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. 2025 Jul 29;44(18):5086–5111. doi: 10.1038/s44318-025-00517-x

Figure 3. ATXN3 is required for completion of lysophagy, but not for phagophore formation.

Figure 3

(A) Lysophagy assay. HeLa cells stably expressing pH-sensitive mKeima fused to the cytosolic terminus of TMEM192. Cells were transfected with indicated siRNAs and treated with LLOMe for 1 h, followed by a 6 h washout. mKeima fluorescence was assessed at the indicated excitation wavelengths, and the ratio between both intensities was determined. Note that the increase in the 561 nm/458 nm ratio is reduced in ATXN3-, ATG5-, or ATG7-depleted cells, indicating a defect in autolysosome formation. n = 4 biological replicates with >30 cells per condition per experiment. One-way ANOVA with Holm–Sidak’s multiple comparison test. The line indicates the median. (B) Phagophore formation around damaged lysosomes is not affected by ATXN3 depletion. HeLa cells were treated with indicated siRNAs, incubated with LLOMe before fixation as indicated, and immuno-stained for LC3 and the damage marker Gal3. Note that samples were also costained for p62 (shown in Fig. EV3B). Scale bar, 15 µm. (C) Quantification of (B). n = 3 biologically independent experiments with >70 cells quantified per condition per experiment. Two-way ANOVA with Tukey’s multiple comparison test. The graph shows mean ± SD. (D) Lysophagy flux is compromised in ATNX3 KO cells. Western blot of indicated cell lines and time points. Note that initial LC3 lipidation is not affected by ATXN3 KO at 2 h chase, but the lipidated LC3 form accumulates in ATXN3 KO after 8 h chase. (E) Quantification of (D). n = 4 biologically independent experiments. Two-way ANOVA with Tukey’s multiple comparison test. Error bars represent the mean with SD. (F) Lack of colocalization of ATXN3 with LC3. HeLa cells expressing ATXN3-GFP were mock or LLOMe-treated as indicated, fixed, and stained for LC3. Arrowheads indicate ATXN3-positive and LC3-negative lysosomes. Scale bar, 10 µm. (G) Quantification of (F). n = 3 biological replicates with >30 cells per condition per experiment. One-way ANOVA with Tukey’s multiple comparison test. The line indicates the mean. Source data are available online for this figure.