Figure 4. p62 oligomerization is critical in lysophagy.

(A) Schematic of p62 domain architecture, highlighting the N-terminal Phox and Bem1p (PB1) domain, LC3-interacting region (LIR), and ubiquitin-associated domain (UBA).

(B) Representative images of α-LAMP1 and either mCherry vector WT-p62, p62ΔPB1, or p62ΔUBA in p62KO HeLa after addition of 750 μM LLOMe for 2 h. Cell outlines are traced. SBs: 10 μm.

(C) Quantification of α-LAMP1 area overlapping with p62 area across different conditions (one-way ANOVA, p_(WT/mCherry) < 0.001, p_(WT/ΔPB1) < 0.001, p_(WT/ΔUBA) < 0.01, n = 3 experiments. Number of cells analyzed per condition ≥ 33).

(D) Representative images of α-LC3B in p62KO HeLa, which were transiently transfected with Flag-LAMTOR2 and either mCherry vector, WT-p62, p62ΔPB1, or p62ΔUBA after addition of 750 μM LLOMe for 2 h. Cell outlines are traced. SBs: 10 μm.

(E) Quantification of normalized α-LC3B intensity at FLAG-LAMTOR2 puncta across different conditions (Kruskal-Wallis test, p_(WT/mCherry) < 0.0001, p_(WT/ΔPB1) < 0.001, p_(WT/ΔUBA) > 0.9999, p_{(ΔUBA/ΔPB1)} < 0.001, n = 4 experiments. Number of cells analyzed per condition ≥ 36).

(F) Representative images of mCherry and HaloTag-WIPI2b in HeLa, which were co-transfected with control siRNA or p62 siRNA (#1). Cells were transfected with different mCherry rescue constructs, which were mCherry vector, WT-p62, p62ΔPB1, p62-LIR^AAAA, or p62ΔUBA after addition of 750 μM LLOMe for 2 h. Cell outlines are traced. SBs: 10 μm.

(G) Quantification of the number of WIPI2b puncta per cell following LLOMe treatment (one-way ANOVA, p_{(control-si+mCherry/p62-si+mCherry)} < 0.01 (not depicted), p_{(control-si+mCherry/p62-si+WT)} = 0.9996, p_{(p62-si+WT/p62-si+mCherry)} < 0.01, p_{(p62-si+WT/p62-si+ΔPB1)} < 0.05, p_{(p62-si+WT/p62-si+LIRmut)} = 0.6880, p_{(p62-si+WT/p62-si+ΔUBA)} = 0.1794 , n = 3 experiments. Number of cells analyzed per condition ≥ 20).

All error bars reflect mean ± SEM. See also Figures S3, S4, and S5.