Additional file 1 of CircCDYL2 bolsters radiotherapy resistance in nasopharyngeal carcinoma by promoting RAD51 translation initiation for enhanced homologous recombination repair
Creators: name: Qu, Hongke
name: Wang, Yumin
name: Yan, Qijia
name: Fan, Chunmei
name: Zhang, Xiangyan
name: Wang, Dan
name: Guo, Can
name: Chen, Pan
name: Shi, Lei
name: Liao, Qianjin
name: Zhou, Ming
name: Wang, Fuyan
name: Zeng, Zhaoyang
name: Xiang, Bo
name: Xiong, Wei
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 1 Supplemental Fig. 1. circCDYL2 promotes radiotherapy resistance in nasopharyngeal carcinoma cells. A. Clonogenic assays demonstrate that CNE2-IR cells exhibited higher radiotherapy resistance, compared with CNE2 cells, after exposure to 2 Gy radiation. B. The expression of circCDYL2 was detected by RT-qPCR in CNE2 and CNE2-IR cells. C. Transfection efficiency was evaluated using RT-qPCR in HNE2 and CNE2 cells after overexpression or knockdown of circCDYL2. D. The levels of γ-H2AX expression were detected by immunofluorescence in HNE2 and CNE2 cells after overexpression or knockdown of circCDYL2 with 6 Gy irradiation for 2, 6, 12, and 24 hours. The results showed that overexpression of circCDYL2 reduced the accumulation of radiation-induced foci (IRIF) of γ-H2AX post-irradiation, while circCDYL2 knockdown had the opposite effect. Scale bar = 10 μm. The right graph represents the number of radiation-induced foci (IRIF) in 30 cells. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 2. circCDYL2 promotes homologous recombination repair. A. Schematic diagram of the HR and NHEJ repair pathways. B. Efficiency of BRCA1 and 53BP1 knockdown was determined by RT-qPCR and western blotting in DR-GFP-U2OS and EJ5-GFP-U2OS cells. C. The impact of overexpression or knockdown of circCDYL2 on HR and NHEJ repair efficiency was detected by Flow cytometry. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 3. circCDYL2 does not influence the expression of BRCA1, 53BP1, or KU70. A. Immunofluorescence showed that circCDYL2 did not affect the accumulation of BRCA1 IRIF in NPC cells after overexpression or knockdown of circCDYL2. Scale bar = 10 μm. On the right, quantification of IRIF per 30 cells was presented. B. Immunofluorescence showed that circCDYL2 did not affect the accumulation of RPA1 IRIF in NPC cells after overexpression or knockdown of circCDYL2. Scale bar = 10 μm. On the right, quantification of IRIF per 30 cells was presented. C. The expression of DNA repair-related proteins BRCA1, RPA1, and Ku70 was detected by western blotting in NPC cells after overexpression or knockdown of circCDYL2. The results showed that circCDYL2 did not affect their expression. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 4. circCDYL2 does not influence the stability of RAD51. A. RT-qPCR showed that circCDYL2 did not affect the mRNA level of RAD51 in NPC cells after overexpression or knockdown of circCDYL2. B. RT-qPCR showed that circCDYL2 did not affect the stability of RAD51 mRNA in NPC cells after overexpression or knockdown of circCDYL2. Cells were treated with actinomycin D for 0, 0.5, 1, or 2 hours, respectively. C, D. Western blotting demonstrated that circCDYL2 did not affect the half-life and ubiquitination degradation of RAD51 in NPC cells after overexpression or knockdown of circCDYL2. Cells were treated with cycloheximide (CHX) (50 μg/ml) or MG132 (20 μM) for 0, 6, 12, or 24 hours, respectively. E. RNA pull-down revealed that circCDYL2 did not interact with RAD51 protein. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 5. circCDYL2 enhances RAD51 translation to promote DNA homologous recombination repair in nasopharyngeal carcinoma. A. Polysome profiling demonstrated that GAPDH mRNA expression on polysomes was not affected by circCDYL2 in NPC cells after overexpression or knockdown of circCDYL2. Data were represented as mean ± SD. B. Flow cytometry showed that RAD51 could partially reverse the effect of circCDYL2 on homologous recombination repair in DR-GFP U2OS cells after simultaneous knockdown of circCDYL2 with overexpression of RAD51 or overexpression of circCDYL2 combined with knockdown of RAD51, respectively. Each group was co-transfected with the HA-Isce1 overexpression plasmid. