DOI: 10.6084/m9.figshare.24136301
title: Additional file 1 of Circ-RAPGEF5 promotes intrahepatic cholangiocarcinoma progression by stabilizing SAE1 to facilitate SUMOylation

description: Additional file 1: Table S1. Univariate and multivariate COX regression analysis of the 91 ICC patients. Table S2. Primers used in this study. Table S3. Antibodies and reagents used in this study. Table S4. FISH probes used in this study. Table S5. Biotinylated probes used in this study. Figure S1. The expression levels of Circ-RAPGEF5, SAE1 and miR-3185 in indicated cells. A-B qRT-PCR and western blot analysis detected the expression level of Circ-RAPGEF5 and liner RAPGEF5 in RBE cells after treatment with Si-Circ-RAPGEF5 or Si-NC. C-F The transfected efficiency of Si-Circ-RAPGEF5 and Circ-RAPGEF5 overexpression plasmid in RBE and CCLP1 cells. G-J The transfected efficiency of Si-SAE1 and SAE1 overexpression plasmid in RBE and CCLP1 cells. K-L the overexpression efficiency of miR-3184 mimic in RBE and CCLP1cells. M-N qRT-PCR analysis detected the Circ-RAPGEF5 expression of stably transfected Sh-Circ-RAPGEF5 and Circ-RAPGEF5 RBE cells. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S2. Circ-RAPGEF5 inhibits apoptosis and promotes migration in ICC cells. A-B Cell apoptosis analysis detected by flow cytometry in Circ-RAPGEF5 knockdown or overexpression cells. C-D The migration ability was assessed by transwell assay in Circ-RAPGEF5 knockdown or overexpression cells. All data are presented as the means ± SD of three independent experiments. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S3. A Differential expression of SAE1 ICC tumor tissues and adjacent normal tissues in TCGA data. B Kaplan-Meier survival curves of external sequencing data from Dong et al. C qRT-PCR analysis of the relative expression levels of SAE1 in xenografts tissue of groups treated with Sh-Circ-RAPGEF5 and Sh-NC. D Representative IHC images for SAE1 of Sh-NC and Sh-Circ-RAPGEF5 virus treated patient-derived tumor xenograft. E qRT-PCR verified the enrichment efficiency of the Circ-RAPGEF5-biotin probe. F-G qRT-PCR detecting relative SUMO expression in RBE and CCLP1 cells. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001. Figure S4. A-B The remaining two Western blot experiments accessed SAE1 protein level after changing the expression of Circ-RAPGEF5 in RBE cell. C-D The remaining two Western blot experiments accessed SAE1 protein level after changing the expression of Circ-RAPGEF5 in CCLP1 cell. Figure S5. A-B The remaining two Western blot experiments detecting SAE1 protein level in knockdown and overexpression of miR-3185 of RBE cell. C-D The remaining two Western blot experiments detecting SAE1 protein level in knockdown and overexpression of miR-3185 of CCLP1 cell. E-F The remaining two Western blot experiments detecting SAE1 protein level in RBE cell transfected with Si-NC or Si-Circ-RAPGEF5 and inhibitor-NC or inhibitor-miR-3185. G-H The remaining two Western blot experiments detecting SAE1 protein level in CCLP1 cell transfected with Si-NC or Si-Circ-RAPGEF5 and inhibitor-NC or inhibitor-miR-3185. Figure S6. A-B The remaining two Western blot experiments accessed the global SUMOylatoin level of Circ-RAPGEF5 silencing and upregulated in RBE cells. C-D The remaining two Western blot experiments accessed the global SUMOylatoin level of Circ-RAPGEF5 silencing and upregulated in CCLP1 cells. E-F The remaining two Western blot experiments detecting the global level of SUMOylation in Circ-RAPGEF5 knockdown and overexpression RBE cell while miR-3185 was silenced. Figure S7. A-B The remaining two Western blot experiments of CO-IP assays to evaluate AKT SUMOylation level of Circ-RAPGEF5 knockdown and overexpression RBE cell. C-D The remaining two Western blot experiments of CO-IP assays to testing SUMO-1 modified AKT level of Circ-RAPGEF5 knockdown and overexpression RBE cell. E-F The remaining two Western blot experiments of SUMOylation modification analysis to explore the levels of AKT SUMOylation in Circ-RAPGEF5 knockdown and SAE1 overexpression RBE cell. Figure S8. A-B The remaining two Western blot experiments detected the expression level of liner RAPGEF5 in RBE cells after treatment with Si-Circ-RAPGEF5 or Si-NC.