Figure 5.

Compounds do not affect the rate of Parkin translocation to mitochondria nor the number of mitochondria within lysosomes

(A) The half-max time (T₅₀) for W403A-Parkin translocation to mitochondria is within 40 min, the half-max time for WT- Parkin is 90 min, and the half-max time for C431A Parkin is never reached (inactive).

(B) The translocation time for W403A-Parkin is 56% faster than WT-Parkin.

(C) Calculated fold change in T₅₀ relative to compound concentration for active (green), partially active (orange) and inactive (gray) THPP series compounds.

(D–F)(D) BIO-2007817, (E) BIO-2007818 and (F) BIO-1984542 (10 μM) translocation curves relative to control (DMSO with CCCP treatment).

(G) Baseline imaging for MitoQC in SH-SY5Y cells. MitoQC is a pH-sensitive mitochondrial fluorescent probe. 24 h of 5 μM Antimycin +10μM Oligomycin stimulates an increased level of mCherry puncta signal indicating mitolysosomes, whose pH is neutralized with BafA1.

(H) Quantitation of number of mitochondria in lysosomes under different conditions described in (G).

(I) At a fixed timepoint (8 h) there is no dose-dependent increase in mitolysosomes for the active compound (green) relative to the reduced activity compound (orange) or inactive compound (gray). In A, n = 2 technical replicates. In C, n = 3 biological replicates. In (D)–(F) n = 2 technical replicates and n = 3 biological replicates. See also Figure S5.