Figure 4: Cell-penetrating NHR2-containing polypeptides transduce myeloid Kasumi-1 cells. (a) Effect of serum and dextran on protein transduction efficiency into hematopoietic cells. Preincubation of Kasumi-1 cells for 3 hours in dextran (0.1 or 1 mg/mL) containing medium, extensive washing of the cells and incubation with 2 μM TN122 for 4 hours in the presence or absence of 20% FCS. Successfully transduced protein was detected by Western blotting after trypsin treatment of the cells. (b) Incubation of Kasumi-1 cells with 6 μM TN122-eGFP in serum-free X-Vivo 10 medium for 3 h, trypsin treatment and flow cytometry analysis to determine the efficiency of protein transduction. Percentages correspond to the percentage of eGFP positive cells. (c) Intracellular localization of TN122-eGFP in Kasumi-1 cells. Incubation of the cells with 4 μM TN122-eGFP in X-Vivo 10 medium for 1 h, trypsin treatment, fixation and permeabilization, DNA staining with Toto3, and subsequent CLSM analysis (63x magnification). (d) TN122-eGFP is captured inside endosomes upon protein transduction. Incubation of 293T cells with 2 μM TN122-eGFP in the presence of 20 μM chloroquine for 24 h, trypsin treatment, staining of the unfixed cells with a rhodamine-coupled wheat germ agglutinin (red), and CLSM analysis. (e) Cytotoxicity of 20 μM chloroquine on Kasumi-1 cells. The percentage of dead cells was measured by flow cytometry using 7-amino-actinomycin D (7AAD) after a 24 hours incubation of the cells in chloroquine-containing medium. (f) Endosomolytic TAT-HA2 increases the intracellular stability of transduced proteins. Cotreatment of Kasumi-1 cells with 3 μM TN122 in the presence or absence of 5 μM TAT-HA2 for different times, trypsin treatment and detection of the myc-tagged TN122 in the cellular lysates.