Figure 3. Q73 expression in H9C2 cells leads to lysosomal dysfunction.

A. Levels of Mfn2, VDAC and LAMP1 were determined by immunoblotting of lysosomal fraction from H9C2 cardiomyocytes transfected with either Q23 or Q73 repeats for 48 hours and then treated with P110 (1μM once) or Veh, for an additional 24 h in serum free- galactose medium. LAMP-1 was used as lysosomal marker, quantified and presented as ratio vs β-actin B. Levels of aconitase and TOM20 were determined by immunoblotting of total lysates, levels were quantified and presented as ratio vs actin (loading control), from cells treated as in A. C. Aconitase activity in H9C2 cardiomyocytes, treated as in A. D. Lysosomal function was measured using LysoSensor™ yellow/blue DND-160 in H9C2 cardiomyocytes, treated as in A. E. Acid phosphatase activity as a secondary readout for lysosomal function was measured in H9C2 cardiomyocytes, treated as in A. F. Autophagic flux was measured using Cyto-ID™ in H9C2 cardiomyocytes, treated as in A. G. Autophagic flux was measured using Cyto-ID™ in H9C2 cardiomyocytes transfected with either Q23 or Q73 repeats for 48 hours and then treated with 3-MA, chloroquine, rapamycin or Veh, for additional 24 h in serum free/galactose medium. H. LDH release was measured as a marker of cell death in H9C2 cardiomyocytes, treated as in G. Data were evaluated by one-way ANOVA and Holm-Sidak's multiple comparisons test for multiple testing between each treatment group. **** p-value <0.0001; *** p-value <0.001; ** p-value <0.01; * p-value <0.05. All graphs represent mean ± s.d. (n=4/5, as indicated in each panel.)