Fig 2: PARP1 mediates starvation induction of autophagy in cultured cardiomyocytes.Primary neonatal rat cardiomyocytes were starved for the indicated different times. a The protein levels of total poly(ADP-ribosyl)ation were detected by western blot analysis. b Cellular PARP activity was assayed as described in Materials and Methods. N = 8 for each group. **P < 0.01 vs. control. c, j Primary rat cardiomyocytes were treated either with PJ34 (10 μM) or 3AB (10 mM), or infected with PARP1 shRNA or an scramble (Scr) shRNA for 24 h, or infected with Ad-PARP1 or Ad-Null and then starved for 24 h. The protein level of DNA damage marker (p-γ-H2AX, p-ATM, p-p53) (c and f), autophagy marker (Gabarapl1, ATG12, LC3), PARP1 and p62 (e, h, and i) were detected by western blot analysis. Representative electron microscopy and GFP-LC3 fluorescence confocal images are displayed to assess the effect on starvation-induced autophagy (d, g, and j)

Fig 3: The influence of PTEN overexpression on CHMP2A-mediated autophagy flux in HK-2 cells and mice subjected to I/R.A Western blotting analysis showing changes in CHMP2A and autophagy flux-related proteins in I/R-treated HK-2 cells in the absence and presence of LV-PTEN. B Representative dual fluorescence of LC3 and P62 staining in I/R-treated HK-2 cells in the absence and presence of LV-PTEN (scale bar, 20 μm). C Representative immunofluorescence images of RFP-GFP-LC3 in I/R-treated HK-2 cells in the absence and presence of LV-PTEN. RFP+GFP+, yellow puncta represent autophagosomes; and RFP+GFP−, red puncta represent autolysosomes (scale bar, 20 μm). D Representative transmission electron microscopic (TEM) images of autophagic structures in I/R-treated HK-2 cells in the absence and presence of LV-PTEN (scale bar, 500 nm). E Western blotting analysis showing changes in CHMP2A and autophagy flux-related proteins in PTENfl-stop-fl, Cre−/− and PTENfl-stop-fl, Cre+/+ mice subjected to BIRI (*P < 0.05 and ***P < 0.001 compared with values in sham + PTENfl-stop-fl, Cre−/− group; ###P < 0.001 compared with values in BIRI + PTENfl-stop-fl, Cre−/− group; n = 6 mice in each group). F Representative dual fluorescence of LC3 and P62 staining in PTENfl-stop-fl, Cre−/− and PTENfl-stop-fl, Cre+/+ mice subjected to BIRI (scale bar, 20 μm). G Representative TEM images of autophagic structures (green arrows) in PTENfl-stop-fl, Cre−/− and PTENfl-stop-fl, Cre+/+ mice subjected to BIRI (scale bar: upper, 2 μm; lower, 1 μm).

Fig 4: NRF2-mediated autophagy dysfunction and ferroptosis are involved in foam cell formation induced by HUA. THP-1 and RAW264.7 cells were treated with oxLDL (100 μg/ml) or coincubated with UA (15 mg/dl) for 24 h. (a and b) qPCR was used to detect the transcription of lipid metabolism-related genes (CD36, ABCA1, and ABCG1) and ferroptosis-related genes (NFE2L2, SLC7A11, and GPX4) in macrophage-derived foam cells. (c and d) Representative western blot images of the protein level of the NRF2/SLC7A11/GPX4 signaling pathway and autophagy-related proteins (LC3B and p62). (e and f) Quantification of NRF2, SLC7A11, GPX4, LC3B, and p62 protein levels. Data are means ± SD, n = 3. ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1; HUA: high level of uric acid; oxLDL: oxidized low-density lipoprotein; qPCR: quantitative PCR. ns indicates no significance. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Fig 5: Detection of direct interaction between LC3B and ATP7B via LIR3. (a) Structural organization of ATP7B indicating the location of the three potential LIR peptides on ATP7B protein. (bi) Fluorescent polarization curves with a nonlinear curve fitting of the one site-specific binding. (bii) Quantification of Kd values from (bi), Experiments were done in three replications. (c) Protein peptide docking. The structural LC3B/LIR3-ATP7B complex was modeled on the crystal structure of the LC3B-LIR of SQSTM/p62 complex (PDB: 2ZJD) using GalaxyWEB server [30]. The LC3B protein is shown in green, the LIR3-ATP7B is shown in magenta. The hydrophobic residues are shown in yellow and the hydrogen bonds are indicated with the red dashed line. (d) Protein pull-down assay. The resin bound GST-LC3B protein was used as bait to pull WT and variant ATP7BW1452A,L1455A from the cell lysate of transfected HEK293H. The pull-down experiment was repeated two times. The input, flow-through, final wash and elution fractions are indicated as IP, FT, W and E, respectively. ATP7B in the elution fraction is indicated with the black dashed box.