Fig 2: Tid1 knockdown significantly decreases the ability of p53-R9 to induce apoptosis.A qPCR confirming Tid1 knockdown in mCherry and mCh-p53-R9 expressing U2OS TP53-/- cells at the mRNA level. B Immunoblot confirming Tid1 knockdown in mCherry and mCh-p53-R9 U2OS TP53-/- cells at the protein level. Blots were probed with antibodies to Tid1, mCherry, and GAPDH. C U2OS TP53-/- cells expressing mCherry or mCh-p53-R9 were transfected with scramble or Tid1 siRNA and analyzed for Caspase-3/7 activity (Caspase-3/7 green apoptosis assay reagent used with the Incucyte Live-Cell Imaging and Analysis System). Area under the curve (AUC) was calculated for N = 3 experiments; Welch’s t test of AUCs from mCherry scramble siRNA versus mCh-p53-R9 scramble siRNA and mCh-p53-R9 scramble siRNA versus mCh-p53-R9 Tid1 siRNA, *p < 0.05. Welch’s t test of AUCs from mCherry Tid1 siRNA versus p53-R9 Tid1 siRNA, ns p > 0.05. Error bars represent standard deviation of three images/well taken in three wells of a representative experiment.

Fig 3: p53-R9 binds translocation chaperone Tid1, and p53-R9 localization at the mitochondria requires Tid1 expression.A U2OS TP53−/− cells were transfected to express GFP, GFP-p53-R9, GFP-e. p53, or GFP-h. p53. Cells (106) were lysed 8 h after transfection, then immunoprecipitation was performed with GFP-Trap beads. Immunoprecipitates were analyzed for Tid1 and GFP expression via immunoblot. B Representative confocal images of cells expressing mCherry or mCherry-p53-R9 and labelled with mCherry antibody (Green), MitoTracker (Red), and Tid1 antibody (Cyan) show the localization of the inducible protein (mCherry or mCh-p53-R9) in the presence or absence of transport chaperone protein Tid1 after transfection with scramble or Tid1 siRNA. Composite image includes both the green and red channels to show colocalization. Tid1 knockdown results in a punctate pattern in the MitoTracker signal, as previously published (Ng A, 2014). C Colocalization of mCherry antibody and MitoTracker signals determined by Pearson’s Correlation Coefficient after Otsu auto threshold applied to remove background. N = 100 cells from ten separate images visually positive for both mCherry and MitoTracker signal prior to background removal. There is significantly more correlation between the mCherry signal and the MitoTracker signal in mCh-p53-R9 (Scramble siRNA) vs both mCherry (Scramble siRNA) and mCh-p53-R9 (Tid1 KD); Two-Way ANOVA with Tukey’s multiple comparison test and correction *p < 0.0001. Error bars represent standard deviation of ten images collected across three experimental replicates (3 or 4 images/replicate).