Fig 2: SPC25 upregulated CCND1 via its transcription factor E2F1. A. JASPAR database presented the binding motif of E2F1 and the binding sequence of E2F1 on CCND1 promoter. B. E2F1 bind to the promoters of CCND1 and overexpression of SPC25 promoted the binding of E2F1 to the promoter of CCND1 detected by ChIP-qPCR in Eca-109 cells. C. The dual-luciferase reporter assay was carried out with CCND1 promoter (wt or mut) after overexpression of E2F1. D. The dual-luciferase reporter assay was carried out with CCND1 promoter (wt) after overexpression of SPC25 with/without E2F1. **, P < 0.01.

Fig 3: Transcriptomic landscape of SPC25 targets. A. Scatterplot of differentially expressed genes and sample clustering analysis after shSPC25/shCtrl infection. Results show the expression levels of 1480 genes are observed up-regulated and 1876 genes are observed down-regulated. B. Heat map representation of the expression profiles of genes significantly changed after shSPC25/shCtrl infection. The expression level of each transcript is represented by the color range, red indicates high and green indicates low. C. The Ingenuity Pathway Analysis (IPA) analysis of classical pathways analysis. Yellow represented activation and blue represented inhibition D. The IPA analysis of relationship network between classical pathways and target genes. E. The expression of 17 hub genes was validated with RT-qPCR. F. Western blot analysis was performed to examine the protein level of CCND1, CDK6, c-Myc, PRKACB and SIRT1. Each bar in the statistical figures represents the mean ± SD from three independent experiments of at least three replicate wells. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 4: Knockdown of SPC25 restrained ESCC cell proliferation and migration, and regulated the cell cycle and apoptosis. A. The expression of SPC25 mRNA in different ESCC cell lines. B. The efficiency of lentiviral vectors to knockdown the expression of SPC25 in EC9706 and Eca-109 cells was examined by RT-qPCR. C. The efficiency of lentiviral vectors to knockdown the expression of SPC25 was examined by Western blot. D-E. Cell growth of EC9706 and Eca-109 cells after knockdown of SPC25 from day 1 to day 5. Cell growth curves was depicted according to Celigo calculation. F-G. Wound healing assay were assayed in EC9706 and Eca-109 cells with SPC25 knockdown by live cell imaging on an IncuCyte system. H-I. Assessment of invasion of EC9706 and Eca-109 cells with SPC25 knockdown using transwell assay (Original magnification: 200 ×). J-K. Cell cycle of EC9706 and Eca-109 cells with SPC25 knockdown by flow cytometry. L-M. Cell apoptosis of EC9706 and Eca-109 cells with SPC25 knockdown by flow cytometry. Each bar in the statistical figures represents the mean ± SD from three independent experiments of at least three replicate wells. *, P < 0.05, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 5: SPC25 facilitated tumorigenesis through CCND1 in ESCC in vivo. A. Tumor weight. B. Growth curves of tumors were measured weekly. C. Representative images of tumors of shCtrl (NC), CCND1 (OE), shSPC25 (KD) and CCND1+shSPC25 (OE+KD) group (n = 6). D. Immunohistochemistry (IHC) staining was performed to examine the protein levels of ki67 (Scale bar, 100 μm).