Fig. 4

PPAA facilitates endosomal escape and is subsequently trafficked to autophagosomes. a Galectin-8 recruitment measured as the average Galectin-8 intensity per cell 2 h post-treatment with 0 or 10 µM PPAA in the presence or absence of the endosomal acidification inhibitor bafilomycin A and its control nocodazole. Data are presented as means ± 95% CI; two-way ANOVA followed by Tukey’s post hoc test. b Representative microscopy images of the treatment groups presented in A. Images are 712.8 × 712.8 µm. c Tracking in real-time indicates that PPAA causes significant (*p < 0.05) endosomal disruption compared with no treatment after 1 h which increases over the timeframe measured. Data are presented as means ± standard deviation. For a, c, *p < 0.05, ****p < 0.0001 vs. no inhibitor, ^####p < 0.0001. d Comparison of delivery reagent-mediated intracellular peptide bioavailability through a quantitative split-GFP fluorescence transduction assay (numbers above bars denote the fold increase in GFP fluorescence compared with delivery of the peptide corresponding to the eleventh β-strand of the GFP protein alone); *p < 0.05; one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM. e PPAA dose-dependent intracellular delivery of TAT-CRE to Ai9 fibroblasts expressing a loxP flanked stop cassette upstream of a tdTomato transgene. Data are expressed as the percentage of treated cells positive for tdTomato expression as a measure of the intracellular bioavailability of the TAT-CRE cargo (12.5 ng/µL = 0.56 µM, 200 ng/µL = 9.1 µM PPAA). Two-way ANOVA testing of the data revealed a significant effect of PPAA dose on TAT-CRE mediated gene recombination (p < 0.0001). Tukey’s post hoc multiple comparisons testing for simple effects within each TAT-CRE dose revealed significant differences in gene recombination with increasing doses of PPAA; *p < 0.05. Data are presented as means ± standard deviation. Representative images of Ai9 fibroblasts pretreated with f 0 and g 200 ng/mL PPAA prior to treatment with 40 units/mL TAT-CRE. Images are 712.8 × 712.8 µm. h Representative still frame (from Supplementary Movie 4) demonstrating that LC3B (green) co-localizes (white arrows) with PPAA (purple) following cellular uptake