Gabriele Multhoff, Technical University, