Figure 6

Oct4-PTD is able to mediate uptake of protein cargo. (A) Oct4-PTD-Cre fusion proteins are taken up by CV1-5B Cre reporter cells as determined by microscopy of cells stained positive for b-Gal activity. Increasing protein concentrations of recombinant wild type Oct4-PTD-Cre fusion protein (WT) and mutated versions of the Oct4-PTD (R16A as wells as K13A/R16A) in fusion with Cre, respectively, were subjected to a Cre recombinase assay where the intracellular activity of transduced protein is assessed by b-galactosidase activity [45]. TAT-modified Cre protein [47] and unmodified Cre protein carrying no transduction peptide served as controls (first two rows). Quantification of Cre-reporter assay outlined in (B) (n = 5). All values relative to TAT-Cre 1 μM. Error bars and n represent standard deviations and image sections quantified for b-Gal activity, respectively. Two-tailed t-test was used for statistical analysis. *p <0.05, **p <0.01, ***p < 0.001. (C-F) Detection of recombinant human Oct4 protein in mammalian cells after extracellular exposure. CV1-5B (C) and human BJ foreskin fibroblasts (E) were incubated with 100 nM recombinant human Oct4 protein for 3 hours, washed and subsequently stained for Oct4 and DAPI. (D, F) Untreated CV1-5B cells (D) and BJ fibroblasts (F) stained with first (anti-Oct3/4) and secondary antibody (Alexa 488) served as controls. Scale bar: 25 μm.