Additional file 1 of Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy
Creators: name: Xia, Qin
name: Li, Yang
name: Xu, Wanting
name: Wu, Chengwei
name: Zheng, Hanfei
name: Liu, Liqun
name: Dong, Lei
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Evolutionary Biology
subject: Science Policy
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology

description: Additional file 1: Figure S1. Smurf1 promotes the formation of p62-liquid droplets. A LN229 cells were transfected with Flag or Flag-Smurf1, fixed followed by immunofluorescence stained with anti-Smurf1 and anti-p62 antibodies. The nuclear was stained with DAPI. Bar: 5 µm. B LN229 cells with Flag, Flag-Smurf1, or Flag-Smurf1C699A were fixed after treating with MG132 (20 µM, 12 h), then immunofluorescence stained with anti-p62 antibody. 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). The nuclear was stained by DAPI. NS p &gt; 0.05, * p &lt; 0.05, *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. C LN229 cells cultured in glass-bottom plates were transfected with GFP-p62. Bar: 1 µm. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. D LN229 cells cultured in glass-bottom plates were transfected with GFP-Smurf1 and treated with MG132 (20 µM, 12 h). Bar: 1 µm. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. Figure S2. p62 phase separation is required for Smurf1-mediated Nrf2 activation. A LN229 cells were transfected with control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-p62, anti-Smurf1, and anti-β-actin). The right panels show relative intensity of p62 and Nrf2 in total cell (mean ± SD from 3 independent experiments). ** p &lt; 0.01 as determined by unpaired two-tailed Student’s t-test. B 293T cells were transfected with control or Smurf1 siRNA oligos. Cytosolic and nuclear fractions were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-Smurf1, anti-H2B, and anti-β-actin). C LN229 cells were transfected with control or Smurf1 siRNA oligos. Total RNAs were prepared from these LN229 cells. The relative mRNA levels of Smurf1 and NQO1 were performed by qRT-PCR analysis. Values were normalized against the amount of mRNA in LN229 transfected with control siRNA oligos; mean ± SD from 3 independent experiments. *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. D LN229 cells treated with control or p62 siRNA oligos were transfected with GFP, GFP-p62, or GFP-p62K7A/D69A. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-GFP, anti-p62, and anti-β-actin). Figure S3. Smurf1 increases the binding affinity between p62 and Keap1 to activate Nrf2. A LN229 cells were transfected with control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-p-p62S349, anti-p-p62S403, anti-Smurf1, and anti-β-actin). Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin. Values were normalized against the intensity of LN229 transfected with si-Control; mean ± SD from 3 independent experiments. * p &lt; 0.05, *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. B LN229 cells treated with control or PTEN siRNA oligos were treated by treated by control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p-mTOR, anti-mTOR, anti-PTEN, anti-Smurf1, and anti-β-actin). C LN229 overexpressed with Flag or Flag-Smurf1 were transfected with HA or HA-PTEN, fixed after treating with MG132 (20 µM, 12 h) and then performed by immunofluorescence analysis with anti-p62 antibody. The nuclear was stained with 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). NS p &gt; 0.05, *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. D LN229 cells were transfected with Flag or Flag-Smurf1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-Keap1 and anti-p62 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number of Keap+p62+ puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. E LN229 cells overexpressed GFP-p62 were fixed after transfecting with Flag or Flag-Smurf1, and then immunofluorescence stained with anti-Keap1 and anti-Flag antibodies. The nuclear was stained with DAPI. Bar: 5 µm. F LN229 cells were transfected with control, Smurf1 or p62 siRNA oligos, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 and anti-Keap1 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number of Keap+p62+ puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p &gt; 0.05, * p &lt; 0.05 as determined by unpaired two-tailed Student’s t-test. G LN229 cells were transfected with control or p62 siRNA oligos, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-Keap1, anti-p62, and anti-Smurf1 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Figure S4. NBR1 enhances the Smurf1 mediated p62 liquid-droplets accumulation. A LN229 cells were transfected with control or Smurf1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-NDP52, anti-OPTN, anti-BAG3, anti-Smurf1, and anti-β-actin). Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to β-actin. Values were normalized against the intensity of LN229 transfected with si-Control; mean ± SD from 3 independent experiments. NS p &gt; 0.05, * p &lt; 0.05, ** p &lt; 0.01 as determined by unpaired two-tailed Student’s t-test. B LN229 cells treated with control or Smurf1 siRNA oligos were transfected with Flag or Flag-Smurf1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-NBR1 and anti-p62 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number and size of NBR1+p62+ puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p &lt; 0.05, ** p &lt; 0.01 as determined by unpaired two-tailed Student’s t-test. C LN229 cells treated with GFP-p62 cultured in glass-bottom plates were transfected with control, Smurf1, or NBR1 siRNA oligos. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. Figure S5. Smurf1 mediated NBR1 expression in p62 dependent manner A LN229 cells were transfected with control, Smurf1, p62 or NBR1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-NBR1, anti-Smurf1, anti-p62, and anti-β-actin). B LN229 cells treated with control or Smurf1 siRNA oligos were transfected with Myc or Myc-Nrf2. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-Nrf2, anti-p62, anti-NBR1, anti-Smurf1, and anti-β-actin). C LN229 cells were treated with control or Smurf1 siRNA oligos and overexpressed Myc or Myc-Nrf2, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-NBR1 and anti-p62 antibodies. The nuclear was stained by DAPI. Bar: 5 µm. Bar graphs indicate the number of NBR1+p62+ puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p &lt; 0.05, *** p &lt; 0.001 as determined by unpaired two-tailed Student’st-test. D LN229 cells treated with control, NBR1, Nrf2, or p62 siRNA oligos were transfected with HA or HA-NBR1, and then overexpressed Flag or Flag-Smurf1. Total RNAs were prepared from these LN229 cells. Bar graphs indicate the amount of NQO1 mRNA. Values were normalized against the amount of NQO1 mRNA in LN229 transfected with control siRNA oligos, HA, and Flag; mean ± SD from 3 independent experiments. NS p &gt; 0.05, * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. E LN229 cells treated with NBR1 or p62 siRNA oligos were transfected with GFP, GFP-p62WT, GFP-p62S349A or GFP-p62K7A/D69A, and overexpressed Flag, Flag-Smurf1, 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 NQO1 mRNA in LN229 transfected with p62 siRNA oligos, HA-NBR1, GFP, and Flag; mean ± SD from 3 independent experiments. NS p &gt; 0.05, * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test. Figure S6. NBR1 enhances Smurf1-drived Nrf2-mediated oxidative stress response. A LN229 cells transfected with control or p62 siRNA oligos were overexpressed Flag or Flag-Smurf1, and treated with control (PBS) or H2O2 (200 µM, 2 h) after transfecting with control or NBR1 siRNA oligos. Cell lysates were prepared and subjected to western blot analysis with the indicated antibodies (anti-p62, anti-NBR1, anti-Flag, and anti-β-actin). B LN229 cells transfected with control or p62 siRNA oligos were overexpressed Flag or Flag-Smurf1, and treated with control (PBS) or H2O2 (200 µM, 2 h) after transfecting with control or NBR1 siRNA oligos. 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 &gt; 0.05, * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001 as determined by unpaired two-tailed Student’s t-test.