Fig. 6

RPN6 enhances gemcitabine resistance by promoting cell cycle progression and DNA damage repair. A-B The bubble chart shows the significant enrichment of Gene Ontology (GO) term and KEGG pathway via Gene Set Enrichment Analysis (GSEA) grouped by RPN6 expression level based on RNA-seq data of TCGA PDAC datasets. C Gene set enrichment analysis (GSEA) of TCGA datasets showed that higher RPN6 expression was significantly associated with cell cycle, double-strand break repair, and homologous PDAC. D QRT-PCR and Western blot analysis of circ-MTHFD1L in the two gemcitabine-resistant cell lines compared with their parental cells. E-F PANC-1-GR cells with stable RPN6 overexpression or knockdown were constructed. G-J The proliferative ability of stably transfected PANC-1-GR or BxPC-3-GR cells dealing with gemcitabine was investigated via colony formation assays (G, H) and CCK-8 assays (cell viability and IC50) (I, J). Representative colony formation images are shown (G), and the numbers of colonies were summarized (H). K Flow cytometry analysis of the cell cycle progression of stably transfected PANC-1-GR or BxPC-3-GR cells dealing with gemcitabine was performed. Representative images and quantification of the results are presented. L Western blot analysis was performed to assess the protein levels of RAD50, RAD51, BRCA1, BRCA2, PCNA, P53 and P21 after knocking down or overexpressing RPN6 in PANC-1-GR and BxPC-3-GR cells. GAPDH was used as a loading control. M Confocal microscopic analysis of γH2AX staining showed that RPN6 decreased the distribution of γ-H2AX foci. Representative images and quantification of the results are presented. N The comet assay confirmed that RPN6 was closely related to the DNA repair capacity. Representative images and quantification of the results are presented. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, between the indicated groups