Fig. 2

p62 phase separation is required for Smurf1-mediated Nrf2 activation. A, B LN229 cells were transfected with HA or HA-Smurf1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-p62, anti-HA, and anti-β-actin) (A). The panels show relative intensity of p62 and Nrf2 in total cell (mean ± SD from 3 independent experiments). ** p < 0.01 as determined by unpaired two-tailed Student’s t-test (B). C The MEF cells were isolated from Smurf1^+/+ (WT) and Smurf1^−/− (Smurf1^KO)mice. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-p62, anti-Smurf1, and anti-β-actin). D, E LN229 cells overexpressing RFP-Nrf2 were fixed after transfecting with GFP or GFP-Smurf1. The nucleus was stained with DAPI. Bar: 5 µm (D). More than 100 cells were assayed for the nuclear signal of Nrf2; mean ± SD from 3 independent experiments. *** p < 0.001 as determined by unpaired two-tailed Student’s t-test (E). F, G 293T cells were transfected with HA or HA-Smurf1. Cytosolic and nuclear fractions of cells were separated and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-HA, anti-H2B, and anti-β-actin) (F). The panels show the quantification of Nrf2 in nucleus (mean ± SD from 3 independent experiments). *** p < 0.001 as determined by unpaired two-tailed Student’s t-test (G). H LN229 cells treated by control, Nrf2, or p62 siRNA oligos were transfected with Flag or Flag-Smurf1. The relative mRNA of NQO1 was performed by qRT-PCR analysis. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and Flag; mean ± SD from 3 independent experiments. NS p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. I LN229 cells treated by p62 siRNA oligos were transfected with Flag or Flag-Smurf1, then overexpressed GFP-p62 or GFP-p62^K7A/D69A. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-GFP, anti-Flag, and anti-β-actin). J, K LN229 cells treated with p62 siRNA oligos were fixed after transfecting with Flag, Flag-Smurf1, GFP-p62, or GFP-p62^K7A/D69A, and then immunofluorescence stained with anti-Nrf2 and anti-Flag antibodies. The nucleus was stained with DAPI. Bar: 5 µm (J). More than 100 cells were assayed for the nuclear signal of Nrf2; mean ± SD. NS p > 0.05, * p < 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test (K). L LN229 cells treated by p62 siRNA oligos were transfected with GFP-p62 or GFP-p62^K7A/D69A, then overexpressed Flag or Flag-Smurf1. Bar graphs indicate that the relative mRNA of NQO1 was performed by qRT-PCR. Values were normalized against the amount of NQO1 mRNA in LN229 treated with si-p62, Flag, and GFP-p62; mean ± SD from 3 independent experiments. NS p > 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. M Role of Smurf1 in p62 droplet formation and Nrf2 activation. Smurf1 overexpression increased formation and material exchange of p62 liquid droplets. Increased p62 droplets promoted Nrf2 nuclear import