Autophagy regulates cellular senescence by mediating the degradation of CDKN1A/p21 and CDKN2A/p16 through SQSTM1/p62-mediated selective autophagy in myxomatous mitral valve degeneration

Abstract

Myxomatous mitral valve degeneration (MMVD) is one of the most important age-dependent degenerative heart valve disorders in both humans and dogs. It is characterized by the aberrant remodeling of extracellular matrix (ECM), regulated by senescent myofibroblasts (aVICs) transitioning from quiescent valve interstitial cells (qVICs), primarily under TGFB1/TGF-β1 control. In the present study, we found senescent aVICs exhibited impaired macroautophagy/autophagy as evidenced by compromised autophagy flux and immature autophagosomes. MTOR-dependent autophagy induced by rapamycin and torin-1 attenuated cell senescence and decreased the expression of cyclin-dependent kinase inhibitors (CDKIs) CDKN2A/p16^INK4A and CDKN1A/p21^CIP1. Furthermore, induction of autophagy in aVICs by ATG (autophagy related) gene overexpression restored autophagy flux, with a concomitant reduction in CDKN1A and CDKN2A expression and senescence-associated secretory phenotype (SASP). Conversely, autophagy deficiency induced CDKN1A and CDKN2A accumulation and SASP, whereas ATG re-expression alleviated senescent phenotypic transformation. Notably, CDKN1A and CDKN2A localized to autophagosomes and lysosomes following MTOR antagonism or MG132 treatment. SQSTM1/p62 was identified as the autophagy receptor to selectively sequester CDKN1A and CDKN2A cargoes for autophagic degradation. Our findings are the first demonstration that CDKN1A and CDKN2A are degraded through SQSTM1-mediated selective autophagy, independent of the ubiquitin-proteasome pathway. These data will inform development of therapeutic strategies for the treatment of canine and human MMVD, and for the treatment of Alzheimer disease, Parkinson disease and other age-related degenerative disorders.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AKT: AKT serine/threonine kinase; aVICs: activated valve interstitial cells; ATG: autophagy related; baf-A1: bafilomycin A₁; BrdU, bromodeoxyuridine; BSA: bovine serum albumin; CDKIs, cyclin-dependent kinase inhibitors; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; co-IP: co-immunoprecipitation; DMSO: dimethylsulfoxide; ECM, extracellular matrix; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; eGFP: green fluorescent protein; ELISA: enzyme-linked immunosorbent assay; HEK-293T, human embryonic kidney 293T; HRP: horseradish peroxidase; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: MAP1LC3/LC3-interacting region; MFS: Marfan syndrome; MKI67/Ki-67: marker of proliferation Ki-67; MMVD: myxomatous mitral valve degeneration; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; OE: overexpression; PBST, phosphate-buffered saline with 0.1% Tween-20; PCNA: proliferating cell nuclear antigen; PIK3CA/PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PLA: proximity ligation assays; PSMA1: proteasome 20S subunit alpha 1; PSMB5: proteasome 20S subunit beta 5; qVICs: quiescent valve interstitial cells; qRT-PCR: quantitative real-time PCR; SA-GLB1/β-gal: SA-senescence-associated GLB1/β-galactosidase; ROS: reactive oxygen species; SASP: senescence-associated secretory phenotype; RPS6KB1/p70 S6K: ribosomal protein S6 kinase B1; SMAD: SMAD family member; SQSTM1/p62: sequestosome 1; STEM: scanning transmission electron microscopy; TGFB: transforming growth factor beta; TGFBR: transforming growth factor beta receptor; TP53/p53: tumor protein p53; UPS: ubiquitin-proteasome system; WT, wild-type.

Keywords: Autophagic degradation; CDKI; MMVD; SASP; TGFB; ubiquitin-proteasome pathway.

Figure 1.