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 6. circCDYL2 promotes radiotherapy resistance in nasopharyngeal carcinoma by upregulation of RAD51. A. Clonogenic assays showed that RAD51 partially reversed the effect of circCDYL2 on CNE2 cell survival post-irradiation after simultaneous overexpression of circCDYL2 combined with knockdown of RAD51 or knockdown of circCDYL2 with overexpression of RAD51, respectively. These cells were exposed to 0, 2, 4, 6, and 8 Gy X-ray irradiation, respectively. B. Immunofluorescence showed that RAD51 could partially reverse the regulation of γ-H2AX radiation-induced foci (IRIF) by circCDYL2 in CNE2 cells after simultaneous overexpression of circCDYL2 combined with knockdown of RAD51 or knockdown of circCDYL2 with overexpression of RAD51, respectively. These cells were irradiated with 6 Gy X-rays for 6 hours. Scale bar = 10 μm. C. Western blotting demonstrated that RAD51 could partially reverse the regulation of γ-H2AX expression levels by circCDYL2 in NPC cells after simultaneous overexpression of circCDYL2 combined with knockdown of RAD51 or knockdown of circCDYL2 with overexpression of RAD51, respectively. These cells were irradiated with 6 Gy X-rays for 0, 2, 6, 12, or 24 hours, respectively. D. Comet assays showed that RAD51 mediated the effect of circCDYL2 on DNA damage repair in CNE2 cells after simultaneous overexpression of circCDYL2 combined with knockdown of RAD51 or knockdown of circCDYL2 with overexpression of RAD51, respectively. These cells were irradiated with 6 Gy X-rays, and. Scale bar = 20 μm. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 7. circCDYL2 does not influence the expression of CDYL2. A. The expression of CDYL2 was analyzed in NPC GEO datasets (GSE12452, GSE53819, GSE64634, and GSE61218). B. The expression of CDYL2 mRNA was detected by RT-qPCR in HNE2 and CNE2 cells after overexpression or knockdown of circCDYL2. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 8. circCDYL2 interacts with EIF3D but does not regulate its expression. A. The biotin-labeled circCDYL2 probe was used for RNA pulldown, and LC-MS/MS was used to identify proteins interacting with circCDYL2 in CNE2 cells. Pathways regulated by circCDYL2 were enriched using the GO database. B. Translation initiation factors, including EIF4G1, EIF3D, EIF3L, EIF2A, EIF2AK2, EIF4G2, and EIF3C, were ranked based on their score values from mass spectrometry data. C. RT-qPCR showed that circCDYL2 did not regulate the expression of EIF3D mRNA in HNE2 and CNE2 cells after overexpression or knockdown of circCDYL2. D. Western blotting demonstrated that circCDYL2 did not regulate the expression of EIF3D protein in HNE2 and CNE2 cells after overexpression or knockdown of circCDYL2. E. The transfection efficiency of EIF3D was detected by RT-qPCR in NPC cells after overexpression or knockdown of EIF3D. F. The transfection efficiency of EIF3D was detected by western blotting in NPC cells after overexpression or knockdown of EIF3D. Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Fig. 9. circCDYL2 interacts with both EIF3D protein and RAD51 mRNA. A. The binding site of circCDYL2 to EIF3D protein was predicted by the catRAPID software. B. The binding site of circCDYL2 to RAD51 mRNA was predicted by the RNA hybrid software. C. Two mutants of circCDYL2 (DEL1 and DEL2) were designed according to the secondary structure of circCDYL2 indicated by the RNA fold software. D. Schematic diagram of the deleted regions of the circCDYL2 mutants. E. The transfection efficiency of circCDYL2 was examined by qRT-PCR in HNE2 and CNE2 cells after overexpression of circCDYL2 or its deletion mutants (DEL1 and DEL2). Data were represented as mean ± SD. ns, not significant; *, p &lt; 0.05; **, p &lt; 0.01; ***, p &lt; 0.001. Supplemental Table 1. Clinicopathological data of 45 NPC and 23 NPE tissues measured by RT-qPCR. Supplemental Table 2. Clinicopathological data of 203 paraffin-embedded NPC tissues and 56 non-neoplastic nasopharyngeal epithelial tissues for in situ hybridization. Supplemental Table 3. Primers, probes, and siRNA used in this study. Supplemental Table 4. Antibodies used in this study. Supplemental Table 5. Potential circCDYL2 interacting proteins in HNE2 cells using the LC-MS/MS method after pulldown by the biotin-labeled circCDYL2 probe. Supplemental Table 6. Translation initiation factors according to the LC-MS/MS data.