Fig 2: Deletion of ATP6AP2 exacerbates progression of TAC-induced hypertrophy to heart failure in mice accompanying NLRP3 inflammasome expression upregulation.A The gross morphology of mice hearts in different groups is shown. B, D, E, F, G Representative images of echocardiograms and quantitative analysis of LVEF, LVFS, LVEDD, and LVESD between different groups (n = 4 for each group). Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. C, H Representative images of Masson trichrome staining of mice hearts and quantitative analysis. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. C, I, J Representative immunohistochemical staining and quantitative analysis about staining of NLRP3 and immunofluorescence images of SQSTM1/P62. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. K, L quantitative analysis of ELISA measurement of serum levels of IL-1β, TNF-α expression levels. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. Data were presented as medians and quartiles.

Fig 3: ATP6AP2 is required for the completion of autophagy and its knockdown inhibits V-ATPase-driven lysosome acidification.A, B Representative fluorescent images are shown. Manually quantitative analysis indicates the mean number of autophagosomes (yellow) and autolysosomes (red) per cell. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. **P < 0.01. C, D Autophagy and mitophagy levels showed by transmission electron microscopy in NRVMs transfected with shR-ATP6AP2 knockdown vector or Ad-ATP6AP2 overexpression vector. Representative images of normal mitochondria and mitochondria in-taken by vacuole structure can be seen. E, F indicated mitophagy percentage reflected the number of autophagosomes containing mitochondria per total number of mitochondria from a cross-sectional assessment of the NRCMs. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. Data were presented as medians and quartiles. G Representative fluorescent images of LysoTracker Red staining in shR-Scr and shR-ATP6AP2 NRVMs treated with DMSO or bafilomycin A1 for 60 min. H the effect of autophagy inducer or inhibitor on IL-1β mRNA expression in NRVMs while sh-ATP6AP2 was transfected. GAPDH was used as the loading control. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. At least four independent experiments were conducted. *P < 0.05, **P < 0.01.

Fig 4: Phenotype changes in shR-ATP6AP2-TAC mice can be partly relieved through blocking NLRP3 inflammasome activation.C57BL/6 J mice were subjected to TAC, then adenovirus-mediated shR-ATP6AP2 vectors or Scr-control were injected into the tail vein of mice (n = 4 for each group), followed by MCC950 (5 mg/kg/day) or vehicle control. Mice were observed at 28days. A–C Representative images of echocardiograms and quantitative analysis of LVEDD, LVESD between different groups. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. *P < 0.05 compared with others. D Quantitative analysis of heart weight/body weight ratios (HW/BW; mg/g). Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. *P < 0.05 compared to other groups. E, F Representative images of Masson trichrome staining and quantitative analysis. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. E, G, H Representative immunohistochemical staining and quantitative analysis of NLRP3, Caspase1 expression. (scale bar = 20 μm). Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. E, I Representative hematoxylin and eosin (HE) staining and quantitative analysis of cardiomyocytes cross-sectional area (scale bar = 20 μm). Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. *P < 0.05 compared with other groups. E, J Representative immunofluorescence images of TUNEL assay showed apoptosis percentage in mouse heart tissues. Statistical analysis was conducted by Kruskal–Wallis one-way ANOVA with Dunn post-hoc test. Data were presented as medians and quartiles.

Fig 5: PACE4 inhibition, like PRR knockdown, reduces V-ATPase activity.(A) Representative proliferation images of LNCaP exposed to both DMSO or a 100 μM of PACE4 inhibitor [33] LLLRVK-amidinobenzylamide (Amba) (C23) over 0 and 64 hours. (B) Western blot analysis of soluble prorenin receptor (sPRR) secretion in conditioned media of LNCaP exposed to DMSO or a 100 μM C23. (C) Representative LysoTracker images of LNCaP cells transfected with non-silencing control siRNA (NSC), PRR siRNA 1 and 2, PACE siRNA, or treated with 100 nM Bafilomycin A1. (D) Quantification of LysoTracker signal intensity relative to number of cells in brightfield images treated with 1% DMSO (Vehicle), 50 μM C23, 100 nM Bafilomycin A1 (BafA1), or transfected with PRR siPRR 1 or siPRR 2. (E) Schematic showing the PRR-HA, sPRR-HA, and M8.9-HA vectors used in panels F, G and H. (F) Representative LysoTracker images of LNCaP cells transfected with empty vector (EV) and treated with DMSO, treated with 100 μM C23, transfected with PRR-HA and treated with 100 μM C23, transfected with M8.9-HA and treated with 100 μM C23, transfected with sPRR-HA and treated with 100 μM C23, or treated with 100 nM BafA1. (G) Corresponding quantification of LysoTracker intensity relative to cell number in the same treatments (**P ≤ 0.01, ***P ≤ 0.001, **** P < 0.0001, n = 4). (H) Western blot analysis of PRR and HA expression in the same treatments. Total lane protein (TLP) was used as loading control. Data are presented as the mean ±SEM. Statistical tests were conducted using Student’s t test. Scale bar measures 100.