Senescent aVICs exhibit impaired autophagy flux. (A and C) Representative images of MAP1LC3/LC3 and ACTB (loading control) immunoblots of aVICs and qVICs following amino acid starvation for the indicated time periods (n = 3). (B and D) MAP1LC3/LC3-II:ACTB ratio normalized to 0 h for aVICs and qVICs treated as in (A and B). (E) Representative images of immunoblots in aVICs and qVICs treated with or without 200 nM rapamycin for 48 h in the presence or absence of 10 µM baf-A1 for 4 h (n = 4). (F) Ratio of (MAP1LC3/LC3-II + baf-A1/ACTB):(MAP1LC3/LC3-ii + baf-A1/ACTB following 48 h rapamycin treatment), corresponding to MAP1LC3/LC3-II turnover (n = 6). (G-I) Ratio of ATG7 (G), CDKN2A/p16 (H), CDKN1A/p21 (I) to ACTB in cells, normalized to aVICs in the absence of rapamycin and baf-A1, that were treated with or without 200 nM rapamycin and 10 µm baf-A1 as in (E). (J) Percentage of aVICs and qVICs (treated with or without AA starvation and baf-A1) with > 50 MAP1LC3/LC3-II puncta as shown in (L). (K) Percentage of aVICs and qVICs (treated with or without AA starvation and baf-A1) with > 20 CDKN1A/p21 puncta as shown in (L). (L) Representative images of MAP1LC3/LC3 and CDKN1A/p21 immunostaining in aVICs and qVICs treated with or without amino acid deprivation for 72 h in the presence or absence of 10 µM baf-A1 for 4 h (n = 3). Scale bars: 50 µm. These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 2.

Senescent aVICs show immature autophagosomes. (A) Representative confocal images of MAP1LC3/LC3 and SQSTM1/p62 immunostaining in aVICs and qVICs in full medium and following amino acid starvation for 48 h in the presence or absence of 10 µm baf-A1 for 6 h. The white arrow locates MAP1LC3/LC3-positive ring-like structures. Scale bars: 50 µm. (B) Quantification of MAP1LC3/LC3-positive ring-like structures per cell in VICs following amino acid deprivation for 48 h as in (A) (n = 6). (C) Quantification of MAP1LC3/LC3 colocalizing with SQSTM1/p62 puncta in VICs as described in (A) (n = 3). (D) Quantification of VICs (%) with > 10 LAMP1 puncta (>1 µm) in the presence of 200 nM rapamycin with or without baf-A1 as shown in (E). (E) Representative images (top) of LAMP1 immunostaining in aVICs and qVICs treated with or without 200 nM rapamycin for 48 h (n = 5). Scale bars: 50 µm. Representative images (bottom) of LysoBrite staining in VICs in the presence or absence of 200 nM rapamycin for 48 h. Scale bars: 50 µm (n = 3). (F) Quantification of VICs (%) with > 10 LAMP1 puncta in the presence of 200 nM rapamycin with or without baf-A1as shown in (E). (G) Quantification of VICs (%) with > 5 LysoBrite puncta in the presence of 200 nM rapamycin with or without baf-A1as shown in (E). (H) Quantification of VICs (%) with > 10 LysoBrite puncta (>0.5 µm) in the presence of 200 nM rapamycin with or without baf-A1as shown in (E). These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 3.

Rapamycin reverses cell senescence and improves autophagy. (A) Representative images of CDKN1A/p21, CDKN2A/p16 and ACTB (loading control) immunoblots of aVICs treated with the indicated dose ranges of rapamycin (Rapa, 25, 50, 100, 200, or 300 nM) for 48 h (n = 3). (B) CDKN1A/p21 (left panel) and CDKN2A/p16 (right panel):actb ratio in cells, normalized to aVICs in the absence of rapamycin, that were treated as in (A). (C) Representative images of CDKN1A/p21, CDKN2A/p16 and ACTB immunoblots of aVICs treated with rapamycin for the indicated time periods (n = 3). (D) CDKN1A/p21 (left panel) and CDKN2A/p16 (right panel):actb ratio in cells, normalized to 0 h for aVICs treated as in (C). (E) Representative images of SA-GLB1/β-gal staining in aVICs treated with either 200 nM rapamycin for 72 h or 80 nM torin-1 for 48 h. Scale bars: 100 µm (n = 3). (F) Percentage of SA-GLB1/β-gal-positive aVICs treated with either rapamycin or torin-1 as shown in (E). (G) Quantification of SASP cytokine expression by qRT-pcr in aVIC cultures treated with either rapamycin or torin-1 (n = 4). (H) Representative images of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3 and ACTB immunoblots of aVICs treated with rapamycin for the indicated time periods in the absence or presence of 5 µm baf-A1 for 4 h (n = 3). (I-K) Quantification of CDKN1A/p21 (I), CDKN2A/p16: (J) and MAP1LC3/LC3: (K) ACTB ratio in cells, normalized to 0 h for aVICs in the presence of baf-A1, that were treated as in (H). (L and M) Representative images (left panel) of immunoblots for the indicated proteins in the cytoplasm and nucleus fractions isolated from aVICs treated with 200 nM rapamycin for 6 h (L) and 48 h (M) (n = 3). The graphs (right panel) show the ratios from the densitometry analysis of the displayed experiment for each protein to TUBB/β-tubulin (cytoplasmic fractions) or histone-H3 (nuclear fractions) normalized to the corresponding aVICs in the absence of rapamycin. These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 4.

ATG overexpression alleviates aVIC senescence. (A and B) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, ATG7, ATG3, MAP1LC3/LC3 and ACTB (loading control) immunoblots of aVICs transfected with empty vectors, pcDNA3.1-Flag-ATG7 (A) or pcDNA3.1-Flag-ATG3 (B) plasmids in the presence or absence of 5 µm baf-A1 for 4 h (n = 3). The graphs (right panel) show the ratios of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3-II to ACTB normalized to aVICs transfected with empty vectors in the absence of baf-A1. (C-E) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, ATG7, MAP1LC3/LC3 and ACTB (loading control) immunoblots of aVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids and then treated with or without 10 ng/mL TGFB1 for 72 h (C), 200 nM rapamycin for 72 h (D) or RPS6KB1-specific siRNA for 24 h (E) in the presence of 5 µm baf-A1 for 4 h (n = 3). The graphs (right panel) show the ratios of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3-II to ACTB normalized to aVICs transfected with empty vectors in the presence of baf-A1. (F) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, ATG7, MAP1LC3/LC3 and ACTB (loading control) immunoblots of HEK293T cells transfected with empty vectors or pcDNA3.1-Flag-RPS6KB1 plasmids and then re-expressed with or without pcDNA3.1-Flag-ATG7 plasmids in the presence of 5 µm baf-A1 for 4 h (n = 3). The graphs (right panel) show the ratios of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3-II to ACTB normalized to aVICs transfected with empty vectors in the presence of baf-A1. (G) Representative confocal images of CDKN1A/p21 and MAP1LC3/LC3 immunostaining in aVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids for 24 h in the absence or presence of 5 µm baf-A1 for 4 h (n = 3). Scale bars: 50 µm. (H and I) Quantification of aVICs (%) with > 10 CDKN1A/p21 puncta (H) or > 25 MAP1LC3/LC3-II puncta (I) transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids with or without baf-A1 as shown in (G). (J and K) Cells were transfected with either empty vectors (vector), canine pcDNA3.1-Flag-ATG7, or subjected to mock transfection (mock) without vectors. The mRNA expression levels of CDKN2A/p16 (J) and CDKN1A/p21 (K) were quantified at 6 h, 24 h, 48 h, 4 day, 7 day and 9 day post-transfection (n = 4). (L) Representative images (left panel) of SA-GLB1/β-gal staining in aVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids. Scale bars: 100 µm (n = 3). Percentage (right panel) of SA-GLB1/β-gal positive cells. (M) Quantification of ROS fluorescence intensity for cells transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids (n = 3). (N) Quantification of secreted TGFB1, IL6 and MMP9 cytokine expression detected by ELISA in collected supernatant from VIC cultures transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids (n = 3). (O) Quantification of BrdU incorporation for aVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids (n = 4). These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 5.

ATG deficiency induces the transformation of VIC senescent phenotype. (A-C) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, ATG7, ATG3, MAP1LC3/LC3 and ACTB (loading control) immunoblots of qVICs transfected with empty vectors, eSpcas9-2A-Puro (PX459)-ATG7 gRNA V2.0 plasmids (A), scramble control (SC), ATG7-specific siRNA (B) or ATG3-specific siRNA (C) in the presence or absence of 5 µm baf-A1 for 4 h (n = 3). The graphs (right panel) show the ratios of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3-II to ACTB normalized to qVICs transfected with empty vectors in the absence of baf-A1. (D and E) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, ATG7, MAP1LC3/LC3 and ACTB (loading control) immunoblots of qVICs transfected with scramble or ATG7-specific siRNA and then treated with or without 200 nM rapamycin for 48 h (D) or RPS6KB1-specific siRNA for 24 h (E) (n = 3). The graphs (right panel) show the ratios of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3-II to ACTB normalized to aVICs transfected with scramble siRNA. (F) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, ATG7, MAP1LC3/LC3 and ACTB immunoblots of HEK293T cells transfected with empty vectors or eSpcas9-2A-Puro (PX459)-ATG7 gRNA V2.0 plasmids and then re-expressed with or without pcDNA3.1-Flag-ATG7 plasmids in the presence or absence of 5 µm baf-A1 for 4 h (n = 3). The graphs (right panel) show the ratios of CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3-II to ACTB. (G) Representative confocal images of CDKN1A/p21 and MAP1LC3/LC3 immunostaining in ATG7 wild-type (WT) and ATG7 knockout (KO) qVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids for 24 h (n = 3). Scale bars: 50 µm. (H and I) Quantification of ATG7 WT and ATG7 KO qVICs (%) with > 5 CDKN1A/p21 puncta (H) or > 5 MAP1LC3/LC3-II puncta (I) transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids as shown in (G). (J) Representative images of SA-GLB1/β-gal staining in ATG7 WT and ATG7 KO qVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids. Scale bars: 100 µm (n = 5). (K) Percentage of SA-GLB1/β-gal positive ATG7 WT and ATG7 KO cells transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids as shown in (J). (L) Quantification of ROS fluorescence intensity for ATG7 WT and ATG7 KO qVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids (n = 3). (M) Quantification of secreted TGFB1, IL6 and MMP9 cytokine expression in collected supernatant from ATG7 WT and ATG7 KO qVIC cultures transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids (n = 3). (N) Quantification of BrdU incorporation for ATG7 WT and ATG7 KO qVICs transfected with empty vectors or pcDNA3.1-Flag-ATG7 plasmids (n = 4). These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 6.

CDKN2A/p16 and CDKN1A/p21 localize on phagophores during autophagy promotion. (A) Representative confocal images (top) of CDKN1A/p21 colocalization with MAP1LC3/LC3 in aVICs treated with or without 200 nM rapamycin for 18 h. The graphs (bottom) display the fluorescence intensity in each channel over the distance (µm) depicted by the arrows. Scale bars: 50 µm. (B) Mean fluorescence of CDKN1A/p21 colocalizing with MAP1LC3/LC3 from the experiments performed in (A) (n = 6). (C) Quantification of MAP1LC3/LC3 puncta colocalizing with CDKN1A/p21 signal in cells as described in (A) (n = 6). (D) Quantification of PLA puncta per cell as described in (E). (E) Representative images of PLA for CDKN1A/p21 and the autophagosome marker MAP1LC3/LC3 in aVICs treated with or without 200 nM rapamycin for 18 h in the presence or absence of 5 µm baf-A1 for 4 h (n = 3). F-actin was stained with phalloidin. Scale bars: 50 µm. The PLA images of CDKN2A/p16 colocalization with MAP1LC3/LC3 are shown in Fig. S6. (F) Co-immunoprecipitation (Co-ip) of MAP1LC3/LC3 from aVICs treated with or without 200 nM rapamycin for 18 h in the presence or absence of 5 µm baf-A1 for 4 h. Whole-cell lysate (WCL) and immunoprecipitation complexes were analyzed by immunoblotting (left panel) with anti-CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3 and ACTB (loading control) antibodies. Quantification (right panel) of CDKN2A/p16 and CDKN1A/p21 bound to MAP1LC3/LC3, normalized to the condition in the presence of rapamycin and baf-A1, from the experiments performed in left panel. (G and H) the cells were immunoprecipitated with an anti-CDKN2A/p16 antibody (G) or anti-CDKN1A/p21 antibody (H). Immunoblots (top) for anti-CDKN1A/p21, CDKN2A/p16, MAP1LC3/LC3 and ACTB. Quantification (bottom) of MAP1LC3/LC3 bound to CDKN2A/p16 or CDKN1A/p21, normalized to the condition in the presence of rapamycin and baf-A1, from the experiments performed in left panel. (I) Schematic summarizing the binding of MAP1LC3/LC3 with CDKN1A/p21 and CDKN2A/p16. These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 7.

