Fig 2: Caspase-6-mediated N-terminal p62 cleavage fragment plays a dominant-negative role in p62-droplet formation.A, B Caspase-6-mediated p62 cleavage blocked p62-droplet formation. A HeLa cells stably Tet-on expressing p62-GFP were transfected with vector or mCherry-p62-N (mCh-p62). p62-GFP-droplet formation was assessed (see below). B HeLa cells transfected with vector or mCh-p62-N, were stained with anti-p62 (C-terminus) antibody (guinea pig) for endogenous p62 proteins. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Endogenous p62-droplet formation was assessed as below. Confocal images were acquired. Bar: 10 µm. The diameter of the biggest p62 puncta (µm) in each cell and the number of p62 puncta > 0.5 µm in each cell were assessed by ImageJ. n = the number of cells, as shown in each plot. Data are shown as mean ± sem. Statistical analysis was performed by unpaired/two-tailed T-test. ***P < 0.0001. C Coomassie blue staining of the purified bacteria-expressed His-tagged p62 and p62-N. D Caspase-6-mediated p62 cleavage fragment (p62-N) inhibited p62-droplet formation in vitro. Upper panels: The indicated individual proteins, or the multiple protein mixtures were subjected to in vitro phase separation in a microcentrifuge tube for 3 h in the buffer: 40 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 10% glycerol. The phase separation buffer only was used as a control. Where appropriate, 3 µM final concentration of p62 was maintained; 3 µM final concentration of p62-N, or 1 µM final concentration of polyubiquitinated proteins (Ub) was added. For the phase separation of the mixture of p62, p62-N and isolated polyubiquitinated proteins, p62 and p62-N were mixed and incubated for 1 h prior to the addition of polyubiquitinated proteins (Ub). Imaging was acquired on a glass-bottomed 384-well plate. The phase-contrast images were acquired with Leica DMi8 microscopy. Scale bar: 10 µm. Lower panels: The number of p62 droplets in each image (232 µm × 310 µm) was scored (LAS-X). n = 9–22 images for each group, as indicated in the plot. The size of p62 droplets was assessed (LAS-X). n = 21–78 droplets, as indicated in the plot. Data are shown as mean ± sem. Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test. The F/degree of freedom/post hoc P values are indicated in each plot. ***P < 0.0001.

Fig 3: Caspase-6-mediated p62 N-terminal cleavage fragment colocalizes with full-length p62 via the PB1 domain.A, B Caspase-6-mediated p62 C-terminal fragment was undetectable. A N-terminally Myc-tagged p62 (Myc-p62) or C-terminally HA-tagged p62 (p62-HA) was transfected into HeLa cells. After 20 h, the cells was subjected to cleavage with the treatment of TNFa (10 ng/ml) + CHX (50 µg/ml) (T + C) for 4 h. The cell lysates were used for immunoblot with anti-Myc or HA antibody, and GAPDH antibody successively. An asterisk symbol (*) denotes Myc-p62-1-329aa cleavage fragment (left). N-256: Myc-p62 N-terminus 1-256aa (left). B HeLa cells was subjected to cleavage with the treatment of TNFa (10 ng/ml) + CHX (50 µg/ml) for 4 h. The cell lysates were used for immunoblot with anti-p62 N-terminus antibody or anti-p62-C-terminus antibody, and GAPDH antibody successively. An asterisk symbol (*) denotes p62-1-329aa cleavage fragment (left). N-256: p62 N-terminus 1-256aa (left). C The colocalization between mCherry-p62-N and GFP-p62. Hela cells expressing GFP-p62 and mCherry-p62-N were fixed, and images were acquired with Leica confocal microscopy. Bar: 10 µm. Boxed areas are magnified. Bar (inset): 2 µm. D, E The physical interaction between p62 and p62-N. D Myc-p62-N/vector or Myc-p62-N/p62-Flag were transfected into HeLa cells. After 20 h, cells were lysed and subjected to immunoprecipitation (IP) with anti-Flag (M2) agarose beads (Sigma). The immunoprecipitates and whole cell lysates (WCL) were used for immunoblot with anti-Myc and anti-Flag antibody successively. E Myc-p62-N/vector or Myc-p62-N/p62-HA were transfected into HeLa cells. After 20 h, cells were lysed and subjected to immunoprecipitation with anti-HA agarose beads (Sigma). The immunoprecipitates (IP) and whole cell lysates (WCL) were used for immunoblot with anti-Myc and anti-HA antibody successively. F, G The PB1 domain was required for the interaction between p62 and p62-N. F GFP-p62/vector, GFP-p62?PB1/p62-N-Flag or GFP-p62/p62-N-Flag were transfected into HeLa cells. After 20 h, cells were lysed and subjected to immunoprecipitation (IP) with anti-Flag (M2) agarose beads (Sigma). The immunoprecipitates and whole cell lysates (WCL) were used for immunoblot with anti-GFP and anti-Flag antibody successively. G Hela cells were transfected with GFP vector/mCherry-p62-N (control), GFP-p62/mCherry vector (control), GFP-p62/mCherry-p62-N or GFP-p62?PB1/mCherry-p62-N. After 20 h, cells were fixed and images were acquired with Leica confocal microscopy. Bar: 10 µm. No significant colocalization between GFP-p62?PB1 and mCherry-p62-N was observed. Boxed areas are magnified. Bar (inset): 2 µm.

