Fig 2: MG132 reduces progerin transcripts and splicing factor SRSF-1 protein expression, while SRSF-5 protein levels are increased Upper panel, lamin A/C, progerin, and GAPDH expressions in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (-) or with (+) the indicated drug (s) (MG132 (5 µM), chloroquine (50 µM), bafilomycin A1 (Baf. A1) (100 nM), caspase-6 inhibitor (Casp-6 inh.) (50 µM) or leptomycin B (Lept. B) (20 ng/ml). Lower panels, progerin expression levels in drug (s)-treated cells relative to DMSO-treated cells (well no. 1) were normalized to GAPDH values. (n = 5).Downregulation of progerin transcripts in response to MG132. Quantitative real-time PCR analyses of progerin mRNA levels in HGPS fibroblasts treated with 5 µM MG132 relative to DMSO-treated cells (Control: “C”). (n = 3).A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, lamin A, lamin C, actin, GAPDH and SRSF-1 expression in MG132-treated HGPS cells up to 96 h and relative to DMSO-treated cells “C”. Urea was used to lyse cells. (n = 4).Proteasome activities in HGPS fibroblasts treated with 5 µM MG132 relative to DMSO-treated cells, (Control: untreated cells). (n = 3).MG132-mediated SRSF-5 accumulation and SRSF-1 downregulation correlate with progerin clearance. A representative Western blotting experiment in whole lysates of HGPS fibroblasts showing progerin, SRSF-5, GAPDH, SRSF-1, LC3BI, and LC3BII expression in MG132-treated HGPS cells up to 96 h and relative to DMSO-treated cells (Control: “C”). The medium was replaced with new drug every 48 h. Urea was used to lyse cells. (n = 4).siRNA inactivation of SRSF-1 reduces progerin levels in HGPS fibroblasts. HGPS fibroblasts were transfected with control siRNA or with siRNA specific for SRSF-1 and 48 h later cells were treated for 24 h with DMSO control (-) or with (+) MG132 (5 µM). (n = 3).Left panels, caspase-mediated downregulation of SRSF-1, in addition to autophagy, contribute to progerin clearance. Western blotting evaluation of lamin A/C, progerin, actin and SRSF-1 in whole lysates from HGPS fibroblasts treated for 48 h either with DMSO control (-) or with (+) the indicated drug (s) [MG132 (5 µM), chloroquine (50 µM), bafilomycin A1 (100 nM) or pan-caspase inhibitor (50 µM)]. Rihgt panels, progerin and SRSF-1 expression levels relative to DMSO-treated cells (well no. 1) were normalized to actin values using ImageJ software. (n = 6).Data information: Results are expressed as mean ± SEM, Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, experimental vs. control; the exact P-values are indicated in Appendix Table S1.Source data are available online for this figure.

Fig 3: Caspase-6-mediated N-terminal p62 cleavage fragment negatively regulates p62 droplets associated autophagosome formation.A Caspase-6-mediated N-terminal p62 cleavage fragment reduced LC3-positive puncta formation. Cells transfected with mCherry empty vector (mCh-vec) or mCherry-p62-N (mCh-p62-N) were stained with anti-LC3 and p62 (C-terminus) antibody (guinea pig) for endogenous LC3 and p62 proteins. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Confocal images were acquired. Bar: 10 µm. Total LC3 puncta in each cell or p62 droplets associated LC3 puncta in each cell were quantified. The diameter and the count of puncta in each cell were assessed by ImageJ. For the size of LC3 puncta, each point in the plot represents the average size of LC3 puncta in each cell. n = 50 cells from three independently plated wells. More than 10,000 LC3 speckles were analyzed. Data are shown as mean ± sem. Statistical analysis was performed by Two-way ANOVA with Bonferroni post-tests. The F/degree of freedom/post hoc P values are indicated in each plot. *P < 0.05; ***P < 0.0001. B Caspase-6-mediated N-terminal p62 cleavage fragment reduced ATG16L1-positive puncta formation. Cells transfected with mCherry empty vector (mCh-vec) or mCherry-p62-N (mCh-p62-N) were stained with anti-ATG16L1 and anti-p62 (C-terminus) antibody (guinea pig) for endogenous ATG16L1 and p62 proteins. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Confocal images were acquired. Bar: 10 µm. Total ATG16L1 puncta in each cell or p62 droplets associated ATG16L1 puncta in each cell were quantified. The diameter and the count of puncta in each cell were assessed by ImageJ. For the size of ATG16L1 puncta, each point in the plot represents the average size of ATG16L1 puncta in each cell. n = 50 cells from three independently plated wells. Data are shown as mean ± sem. Statistical analysis was performed by Two-way ANOVA with Bonferroni post-tests. The F/degree of freedom/post hoc P values are indicated in each plot. *P < 0.05; **P < 0.01; ***P < 0.0001. C Caspase-6-mediated N-terminal p62 cleavage fragment enhanced the levels of puromycin-induced ubiquitin puncta. HeLa cells were transfected with mCherry empty vector /HA-ubiquitin or mCherry-p62-N/HA-ubiquitin (HA-ub). After 20 h, cells were treated with vehicle or puromycin (5 µg/ml) for 5 h. Cells were stained with anti-HA and p62 (C-terminus) (guinea pig) antibody. Note that anti-p62 C-terminus antibody does not react with mCh-p62-N. Confocal images were acquired. Bar: 10 µm. The diameter and the count of puncta in each cell were quantified by ImageJ. n = 50 cells from three independently plated wells. 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. ns not significant; **P < 0.01; ***P < 0.0001.

Fig 4: 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 TNFα (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 TNFα (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-p62ΔPB1/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-p62ΔPB1/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-p62ΔPB1 and mCherry-p62-N was observed. Boxed areas are magnified. Bar (inset): 2 µm.

Fig 5: Caspase-6 mediates p62 cleavage at D256.A Caspase-6 inhibitor blocked p62 cleavage at D256. HeLa cells were treated with vehicle control, or TNFα (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), TNFα (10 ng/ml) + CHX (50 µg/ml) (T + C), and TNFα (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 TNFα (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 TNFα (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.