Figure 2. p62 responds to lysosomal damage in human and rat neurons.

(A) Representative max projections of α-p62 and LAMP1-mNeonGreen in human iPSC-derived neurons (i³Neurons), which were treated with EtOH or 1.0 mM LLOMe for 2 h. Cell outlines are traced. White boxes indicate inset image region. Arrowheads indicate areas of co-localization. Inset images are single z-plane images. SBs: 10 αm; insets: 2 αm.

(B) Quantification of the overlapping area from max projections between α-p62 and LAMP1-mNeonGreen in cells treated with EtOH or with LLOMe (unpaired t test, p < 0.001, n = 4 experiments. Number of cells analyzed per condition ≥ 54).

(C) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.5047, n = 4 experiments. Number of cells analyzed per condition ≥ 54).

(D) Representative max projections of images comparing α-TAX1BP1 localization with lysosomes in i³Neurons following treatment with either EtOH or 1.0 mM LLOMe for 2 h. Cell outlines were traced. SBs: 10 αm.

(E) Quantification of the percentage of LAMP1 area occupied by α-TAX1BP1in cells treated with EtOH or with LLOMe (unpaired t test, p < 0.05, n = 4 experiments. Number of cells analyzed per condition ≥ 45).

(F) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.6003, n = 4 experiments. Number of cells analyzed per condition ≥ 45).

(G) Representative max projections of images comparing α-NDP52 localization with lysosomes in i³Neurons following treatment with either EtOH or 1.0 mM LLOMe for 2 h. Cell outlines were traced. SBs: 10 μm.

(H) Quantification of the percentage of LAMP1 area occupied by α-NDP52 in cells treated with EtOH or with LLOMe (unpaired t test, p = 0.0708, n = 4 experiments. Number of cells analyzed per condition ≥ 45).

(I) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.4643, n = 4 experiments. Number of cells analyzed per condition ≥ 45).

(J) Representative images max projections comparing α-OPTN localization with lysosomes in i³Neurons following treatment with either EtOH or 1.0 mM LLOMe for 2 h. Cell outlines were traced. SBs: 10 μm.

(K) Quantification of the percentage of LAMP1 area occupied by α-OPTN in cells treated with EtOH or with LLOMe (unpaired t test, p = 0.2476, n = 4 experiments). Number of cells analyzed per condition ≥ 50).

(L) Quantification of LAMP1 area from max projections in the different conditions (unpaired t test, p = 0.8482, n = 4 experiments. Number of cells analyzed per condition ≥ 50).

(M) Representative images of rat hippocampal neurons 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 Figure 1N. SBs: 10 μm.

(N) Inset images of LAMP2-BFP and EGFP-p62 from Figure 1M of rat hippocampal neurons following 45-min post-laser activation. SBs: 2 μm.

(O) Quantification of LAMP1-KillerRed intensities within irradiated or untreated regions.

(P) Quantification of EGFP-p62 intensities within irradiated or untreated regions.

All error bars reflect mean ± SEM. See also Video S2.