Fig 1: Poly-GA exposure does not lead to major lysosomal damage but alters lysosomal acidification.(A) Quantification of relative expression levels after RT-qPCR for genes involved in lysosomal metabolism. RNA was isolated from primary mouse cortical neurons (10 d in culture) exposed or not to 1 µM poly-GA oligomers or 1 µM poly-GA fibrils for 24 h, retrotranscribed and subjected to quantitative real-time PCR. Only the transcriptional levels of ATP6V0E1 (a component of the V-ATPase) were increased upon treatment with poly-GA fibrils compared with control untreated neurons. One-way ANOVA with Tukey’s multiple-comparisons test. **P = 0.01. The data were collected from three independent biological replicates. (B) Immunofluorescence assays were used to compare the protein levels of cathepsins L, B, and D in primary mouse cortical neurons exposed to 1 µM dipeptide repeats (DPRs) for 24 h vs untreated neurons. The cathepsin levels in DPR-treated and untreated cells were comparable. One-way ANOVA with Tukey’s multiple-comparisons test. ns = P > 0.05. The data were collected from two independent biological replicates. (C) Primary mouse cortical neurons were exposed to 1 µM poly-GA DPRs for 24 h (or were left untreated), and Lysosome-Specific Self-Quenched Substrate (#ab234622; Abcam) was added during the final 1 h of the 24-h period to measure lysosomal in situ enzyme activity. Scale bars = 5 µm. (D) Mean fluorescence intensity of Lysosome-Specific Self-Quenched Substrate was quantified per neuronal cell. Bar graphs of mean ± SEM. One-way ANOVA with Tukey’s multiple-comparisons test. ns = P > 0.05. The data were collected from three independent biological replicates. (E) The galectin-3 immunofluorescence assay was used to compare galectin-3 protein levels in HeLa cells exposed or not exposed to 1 µM poly-GA fibrils for 24 h. During the last 1 h of the 24-h span, the lysosomotropic drug LLoMe (at 1 mM) was used to induce lysosomal damage, which is visible by galectin-3 puncta formation (positive control). Scale bars = 10 µm. (F) HeLa cells were exposed to 1 µM poly-GA DPRs for 24 h (or were left untreated), and LysoSensor Green DND-189 was added during the final 1 h of the 24-h period to measure changes in lysosomal pH (this dye exhibits increasing fluorescence as pH decreases). Scale bars = 20 µm. (G) Regardless of LLoME treatment, the levels of galectin-3 did not differ between HeLa cells exposed or not exposed to poly-GA fibrils. Bar graphs of mean ± SEM. Unpaired two-tailed t test with Welch’s correction. ns = P > 0.05. The data were collected from three independent biological replicates. (H) Mean fluorescence intensity of LysoSensor was quantified in live HeLa cells. Bar graphs of mean ± SEM. One-way ANOVA with Tukey’s multiple-comparisons test. ****P = 0.0001. The data were collected from three independent biological replicates.