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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 1. p62 is dynamically recruited to damaged lysosomes in HeLa cells.

Figure 1.

(A) Western blot analysis of lysosomal immunoprecipitation following either no treatment (−) or treatment with 1.0 mM LLOMe (+) for 1 h.

(B–F) Quantification of lysosomal immunoprecipitation following normalization to α-LAMP1 intensity in either untreated (Unt) or LLOMe-treated HeLa cells. (B) α-FIP200 band intensities (unpaired t test, p < 0.05, n = 3). (C) α-TAX1BP1 band intensities (unpaired t test, p < 0.001, n = 3). (D) α-p62 band intensities (unpaired t test, p < 0.01, n = 3). (E) α-GABARAP band intensities (unpaired t test, p < 0.05, n = 3). (F) α-LC3B band intensities (unpaired t test, p < 0.001, n = 3).

(G) Single z-plane representative images of endogenous α-LAMP1 and α-p62 in HeLa cells treated with ethanol (EtOH) as a vehicle control or LLOMe (at 250 or 750 μM) for 2 h. Cell outlines are traced. White boxes indicate inset image region. Arrowheads indicate regions of co-localization. Scale bars (SB): 10 μm; insets: 2 μm.

(H) Quantification of the overlapping area from maximum (max) projections between endogenous α-p62 and α-LAMP1 in cells treated with EtOH or different concentrations of LLOMe (one-way ANOVA, p(EtOH/250) < 0.001, p(EtOH/750) < 0.0001, n = 3 experiments. Number of cells analyzed per condition ≥ 54).

(I) Quantification of LAMP1 area from max projections for the experiments shown in (H) (one-way ANOVA, p(EtOH/250) = 0.96, p(EtOH/750) = 0.9766, n = 3 experiments. Number of cells analyzed per condition ≥ 54).

(J) Representative images of HeLa cells transiently transfected with LAMP1-KillerRed, LAMP2-BFP, and EGFP-p62. Cell outlines are traced. Blue boxes reflect regions irradiated. Arrowheads reflect location of inset images in (K). SBs: 10 μm.

(K) Inset images from (J), showing EGFP-p62 recruitment to lysosomes in HeLa cells at 60 min post-laser irradiation. SBs: 2 μm.

(L) Quantification of LAMP1-KillerRed intensities within irradiated or Unt regions from the same cells.

(M) Quantification of EGFP-p62 intensities within irradiated or Unt regions.

(N) Single z-plane representative images of endogenous α-LAMP1, α-p62, and α-GABARAP/L1/L2 in HeLa cells, which were treated with EtOH or 750 μM LLOMe for 2 h. Cell outlines are traced. White boxes indicate inset region. Arrowheads in inset regions indicate co-localization. SBs: 10 αm; insets: 2 αm.

(O) Quantification of the percentage of α-LAMP1 occupied by α-GABARAPs (unpaired t test, p < 0.01, n = 3 experiments. Number of cells analyzed per condition ≥ 29).

(P) Quantification of the percentage of α-GABARAPs rings (in single z-plane) co-localizing with α-p62 (% p62+: 94% ± 0.49%). Data are represented as mean ± SEM.

(Q) Quantification of α-LAMP1 area per cell (unpaired t test, p = 0.9117, n = 3 experiments. Number of cells analyzed per condition ≥ 29).

All error bars reflect mean ± SEM. See also Video S1.