Fig. 2

CSN8 is essential for inducing EMT and dormancy under hypoxic environments, and for developing stress resistance. a HCT116 and DLD-1 cells were cultured under 20% O₂ or 1% O₂ conditions, or treated with TNF-α (20 ng/mL) or IL-1β (20 ng/mL) for 24 h, and then real-time PCR was used to analyze CSN8 mRNA expression. b CSN8-silenced CRC cells and control cells were cultured under 20% O₂ or 1% O₂ conditions for another 48 h. Photographs were taken using a phase-contrast microscope. c Annexin V–FITC/PI staining assay was used to analyze the apoptotic and necrotic cells. d Real-time PCR was used to analyze the mRNA expression of the hypoxic response, EMT, and dormancy-related genes. e CSN8-overexpressed CRC cells and control cells were transfected with NF-κB dual-luciferase plasmid or control plasmid. After being transfected for 48 h, luciferase activity was detected with the dual-luciferase reporter assay system. f CSN8-overexpressed CRC cells were treated with NF-κB inhibitor DHMEQ (25 μM) or DMSO solvent control for 24 h, and then real-time PCR was used to analyze the HIF-1α mRNA levels. g Western-blot analysis of the HIF-1α-immunoprecipitated lysates with the anti-ubiquitin antibody. h CSN8-overexpressed CRC cells were treated with the HIF-1α inhibitor LW6 (20 μM) or DMSO solvent control for 24 h, and then real-time PCR was used to analyze the mRNA levels of EMT and dormancy-related genes. i The model depicts the pivotal role of CSN8 in the EMT and dormancy process of CRC cells under the hypoxic microenvironment. ^*P < 0.05; ^**P < 0.01; ns, P ≥ 0.05