Figure 5. The small heat shock protein HSP27 and p62 dynamically interact in lysophagy.

(A) Representative images of mCherry-LAMTOR2 and EGFP-HSP27 puncta following addition of 750 μM LLOMe in HeLa cells over 2 h. Arrowheads indicate co-localization over time. SBs: 2 μm.

(B) Quantification of total mCherry-LAMTOR2 puncta per cell in HeLa cells following treatment with 750 μM LLOMe (n = 7 cells).

(C) Quantification of total EGFP-HSP27 puncta per cell in HeLa cells following treatment with 750 μM LLOMe (n = 7 cells).

(D) Quantification of total mCherry-LAMTOR2 puncta and the fraction of mCherry-LAMTOR2 puncta that co-localize with EGFP-HSP27 in HeLa cells (two-way ANOVA, p < 0.0001, n = 7 cells).

(E) Western blot analysis of HeLa lysates for HSP27, phospho-S15 HSP27, and α/β-tubulin that were untreated (Unt) or treated with 750 μM LLOMe for 2 h.

(F) Quantification of α-phospho-S15 HSP27 band intensities normalized to α-HSP27 intensity (unpaired t test, p < 0.0001, n = 6 experiments).

(G) Western blot analysis of HeLa lysates for HSP27, phospho-S78 HSP27, and α/β-tubulin that were Unt or treated with 750 μM LLOMe for 2 h.

(H) Quantification of α-phospho-S78 HSP27 band intensities normalized to α-HSP27 intensity (unpaired t test, p < 0.0001, n = 6 experiments).

(I) Western blot analysis of HeLa lysates for HSP27, phospho-S82 HSP27, and α/β-tubulin that were Unt or treated with 750 μM LLOMe for 2 h.

(J) Quantification of α-phospho-S82 HSP27 band intensities normalized to α-HSP27 intensity (unpaired t test, p < 0.0001, n = 6 experiments).

(K) Representative max projections of α-LAMP1, α-p62, or α-phospho-S82 HSP27 in HeLa cells, following treatment with either EtOH or 750 μM LLOMe. White boxes indicate inset image region. Arrowheads indicate co-localization. SBs: 10 μm; insets: 2 μm.

(L) Quantification of the overlapping area from max projections between μ-phospho-S82 HSP27 and μ-LAMP1 in cells treated with EtOH or with LLOMe (unpaired t test, p < 0.05, n = 3 experiments. Number of cells per condition ≥ 42).

(M) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.8827, n = 3 experiments. Number of cells per condition ≥ 42).

(N) Quantification of the overlapping area from max projections between α-p62 and α-phospho-S82 HSP27 in the different conditions (unpaired t test, p < 0.001, n = 3 experiments. Number of cells per condition ≥ 42).

(O) Representative images of mCherry-p62 and EGFP-HSP27 in HeLa cells following addition of 750 μM LLOMe over 2 h. Arrowheads indicate co-localization over time. SBs: 2 μm.

(P) Quantification of total EGFP-HSP27 puncta and the fraction of EGFP-HSP27 puncta that co-localize with mCherry-p62 over time (two-way ANOVA, p < 0.01, n = 4 cells).

(Q) Quantification of EGFP-HSP27 and mCherry-p62 fluorescence intensities normalized to the fluorescence at last frame analyzed. Puncta were selected if they remained in z-plane for 1 h (two-way ANOVA, p < 0.0001, n = 9 puncta per condition).

(R) Schematic of p62 domain architecture, highlighting K7 and D69, in the PB1 domain of p62. Alanine substitution of these residues prevents homo-oligomerization.

(S) Representative single z-plane images of EGFP-HSP27 in p62KO HeLa cells that were co-transfected with either WT, p62ΔPB1, or K7A/D69A p62. Cells were treated with 750 μM LLOMe for 2 h. Cell outlines are traced. SBs: 10 μm.

(T) Quantification of the number of EGFP-HSP27 puncta per cell (one-way ANOVA, p_(WT/ΔPB1) < 0.05, p_{(WT/(K7A/D69A))} < 0.01, p_{(ΔPB1/(K7A/D69A)} = 0.2131, n = 3 experiments. Number of cells per condition ≥ 21).

(U) Quantification of the cell area per cell (one-way ANOVA, p_(WT/ΔPB1) = 0.5725, p_{(WT/(K7A/D69A))} = 0.5899, p_{(ΔPB1/(K7A/D69A)} = 0.9995, n = 3 experiments. Number of cells per condition ≥ 21).

(V) Quantification of GFP fluorescence intensity per cell (one-way ANOVA, p_(WT/ΔPB1) = 0.9997, p_{(WT/(K7A/D69A))} = 0.8625, p_{(ΔPB1/(K7A/D69A)} = 0.8506, n = 3 experiments. Number of cells per condition ≥ 21).

(W) Representative Western blot demonstrating EGFP-HSP27 co-immunoprecipitation with either WT, p62ΔUBA, p62ΔPB1, K7A/D69A p62, or mCherry vector.

(X) Quantification of co-immunoprecipitation of mCherry-tagged constructs with EGFP-HSP27 (Kruskal-Wallis test, p_{(mCherry/WT-p62)} < 0.05, p_{(mCherry/ΔUBA)} < 0.01, p_{(mCherry/ΔPB1)} = 0.1922, p_{(mCherry/(K7A/D69A))} < 0.05, n = 5 experiments).

All error bars reflect mean ± SEM. See also Videos S4 and S5 and Figure S6.