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. 2016 Jan 5;113(3):E291–E299. doi: 10.1073/pnas.1518634113

Fig. 1.

Fig. 1.

P21 improves PTD-mediated cellular uptake. (A) Schematic of the proteins created after screening domains that improve efficiency of protein delivery to cells. mR and mR-8R are described in Fig. S1. P21-mR is mRFP with an N-terminal fusion of the P21 domain of HB-EGF. P21-mR-8R is mRFP with N-terminal fusion of P21 and C-terminal fusion of 8R. (B) Fusion of P21 to mR-8R significantly improves uptake into NIH3t3 cells. Fluorescence microscopy images of NIH3t3 cells treated with proteins (20 µg/mL) for 12 h in standard media conditions. (Scale bar, 100 µm.) (C) P21-mR-8R is efficiently taken into hESCs and mESCs (HUES7 and CGR-8, respectively) and hiPSCs (IPS2) and mouse cardiomyocyte cell line HL1. Flow cytometry analyses of the mR-8R inefficiently delivered cell lines treated with proteins mR-8R (20 µg/mL) for 12 h. (D) P21-mR-8R initially strongly interacts with cell membranes and progressively is taken up and localized perinuclearly. Fluorescence (Top) and confocal laser scanning microscopy (Bottom) images of NIH3t3 cells treated with P21-mR-8R (20 µg/mL) for 1 h, 1 h with washes and a further 5 h incubation (in serum-free media), or 6 h treatment. Cells were preincubated for 1 h in serum-free media and transduced for the desired time in serum-free media. (Scale bars, top, 50 µm; bottom, 10 µm.) (E) Enhancement of cellular uptake mediated by P21 and 8R are affected by Trypsin proteolysis. Flow cytometry analyses NIH3t3 cells treated with proteins (20 µg/mL) for 1 h and a further 5 h incubation (in serum-free media), with or without 10 min predigestion with Trypsin or treatment with nonproteolytic cell dissociation solution (CDS). Cells were preincubated for 1 h in serum-free media, treated with Trypsin, and transduced for 1 h in serum-free media. (F) Cell surface interaction of P21-containing proteins is disrupted by Tritonx100 treatment. Flow cytometry analyses of NIH3t3 cells treated with proteins (20 µg/mL) for 1 h and a further 5 h incubation (in serum-free media) with 10 min pretreatment of PBS or PBS containing 0.1% (vol/vol) Tritonx100 (Tx100). Cells were preincubated for 1 h in serum-free media, treated with PBS or PBS with Tx100, and transduced for 1 h in serum-free media. Error bars indicate SD. n = 6.