Fig 4: Caspase-6 mediates p62 cleavage at D256.A Caspase-6 inhibitor blocked p62 cleavage at D256. HeLa cells were treated with vehicle control, or TNFa (10 ng/ml) + CHX (50 µg/ml) along with DMSO, caspase-3 inhibitor (C3-I) (20 µM), caspase-6 inhibitor (C6-I) (20 µM), or z-VAD-fmk (z-VAD) (20 µM) for 4 h. Cell lysates were subjected to immunoblot with anti-p62 or GAPDH antibodies successively. An asterisk symbol (*) denotes p62-1-329aa cleavage fragment. N-256: p62 N-terminus 1-256aa. B Caspase-6 inhibitor or pan-caspase inhibitor restored p62-droplet formation. HeLa cells were treated with vehicle (DMSO), TNFa (10 ng/ml) + CHX (50 µg/ml) (T + C), and TNFa (10 ng/ml) + CHX (50 µg/ml) along with DMSO, caspase-3 inhibitor (C3-I) (20 µM), caspase-6 inhibitor (C6-I) (20 µM) or z-VAD-fmk (z-VAD) (20 µM), for 3 h. The cells were stained with anti-p62 antibody (guinea pig). Confocal images were acquired with confocal microscopy. Bar: 10 µm. The diameter of the biggest p62 puncta (µm) in each cell was measured, and the number of p62 puncta > 0.5 µm in each cell was assessed (ImageJ). n = the number of cells, as shown in each plot. Data are shown as mean ± sem. Statistical analysis was performed by one-way ANOVA with Dunnett’s multiple comparison test. The F/degree of freedom/post hoc P values are indicated in each plot. ns not significant; ***P < 0.0001. C, D In vitro cleavage of p62 by caspase-6. For caspase-6 in vitro cleavage assay, recombinant 6× His-tagged p62 (del 1-84aa) was cleaved by control (buffer only) (Ctrl), caspase-3 (C3), caspase-6 (C6) or caspase-9 (C9) in vitro. The reaction mixture was resolved by SDS-PAGE (the arrows mark N- or C-terminal p62), and subjected to Coomassie blue staining (C) or subjected to immunoblot with anti-p62 (N-terminus) (D). Note that the mobility shift of the p62 N-terminal fragment here differs from those in other occasions of p62 N-terminus, as p62 del 1-84aa was used here. E Caspase-6 knockdown blocked p62 cleavage. HeLa cells were knocked down with siRNA for control (Ctrl), caspase-3 (C3), caspase-6 (C6), or caspase-9 (C9) in duplicate, respectively. After 48 h, cells were treated with DMSO (-) or TNFa (10 ng/ml) + CHX (50 µg/ml) (+), as indicated, for 4 h. Cell lysates were subjected to immunoblot with anti-p62 (N-terminus) or GAPDH successively. In parallel (lower panels), samples for control knockdown, caspase-3 knockdown, caspase-6 knockdown, or caspase-9 knockdown were probed with anti-caspase-3 (left), anti-caspase-6 (middle) or anti-caspase-9 antibody (right) to validate knockdown effectiveness. An asterisk symbol (*) denotes p62-1-329aa cleavage fragment. N-256: p62 N-terminus 1-256aa. F Caspase-6 knockdown restored p62-droplet formation. HeLa cells were knocked down (KD) with siRNA for control (Ctrl), caspase-3 (C3), or caspase-6 (C6) in duplicate, respectively. After 48 h, cells were treated with DMSO or TNFa (10 ng/ml) + CHX (50 µg/ml) (T + C), as indicated, for 3 h. The cells were stained with anti-p62 antibody (guinea pig). Confocal images were acquired with confocal microscopy. Bar: 10 µm. The diameter of the biggest p62 puncta (µm) in each cell, and the number of p62 puncta > 0.5 µm in each cell were assessed by ImageJ. n = the number of cells, as shown in each plot. Data are shown as mean ± sem. Left panel: Statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test. The F/degree of freedom/post hoc P values are indicated in each plot. ns not significant; **P < 0.01; ***P < 0.0001. Right panel: Statistical analysis was performed by unpaired/two-tailed T-test. ns not significant; **P = 0.006; ***P < 0.0001.