Fig 1: RCN1 contributes to selective activation of the unfolded protein response.A qRT-PCR and western blot analysis showing the mRNA and protein levels of IRE1, CHOP, GRP78, and XBP1s in sorafenib-resistant and sorafenib-sensitive Huh7 cells. B Western blot analysis showing protein levels of XBP1 and p-IRE1α in sorafenib-resistant Huh7 cells with or without RCN1 silencing. C Total mRNA levels of XBP1 in RCN1-knockdown drug-resistant cells. D, E Western blot analysis of RCN1, IRE1α, and XBP1 expression in sorafenib-resistant Huh7 cells with downregulated or overexpressed IRE1. Data are presented as the means ± SEM of three independent experiments; data for western blot have undergone quantitative analysis. ns not significantly different. *P < 0.05; **P < 0.01; ***P < 0.001, t-test.
Fig 2: The IRE1α–XBP1s pathway is crucial for drug resistance and HCC malignancy.A Representative flow cytometry analysis of Annexin V-PI staining in sorafenib-resistant Huh7 cells with or without RCN1 silencing, in the presence of 5 μM sorafenib. B Representative flow cytometry analysis of Annexin V-PI staining in sorafenib-resistant Huh7 cells in the presence of 5 μM sorafenib and different doses of MKC8866. C Cell proliferation in sorafenib-resistant Huh7 cells with IRE1a knockdown assessed using an EdU assay. D Transwell assays of cell migration and invasion in sorafenib-resistant Huh7 cells after IRE1α knockdown. E Cell proliferation in sorafenib-resistant Huh7 cells assessed using EdU assays after treatment with 0.3 μM MKC8866. F Transwell assays of cell migration and invasion in sorafenib-resistant Huh7 cells in the presence of 0.3 μM MKC8866. Data are presented as the means ± SEM of three independent experiments. ns not significantly different. **P < 0.01; ***P < 0.001; ****P < 0.0001, t-test.
Fig 3: RCN1 activates c-MYC signaling through the IRE1α-XBP1s pathway in sorafenib-resistant cells.A c-MYC expression in sorafenib-resistant Huh7 cells with downregulated RCN1. B Flow cytometry analysis of Annexin V-PI staining in Huh7 RCN1-knockdown sorafenib-resistant cells with or without MYC overexpression, in the presence of 5 μM sorafenib. C, D MYC overexpression rescues the inhibitory effect of RCN1 shRNA on the. proliferation, migration, and invasion of sorafenib-resistant cells. E Sorafenib-resistant Huh7 cells were transfected MYC luciferase reporter plasmids. with or without a flag-XBP1s plasmid. After 24 h, the cells were harvested for a luciferase activity assay. F Schematic model. The increasing expression of RCN1 results in an activation of the IRE1α–XBP1s–c-MYC axis in sorafenib-resistant cells. Data are presented as the means ± SEM of three independent experiments; data for western blot have undergone quantitative analysis. ns: not significantly different. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, t-test.
Fig 4: The RCN1 EFh1/2 domain binds with GRP78 and promotes the dissociation of GRP78 from IRE1.A Immunoblot analysis of the interaction between GRP78 and IRE1α using immunoprecipitates from sorafenib-resistant Huh7 cells with or without RCN1 silencing. B Immunoblot analysis of complex formation between endogenous RCN1 and GRP78 immunoprecipitated from sorafenib-resistant Huh7 cells. C Schematic representation of the whole-length and deletion RCN1 mutants. D Immunoblot analysis of complex formation between endogenous GRP78 and HA-tagged mutant RCN1. E Immunoblot analysis of IRE1α phosphorylation and XBP1s protein levels in sorafenib-resistant Huh7 cells transfected with whole-length and deletion RCN1 mutants. Data are presented as the means ± SEM of three independent experiments. ns not significantly different. **P < 0.01; ***P < 0.001; ****P < 0.0001, t-test.
Fig 5: RCN1 is upregulated in sorafenib-resistant HCC cells and may predict a poor prognosis.A Representative flow cytometry analysis of Annexin V-PI staining in Huh7 and HepG2 sorafenib-sensitive and -resistant cells treated with 5 μM sorafenib. B Subcutaneous injection of sorafenib-sensitive and -resistant Huh7 cells into nude mice, and ex vivo images of resected xenografts. C Self-renewal ability of sorafenib-resistant HCC cells examined using the sphere formation assay. D Heatmap of 1145 genes differentially expressed between sorafenib-sensitive cells and cells with acquired sorafenib resistance from the GSE94550 dataset. E Heatmap of the 20 most variable genes (|logFC|>3). The abscissa in panels A and B represents the sample number, the ordinate represents the differentially expressed genes, the histogram at the upper right represents the color level, and each cell in the matrix represents a specific expression level. F Genomic RCN1 expression in Huh7 and HepG2 sorafenib-sensitive and -resistant cells. G Proteomic RCN1 expression in Huh7 and HepG2 sorafenib-sensitive and -resistant cells. H RCN1 expression was upregulated in sorafenib-resistant HCC when compared with sorafenib-sensitive HCC. I RCN1 mRNA expression was upregulated in HCC tumor tissues (n = 68) compared with the corresponding adjacent tissues (n = 68). GAPDH served as an internal reference. J RCN1 expression was significantly higher in tumor tissues than in the adjacent tissues, according to the GEPIA website. K Association of RCN1 expression with overall survival (top) and disease-free survival (bottom). Data are presented as the means ± SEM of three independent experiments; data for western blot have undergone quantitative analysis. ns not significantly different. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, t-test.
Supplier Page from Abcam for Anti-RCN1/RCN antibody [EPR17163-117]