CDKN2A/p16 and CDKN1A/p21 interacts with autophagy receptor SQSTM1/p62. (A and B) Co-immunoprecipitation of CDKN1A/p21 and CDKN2A/p16 from aVICs treated with 10 µm MG132 for 36 h, 200 nM rapamycin for 24 h, or 80 nM torin-1 for 18 h following 5 µm baf-A1 treatment for 4 h. Whole-cell lysate (WCL) and immunoprecipitation complexes were analyzed by immunoblotting with anti-CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, MAP1LC3/LC3 and ACTB (loading control) antibodies (n = 2). (C and D) the cells were immunoprecipitated with an anti-MAP1LC3/LC3 antibody and anti-SQSTM1/p62 antibody. WCL and immunoprecipitation complexes were analyzed by immunoblotting with anti-CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, MAP1LC3/LC3 and ACTB (n = 2). (E) HEK293T cells were co-transfected with pcDNA3.1-HA-CDKN1A/p21, pcDNA3.1-MYC-MAP1LC3/LC3 and pcDNA3.1-Flag-SQSTM1/p62 for 24 h in the presence of 5 µm baf-A1 for 4 h. Flag, HA, MYC and ACTB (loading control) immunoblots for IP of Flag from HEK-293T cells and the corresponding WCL (n = 3). (F) IP of HA from aVIC co-transfected with pcDNA3.1-HA-CDKN1A/p21 and pcDNA3.1-Flag-SQSTM1/p62 in the presence of 5 µm baf-A1 for 4 h. Immunoblots for anti-ha, Flag, ubiquitin (Ub), and ACTB (n = 3). (G) Representative confocal images of CDKN1A/p21 colocalization with SQSTM1/p62 and MAP1LC3/LC3 in aVICs treated with or without 200 nM rapamycin for 48 h in the absence or presence of 5 µm baf-A1 for 4 h (n = 3). (H) PLA for CDKN1A/p21, CDKN2A/p16 and SQSTM1/p62 on aVICs treated with or without 200 nM rapamycin for 18 h in the presence of 5 µm baf-A1 for 4 h (n = 3). F-actin was stained with phalloidin. Scale bars: 50 µm. (I) Number of PLA dots (CDKN1A/p21-SQSTM1/p62 and CDKN2A/p16-SQSTM1/p62) per cell. (J) the graphs display the fluorescence intensity in each channel (as shown in G) over the distance (µm) depicted by the arrows. (K-M) HEK293T cells were co-transfected with pcDNA3.1-SQSTM1/p62, pcDNA3.1-HA-CDKN1A/p21 and pcDNA3.1-Flag-MAP1LC3/LC3. Flag, HA, SQSTM1/p62 and ACTB (loading control) immunoblots for the three-way IP of SQSTM1/p62, MAP1LC3/LC3, CDKN1A/p21 from HEK-293T cells in the presence of baf-A1 and the corresponding whole-cell lysate (WCL) (n = 2). (N) Schematic summarizing the recruitment of cargoes CDKN2A/p16 and CDKN1A/p21 to phagophores by SQSTM1/p62 sequestration. These experiments were repeated at least two times (n ≥ 2), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 8.

