Research Paper Volume 16, Issue 8 pp 7311—7330

Figure 3. Exploration and verification of underlying mechanism of EIF3B regulating cholangiocarcinoma. (A) Gene expression profile from PrimeView Human Gene Expression Array analysis in shCtrl/shEIF3B-transfected HCCC-9810 cells. (B, C) The enrichment of the differentially expressed genes (DEGs) in canonical signaling pathways was analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) (B) and IPA (C). (D) Interaction network based on IPA illustrating the impact of EIF3B on key genes. (E, F) qRT-PCR (E) and western blot (F) analysis were used to detect the levels of candidate DEGs in HCCC-9810 cells with shEIF3B or shCtrl. (G, H) Co-immunoprecipitation (Co-IP) experiment demonstrating the exogenous interaction between EIF3B and PCNA in 293T cells (G), and the endogenous interaction between SYVN1 and EIF3B in HCCC-9810 cells (H). (I) Analysis of SYVN1 protein levels in HCCC-9810 cells upon EIF3B depletion. (J) Investigation of PCNA protein stability in HCCC-9810 cells with EIF3B knockdown or SYVN1 overexpression following 0.2 mg/mL CHX treatment for indicated times. (K) Impact of the proteasome inhibitor MG-132 on the degradation of PCNA protein following EIF3B knockdown or SYVN1 overexpression. (L) The lysates of HCCC-9810 cells with EIF3B knockdown were immunoprecipitated, and western blot was performed to examine the ubiquitination of PCNA. All the experiments were in triplicate.