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. Author manuscript; available in PMC: 2023 Oct 23.
Published in final edited form as: Cell Rep. 2023 Jan 25;42(2):112037. doi: 10.1016/j.celrep.2023.112037

Figure 6. The small heat shock protein HSP27 regulates p62 condensates to promote lysophagy.

Figure 6.

(A) Representative images of HaloTag-DFCP1 and EGFP-HSP27 puncta in HeLa cells after addition of 750 μM LLOMe over 2 h. Arrowheads indicate co-localization. SBs: 2 μm.

(B) Quantification of total EGFP-HSP27 puncta and the fraction of EGFP-HSP27 puncta that co-localize with HaloTag-DFCP1 in HeLa cells (two-way ANOVA, p = 0.1160, n = 7 cells per condition).

(C) Representative max projections of endogenous galectin-3 flux assay. Cells were fixed immediately following LLOMe treatment or 15 h post-washout of LLOMe. Cells were transiently transfected with one of two ATG5 siRNAs, one of two HSP27 siRNAs, or a control siRNA. Cell outlines are traced. SBs: 10 μm.

(D) Quantification of the galectin-3 flux assay, indicating the fraction of cells with n > 3 galectin-3 puncta per cell (one-way ANOVA, post-LLOMe: p(control/ATG5#1) =0.9283, p(control/ATG5#2) > 0.9999, p(control/HSP27#1) > 0.9999, p(control/HSP27#2) > 0.9991; post-washout p(control/ATG5#1) < 0.0001, p(control/ATG5#2) < 0.0001, p(control/p62#1) < 0.0001, p(control/p62#2) < 0.0001 p(ATG5 #1/ATG5#2) > 0.9999, p(HSP27 #1/HSP27#2) = 0.0571, n = 3 experiments).

(E) Representative images of α-LAMP1, α-p62, and α-GABARAPs in HeLa cells following transient transfection of control siRNA or HSP27 siRNA (#1). White boxes indicate inset image region. Arrowheads indicate co-localization. SBs: 10 μm; insets: 2 μm.

(F) Quantification of the percentage of area of α-p62 that is occupied by α-GABARAPs (Mann-Whitney U test, p < 0.05, n = 4 experiments. Number of cells analyzed per condition ≥ 57).

(G) Quantification of α-GABARAPs fluorescence intensity at α-p62 segmented area across conditions (Mann-Whitney U test, p < 0.0001, Number of cells analyzed per condition ≥ 57).

(H) Representative images of fluorescence recovery of EGFP-p62 following photo-bleaching in cells transiently transfected with control siRNA or HSP27 siRNA (#1). Cells were treated with LLOMe for 1 h before photo-bleaching, and cells were imaged for 20 min following photo-bleaching. SBs: 2 μm.

(I) Quantification of fluorescence recovery after photo-bleaching of EGFP-p62 (two-way ANOVA, p < 0.0001, n = 9 puncta per condition).

(J) Quantification of the immobile fraction of EGFP-p62 across conditions (unpaired t test, p < 0.05, n ≥ 8 puncta per condition).

All error bars reflect mean ± SEM. See also Videos S6 and S7 and Figure S6.