Fig 2: ADAMTS-4 moves to the nucleus and directly cleaves PARP-1. Representative images of confocal microscopy of immunofluorescence immunostaining (A) and western blot analysis (B) showing that PA induced ADAMTS-4 nuclear translocation. (C) Representative images of double-staining of ADAMTS-4 and SM22-α showing that ADAMTS-4 was seen in the nuclei of SMCs. (D) Representative confocal microscopy images of immunofluorescence staining showing that ADAMTS-4 was highly abundant in the condensed nuclei of dying cells. (E) Representative images of double-staining of ADAMTS-4 and TUNEL in PA treated SMCs showing ADAMTS-4 in the nuclei of TUNEL+ apoptotic cells.(F) Representative confocal microscopy images of immunofluorescence staining showing that ADAMTS-4 colocalized with PARP-1 in the nuclei of PA-treated cells. (G) Co-immunoprecipitation analysis indicating that ADAMTS-4 and PARP-1were in the same complex in SMCs. (H) Recombinant human PARP-1 was incubated with affinity-purified recombinant human ADAMTS-4 in the presence or absence of ADAMTS-4 inhibitor. Western blot analysis showing that ADAMTS-4 directly cleaved PARP-1. Data shown in (A–F) are representative of 3 independent experiments.

Fig 3: Atherosclerotic plaques of Atg7F/FTagln-Cre+,apoe-/- mice show several features of VSMC senescence. (A) Sections of the aortic root were stained for senescence-associated (black arrowheads) and compared with serial TAGLN staining (not shown) to locate the fibrous caps. Scale bar: 10 µm. (B) Consecutive sections of the aortic root were double stained for phospho RB (red; white arrowheads) and ACTA2 (green). Scale bar: 25 µm (C) Sections of the brachiocephalic artery were double stained with phospho RB and periodic acid-Schiff (PAS) to quantify phospho RB-positive VSMC nuclei in the media (**, P < 0.01; Mann Whitney test). Scale bar: 50 µm.

Fig 4: Defective VSMC autophagy accelerates atherogenesis after 10 wk of western-type diet. (A) Atg7+/+Tagln-Cre+ apoe-/- (+/+) and Atg7F/FTagln-Cre+,apoe-/- (-/-) mice (n = 16) were fed a western-type diet for 10 wk. Sections of the brachiocephalic artery were stained with H&E to quantify plaque size and percentage of necrosis. (*, P<0.05; Student t test). (B to D) Consecutive sections were immunostained for cleaved CASP3 (B), LAMP2 (C) and ACTA2 (D) to measure the percentage of apoptosis (indicated by a black arrowhead in the high-power photograph of the boxed area in the left corner of each image), percentage of macrophages and fibrous cap thickness (indicated by black arrows), respectively (*, P < 0.05; Student t test (B); ***, P < 0.001; Student t test (C); *P < 0.05; n = 10 measurements/mouse, Repeated Measure (D)). (E) Consecutive sections were stained with Sirius red to quantify total collagen. (*, P < 0.05; Student t test). *, necrotic core. Scale bar: 100 µm.

Fig 5: Defective VSMC autophagy promotes formation of a thick fibrous cap and enhances total collagen deposition after 14 wk of western-type diet. (A) Atg7+/+Tagln-Cre+,apoe-/- (+/+) and Atg7F/FTagln-Cre+,apoe-/- (-/-) mice (n = 16) were fed a western-type diet for 14 wk. Sections of the brachiocephalic artery were stained with H&E to quantify plaque size and percentage of necrosis. (NS, not significant; Student t test). (B to D) Consecutive sections were immunostained for cleaved CASP3 (B), LAMP2 (C) and ACTA2 (D) to measure the percentage of apoptosis, percentage of macrophages and fibrous cap thickness (indicated by black arrows), respectively. (NS, not significant; Student t test (B,C); **, P < 0.01; n = 10 measurements/mouse, Repeated Measure (D)). (E) Consecutive sections were stained with Sirius red to quantify total collagen. (*, P < 0.05; Student t test). Scale bar: 100 µm. *, necrotic core.