Fig. 7

NBR1 enhances Smurf1-drived Nrf2-mediated oxidative stress response. A LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H₂O₂ (200 µM, 2 h). Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Smurf1, anti-NBR1, anti-p62, and anti-β-actin). B LN229 cells transfected with control, Nrf2 or p62 siRNA oligos were treated with control (PBS) or H₂O₂ (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1 and NBR1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (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. C LN229 cells were fixed after treating with control (PBS) or H₂O₂ (200 µM, 2 h), and then immunofluorescence stained with anti-Smurf1, anti-p62, and anti-NBR1 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). ** p < 0.01 as determined by unpaired two-tailed Student’s t-test. D LN229 cells were transfected with control or Smurf1 siRNA oligos. Thereafter, the cells were divided into three groups: (i) cultured in regular medium, (ii) treated with H₂O₂ (200 µM, 2 h), and (iii) after treating with H₂O₂ (200 µM, 2 h), cultured in regular medium for another 12 h. Cell lysates were prepared and subjected to western blot analysis with indicated antibodies (anti-NBR1, anti-p62, anti-p-p62^S349, anti-p-p62^S403, anti-Smurf1, and anti-β-actin). E LN229 cells transfected with control or Smurf1 siRNA oligos were treated with control (PBS) or H₂O₂ (200 µM, 2 h). Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of Smurf1, p62, NBR1, and NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 treated with control siRNA oligos and control (PBS) (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. F LN229 cells treated with control or Smurf1 siRNA oligos and fixed after treating with control (PBS) or H₂O₂ (200 µM, 2 h), and then immunofluorescence stained with anti-NBR1, anti-Smurf1 and anti-p62 antibodies. The nucleus was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test. G LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H₂O₂ (200 µM, 2 h) after overexpressing HA or HA-NBR1. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-Flag, and anti-β-actin). H LN229 cells transfected with control or p62 siRNA oligos were transfected with Flag or Flag-Smurf1, and treated with control (PBS) or H₂O₂ (200 µM, 2 h) after overexpressing HA or HA-NBR1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of mRNA in LN229 transfected 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 Role of Smurf1 in stress response. Stress upregulates the level of Smurf1, leading to the increased formation and material exchange of p62 liquid droplets. Smurf1 promotes the phosphorylation of p62^S349 by activating mTORC1 signalling pathway. These further promote the transcription of anti-stress proteins by competitively binding Keap1 and mediating Nrf2 nuclear import. The activated Nrf2 increases the mRNA level of Smurf1, p62, and NBR1 to promote the formation and enlarging of p62 liquid droplets as positive feedback