Additional file 2 of Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

Additional file 2: Fig. S1. CircRNF13 is lowly expression in NPC. A The top 20 circRNAs in the RNA-seq data were tested in NPC (n = 36) and NPE (n = 12) tissues using RT-PCR. NPE, non-tumor nasopharyngeal epithelial tissues. Data have been represented as mean ± standard deviation (SD). *, p < 0.05; **, p < 0.01; ****, p < 0.0001. B Expression of circRNF13 was verified using qPCR in NPC cell lines 5-8F, CNE2, HNE2,HONE1, and 6-10B and normal immortal nasopharyngeal epithelial NP69 cells. Fig. S2.CircRNF13 inhibits proliferation, migration, and invasion of NPC. The overexpression or knockdown efficiencies of circRNF13 were measured in HNE2 and CNE2 cells after transfection with circRNF13 overexpression vector or circRNF13 siRNAs. RNF13 mRNA expression was not affected by transfection. All experiments were repeated at least three times. Data have been represented as mean ± SD. ***, p <0.001. Fig. S3.CircRNF13 inhibits glycolysis in NPC cells. A The proteomics profile in CNE2 cells was analyzed upon circRNF13 knockdown. A total of 294 differentially expressed proteins between sicircRNF13 and scrambled siRNAs were screened, including 100 proteins upregulated and 194 proteins downregulated by sicircRNF13. B The glycolysis pathway was enriched according to the 294 differential proteins screened from the LC-MS/MS data, using gene set enrichment analysis with the IPA software. C The extracellular acidification rate (ECAR) in HNE2 and CNE2 cells was measured using Seahorse XF assays, in response to circRNF13 overexpression or knockdown. D Representative images of wound-healing assay showed that the glycolysis inhibitor 2-DG attenuated the effect of circRNF13 on NPC cell migration when HNE2 and CNE2 cells transfected with sicircRNF13 were treated with it. Fig. S4. SUMO2 inhibits proliferation, migration, invasion, and glycolysis in NPC cells. A The overexpression efficiency of SUMO2 was measured in HNE2 and CNE2 cells using RT-PCR and western blotting, after transfection of the SUMO2 overexpression vector. Data have been represented as mean ± SD. ***, p <0.001. B Overexpression of SUMO2 inhibited proliferation of HNE2 and CNE2 cells, as assessed using MTT assay. All experiments were repeated at least three times. Data have been represented as mean ± SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C The migration ability of HNE2 and CNE2 cells was significantly reduced upon overexpression of SUMO2, as assessed using wound-healing assays. All experiments were repeated at least three times. Data have been represented as mean ± SD. **, p < 0.01; ***, p < 0.001. D The invasive ability of HNE2 and CNE2 cells was significantly decreased upon overexpression of SUMO2, as assessed using Transwell assays. All experiments were repeated at least three times. Data have been represented as mean ± SD. ***, p < 0.001. E The extracellular acidification rate (ECAR) was measured using Seahorse XF assays upon overexpression of SUMO2 in HNE2 and CNE2 cells. Glycolysis, glycolytic capacity, and glycolytic reserve were analyzed. All experiments were repeated at least three times. Data have been represented as mean ± SD. **, p < 0.01. Fig. S5. SUMO2 promotes degradation of GLUT1. A The binding motifs for SUMOylation on the GLUT1 protein (K245 and K451) were predicted using GPS-SUMO 2.0 website. B Expression of GLUT1 protein was examined in HNE2 and CNE2 cells using western blotting, after overexpression of SUMO2. C Degradation of GLUT1 was detected in HNE2 and CNE2 cells using western blotting, after overexpression of SUMO2 and treatment with 50 μg/mL cycloheximide (CHX).