The degradation of CDKN1A/p21 and CDKN2A/p16 is mediated by autophagy receptor SQSTM1/p62. (A-D) Co-ip of MAP1LC3/LC3, SQSTM1/p62, CDKN2A/p16 and CDKN1A/p21 from SQSTM1/p62 WT and SQSTM1/p62 KO aVICs in the presence or absence of 5 µm baf-A1 for 6 h. Whole-cell lysate (WCL) and immunoprecipitation complexes were analyzed by immunoblotting with anti-CDKN1A/p21, CDKN2A/p16, SQSTM1/p62, MAP1LC3/LC3 and ACTB (loading control) antibodies (n = 3). (E) Quantification of aVICs and qVICs (%) with > 20 CDKN1A/p21 puncta transfected with scramble or SQSTM1/p62-specific siRNA as shown in (F). (F) Representative confocal images of CDKN1A/p21 and SQSTM1/p62 in aVICs and qVICs transfected with scramble or SQSTM1/p62-specific siRNA for 24 h (n = 3). (G) Representative images (top) of HA (CDKN1A/p21), SQSTM1/p62, MAP1LC3/LC3 and ACTB (loading control) immunoblots of HEK293T cells transfected with empty vectors or pcDNA3.1-HA-CDKN1A/p21 plasmids in the presence or absence of 200 nM rapamycin for 48 h and/or 5 µm baf-A1 for 4 h (n = 3). CDKN1A/p21:ACTB ratio (bottom) normalized to SQSTM1/p62 knockdown (KD) HEK293T treated with rapamycin and baf-A1. (H) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62 and ACTB (loading control) immunoblots of aVICs transfected with scramble or SQSTM1/p62-specific siRNA in the presence or absence of 200 nM rapamycin for 48 h (n = 6). CDKN1A/p21, CDKN2A/p16:ACTB ratio (right panel) normalized to SQSTM1/p62 knockdown (KD) aVICs treated with rapamycin. (I) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62 and ACTB (loading control) immunoblots of SQSTM1/p62 KO HEK293T cells transfected with empty vector or pcDNA3.1-Flag-SQSTM1/p62 plasmids in the presence or absence of 200 nM rapamycin for 48 h (n = 3). CDKN1A/p21, CDKN2A/p16:ACTB ratio (right panel) normalized to SQSTM1/p62 KO HEK293T cells in the absence of rapamycin. (J) Representative images (left panel) of CDKN1A/p21, CDKN2A/p16, SQSTM1/p62 and ACTB (loading control) immunoblots of aVICs and qVICs transfected with scramble or SQSTM1/p62-specific siRNA 24 h (n = 6). CDKN1A/p21, CDKN2A/p16:ACTB ratio (right panel) normalized to aVICs with scrambles. (K) Representative images of SA-GLB1/β-gal staining in SQSTM1/p62 WT and SQSTM1/p62 KO qVICs transfected with empty vectors or pcDNA3.1-Flag-SQSTM1/p62 plasmids. Scale bars: 100 µm (n = 3). (L) Percentage of SA-GLB1/β-gal positive cells. (M) Quantification of ROS fluorescence intensity for SQSTM1/p62 WT and SQSTM1/p62 KO qVICs transfected with empty vectors or pcDNA3.1-Flag-SQSTM1/p62 plasmids (n = 3). (N) Quantification of secreted TGFB1, IL6 and MMP9 cytokine expression detected by ELISA in collected supernatant from qVIC cultures transfected with empty vectors or pcDNA3.1-Flag-SQSTM1/p62 plasmids (n = 3). (O) Quantification of BrdU incorporation for SQSTM1/p62 WT and SQSTM1/p62 KO qVICs transfected with empty vectors or pcDNA3.1-Flag-SQSTM1/p62 plasmids (n = 4). These experiments were repeated at least three times (n ≥ 3), and results are presented as mean ± SEM. ANOVA followed by Tukey’s range test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ^#p <0.05; ^##p <0.01; ^###p <0.001).

Figure 9.

Schematic illustration of the proposed model for the autophagic degradation of CDKN1A/p21 and CDKN2A/p16.