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. 2021 Feb 2;2021:6610124. doi: 10.1155/2021/6610124

Figure 3.

Figure 3

Bixin activates Nrf2 by the upregulation of P62. (a) LO2 cells were treated with bixin 40 μM for the indicated time. Cell lysates were subjected to immunoblot analyses with the indicated antibodies. Quantification of relative protein expression was determined; results are expressed as the means ± SD (∗p < 0.05, Ctrl vs. bixin treatments). (b) LO2 cells were treated with bixin 40 μM for 4 h and 24 h. mRNA was extracted, and P62 mRNA expression was detected by qRT-PCR assay; results are expressed as the means ± SD (∗p < 0.05, Ctrl vs. bixin treatments). (c) LO2 cells were transfected with nontargeted siRNA or P62-targeted siRNA for 24 h, and then treated with bixin 40 μM for another 24 h. The indicated protein expression was investigated by immunoblot analysis. (d) LO2 cells were treated with bixin (40 μM) for 24 h. Cell lysates were immunoprecipitated with the anti-P62 antibody and analyzed by immunoblot analyses with the anti-Keap1 antibody. The total protein expression was analyzed by immunoblot assay with the anti-P62, anti-Keap1, and anti-GAPDH antibodies (input). (e) LO2 cells were treated with bixin (40 μM) for 24 h. The cells were fixed and subjected to indirect immunofluorescence staining of P62 (red) and Keap1 (green).