0.05.

CPT1A inhibited proliferation and radio-resistance in nude mice.

A. Image of tumours formed in nude mice, with knock out of CPT1A and radiation. B. Scattergram showing the weight of tumours. C. Immunohistochemical staining of Ki67 in tumours. D. Bar chart demonstrating the percentage of positively stained of Ki67 cells. E. Image of tumours formed in nude mice, with overexpression of CPT1A and radiation. F. Scattergram showing the weight of tumours. G. Immunohistochemical staining of Ki67 in tumours. H. Bar chart demonstrating the percentage of positively stained of Ki67 cells. ***, P < 0.001, ** P < 0.01, * P < 0.05.

The effect of CPT1A on ROS related enzyme activity.

A. Enriched KEGG pathway of DEGs in mRNA sequencing. B. Generation and scavenging of ROS in cell. C. ROS of HCT 116-KO cell, SW480-OE cell and their control with DCFH-DA by flow cytometry. D. Bar graph to show the mean of DCFH-DA in HCT 116-KO, HCT 116-NC cells, SW480-NC and SW480-OE cells. E. GSH / GSSG ratio measurement under CPT1A knockout and radiation. F. GSH / GSSG ratio measurement under CPT1A overexpression and radiation. G. Effect of the CPT1A knockout on SOD activity. H. Effect of the CPT1A overexpression on SOD activity. I. Effect of the CPT1A on CAT activity. ***, P < 0.001, ** P < 0.01, * P < 0.05.

CPT1A increases the transcription and protein of ROS scavenge related genes by regulating the transcription factor activity of FOXM1.

A.The protein level of FOXM1, CPT1A, CAT, SOD1, SOD2, SOD3 after knockout and overexpression of CPT1A. B. The mRNA level of FOXM1, CAT, SOD1, SOD2, SOD3 after knockout and overexpression of CPT1A. C. Veen map showing the potential transcription factor of SOD1, SOD2 and CAT. D. The protein level of FOXM1, CPT1A, CAT, SOD1, SOD2 after overexpression of FOXM1 in HCT116-CPT1AKO cells. E. Schematic diagram summarising our working model, namely, decreased CPT1A promotes the transcription factor activity of FOXM1, increasing the mRNA and protein level of CAT, SOD1, and SOD2, followed by increasing ROS scavenge after irradiation and therefore CRC cells become radioresistance. ***, P < 0.001, ** P < 0.01, * P < 0.05.

Potential binding site of SOD1, SOD2, CAT promoter predicted by hTFtarget and JASPAR.

Key Resources Table.

Correlation between clinicopathological features and the expression of CPT1A in tumor paraffin section

The level of CPT1A in different CRC cancer cell lines.

A. Colony-forming assay in different cell lines. B. The map of multi-target, single-hit model. C.The mRNA level of CPT1A in different cell lines. D. The protein level of CPT1A in different cell lines. ***, P < 0.001,

Establishment of radio-resistance cells and overexpression of CPT1A rescues the radiation resistance of HCT-15-25F cells.

A. Workflow of establishing of radio-resistance cells. F, fraction; W, week. B. Colony-forming assay in different cell lines. C. The map of multi-target, single-hit model. D.The protein level of CPT1A in radio-resistance cells. E. Western blot for CPT1A in HCT-15-25F cell line after transfected with overexpression lentivirus. F. Protein expression of γ-H2A.X in HCT-15-25F-NC and HCT-15-25F-OE cell lines. G. Colony-forming assay in different cell lines. H. The map of multi-target, single-hit model. ***, P < 0.001, ** P < 0.01, * P < 0.05.

The result of mRNA sequencing after CPT1A knock-out.

A. Heatmap of all genes. B. Volcanic map of all genes. C. Enriched GO terms of biological process. D. Enriched GO terms of cellular component. E. Enriched GO terms of molecular function.

The effect of CPT1A on ROS and enzyme activity in radio-resistance cell after 6Gy radiation.

A. ROS of HCT-15-25F and HCT-15 with DCFH-DA by flow cytometry. B. ROS of HCT-15-25F-OE cell and its control with DCFH-DA by flow cytometry. C. Bar graph to show the mean of DCFH-DA in HCT-15-25F and HCT-15 cells. D. Bar graph to show the mean of DCFH-DA in HCT-15-25F-OE and HCT-15-25F-NC cells. E. GSH / GSSG ratio after CPT1A overexpression. F. GSH / GSSG ratio after CPT1A overexpression and 6Gy radiation. G. Effect of the CPT1A overexpression on SOD activity. H. Effect of the CPT1A overexpression on SOD activity after 6Gy radiation. I. Effect of the CPT1A overexpression on POD activity. J. Effect of the CPT1A overexpression on POD activity after 6Gy radiation. K. Effect of the CPT1A on CAT activity. ***, P < 0.001, ** P < 0.01, * P < 0.05.

The correlation of FOXM1 with ROS scavenge gene.

A. The correlation of FOXM1 with SOD1. B. The correlation of FOXM1 with SOD2.