Fig. 5

NBR1 enhances Smurf1-mediated p62 liquid-droplets accumulation. 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-p62, anti-NBR1, anti-NDP52, anti-OPTN, anti-BAG3, anti-HA, and anti-β-actin) (A). 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 HA; mean ± SD from 3 independent experiments. NS p > 0.05, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test (B). C, D LN229 cells transfected with RFP-Smurf1 and GFP-p62 were treated with control or NBR1 siRNA oligos, fixed after 24 h transfection with HA or HA-NBR1. The nucleus was stained by DAPI. Bar: 5 µm (C). Bar graphs indicate the ratio of the number of RFP-Smurf⁺GFP-p62⁺ puncta to the number of GFP-p62⁺ puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). * p < 0.05 as determined by unpaired two-tailed Student’s t-test (D). E, F LN229 cells treated with control, NBR1, or Smurf1 siRNA oligos were transfected with GFP, GFP-Smurf1, or GFP-NBR1, fixed after treating with MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 antibody. Bar: 5 µm. The nucleus was stained by DAPI (E). 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 > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by unpaired two-tailed Student’s t-test (F). G LN229 cells overexpressing Flag-Smurf1 were transfected with GFP or GFP-NBR1, fixed after treating with BafA1 (100 nM, 24 h) and/or MG132 (20 µM, 12 h), and then immunofluorescence stained with anti-p62 antibody. 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 > 0.05, * p < 0.05 as determined by unpaired two-tailed Student’s t-test. H, I LN229 cells transfected with RFP-GFP-p62 were treated by control, Smurf1 or NBR1 siRNA oligos, treated with MG132 (20 µM, 12 h), and fixed after transfection with HA, HA-NBR1 or Flag-Smurf1. The nucleus was stained by DAPI. Bar: 5 µm (H). Bar graphs indicate the number of RFP-GFP-p62 puncta in each cell (mean ± SD, n = 10 cells examined over three independent experiments). NS p > 0.05, ** p < 0.01 as determined by unpaired two-tailed Student’s t-test (I). J LN229 cells transfected with GFP-p62 and Flag-Smurf1 cultured in glass-bottom plates were treated with control or NBR1 siRNA oligos, then overexpressed HA or HA-NBR1. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments. K LN229 cells transfected with GFP-p62 were treated with control or Smurf1 siRNA oligos, and overexpressed HA or HA-NBR1. The signal recovery after photobleaching was measured; mean ± SD, n = 20 droplets examined over three independent experiments