Figure 3. TPP1 Is the Relevant Substrate of AAF-CMK.

(A) Known cellular targets of AAF-CMK.

(B and C) Z score of percent motile (B) and KS integrated distance (C) in neurons transfected for at least 3 days with either scrambled or TPP2 shRNA (TPP2-i) and treated with 10 μM AAF-CMK 1 h prior to and during time-lapse imaging. TPP2-i neither mimicked nor occluded the effect of AAF-CMK.

(D and E) Lack of effect of 2-h incubation with TPP2 inhibitor butabindide at 10 μM (D) and proteasome inhibitor carfilzomib at 5 μM (E) on percent motile and lack of dose-dependent response.

(F and G) Z scores for percent motile (F) and KS integrated distance (Id) (G) in wells transduced either with scrambled shRNA or with TPP1 shRNAs and treated with either solvent or 10 μM AAF-CMK for 2 h before time-lapse imaging. Two non-overlapping sequences targeting TPP1 were used (TPP1-i 1 and 2) (Z scores of KS displacement were similar).

(H) Effect of TPP1 over expression (OE) on the three motility descriptors.

At least 4 wells per condition (each well representing the traces collected from more than 10,000 mitochondria) were used for the calculation of Z scores. Statistical significance between groups of Z scores was determined using an unpaired two-tailed t test. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns = p > 0.05. Data in all panels represented as Tukey’s box plots.