Fig. 4: LINC00239 interacts with Keap1 and regulate the stability of Nrf2.

A Western blot analysis of the expression levels of Keap1 and Nrf2. B The mRNA level of Nrf2 encoding gene (NFE2L2) was not significantly different in CRC cells lacking or overexpressing LINC00239. C LINC00239 reduced the interaction between Nrf2 and Keap1. CRC cells were treated with 10 μM erastin for 12 h. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. D, E LINC00239 regulates the subcellular localization of Nrf2. Subcellular fractionation was used to isolate cytoplasmic and nuclear proteins, and immunoblotting was performed to examine the localization of Nrf2 following the downregulation or overexpression of LINC00239. F, G Immunofluorescence was used to localize Nrf2 in cells lacking or overexpressing LINC00239. All cells were treated with 10 μM erastin for 12 h. CRC cells were labeled with anti-Nrf2 (green), anti-Keap1 (red), and DAPI (blue). Scale bar: 10 μm. H LINC00239 reduced the protein degradation of Nrf2. SW620 cells transfected with LINC00239-knockdown and control plasmids were left untreated or treated with 10 μM of MG132 for 6 h to block the degradation of ubiquitinated proteins. I, J LINC00239 stabilized Nrf2 under basal conditions. All cells were left untreated or treated with 50 μg/mL CHX and incubated for the indicated time periods. Lysates were analyzed by western blotting. K LINC00239 reduced the ubiquitination of Nrf2. All cells treated with 10 μM of MG132 for 6 h and subjected to an in vivo ubiquitination assay to detect ubiquitin-conjugated endogenous Nrf2 proteins. Lysates were denatured, immunoprecipitated with anti-Nrf2 and blotted with an anti-ubiquitin antibody. Data shown represent mean ± SD from three independent experiments. ns P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test.