Fig 2: ADPr-enriched p62 bodies contain ubiquitin but lack autophagy marker LC3B.(A) Schematic of p62 domain architecture. (B) p-STAT1, STAT1, HO1, and NQO1 levels were measured in A549 wild-type (WT) and p62 knockdown (KD) cells treated with different combinations of treatments with RBN (10 µM) and IFNγ. (C) IL6 mRNA levels were measured in A549 cells treated with different combinations of RBN (10 μM) pretreatment for 1 h and IFNγ treatment for either 6 or 24 h by RT-qPCR. (D–I) A549 cells after 24-h treatment with or without IFNγ were analyzed for colocalization between (D) ADPr and autophagy marker LC3B, n = 118–182 condensates, (E) ADPr and ubiquitination (FK2), n = 45–65 condensates, (F) PARP14 and Ub-K48, n = 102–125 condensates, (G) ADPr and NBR1, n = 60–61 condensates, (H) ADPr and TAX1BP1, n = 38-79 condensates, and (I) ADPr and FIP200, n = 58 condensates. (J) Heatmap showing proteins identified using mass spectrometry (MS)-based proteomics analysis, following immunoprecipitation of proteins with p62 from A549 cell lysates which were either untreated or treated with IFNγ for 24 h. IgG rabbit antibody was used as a control. Coloring indicates z-score, ranging from black (z < 0), to blue (z = 1), to cyan (z > 2). (K) Volcano plot of MS-identified proteins showing significant changes in the p62 interactome after 24 h of IFNγ treatment. Key p62 interactors mentioned in the text are bolded, while other interactors with increased association (log2 ratio >1.5 and −log10 p value >3) are labeled. Significance testing via two-tailed Student’s t-test, corrected for multiple-hypotheses testing via permutation-based FDR control (corrected p value <0.1%); n = 4 biological replicates. (L) Term enrichment analysis showing UniProt keywords statistically enriched within the p62-specific network after IFNγ treatment. Statistical testing was conducted using the STRING database with default settings. White arrows indicate the position of condensates, and white boxes represent the zoomed-in regions. The total area of condensates and Pearson correlation coefficient were analyzed by CellProfiler. Mean ± SEM, ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unless otherwise stated. (C) One-way ANOVA; (D–I) Unpaired t-test. All p values are provided in Dataset EV2. Data were representative of three biological replicates, unless otherwise stated. Scale bar, 10 µm. Source data are available online for this figure.

Fig 3: Lab-Vpr but not TFV-Vpr associates with NDP52, SQSTM1 and TAX1BP1 autophagy-cargo receptors.HEK293T cells were transfected with 2,000 ng of either Lab-Vpr or TFV-Vpr. 48 h later, cells were harvested, and lysates were immunoprecipitated for BNIP3L (A), OPNT (B), NBR1 (C), SQSTM1 (D), TAX1BP1 (E), or NDP52 (F). The pulldown fraction was analyzed for each of these autophagy receptors and Vpr. The whole cell lysates were also analyzed for these autophagy factors, Vpr and ACTB. Blots are representative of 3 independent experiments. (G) Lab-Vpr was transfected in parental, SQSTM1KO/TAX1BP1KO and SQSTM1KO/TAX1BP1KO/NDP52KO knockout cells. 44 h later, cells were treated with increasing amounts of Torin2 (5-10 nM) for 4 h. Cells were then harvested and analyzed by western blot for Vpr, SQSTM1, TAX1BP1, NDP52 and ACTB. Blots are representative of 3 independent experiments. Graph: Vpr levels were quantified by normalizing to ACTB, and their relative expression compared to WT cells treated with DMSO (D on the x axis) were calculated from 3 independent experiments. Data correspond to the mean and the SEM of 3 independent experiments. *: p < 0.05.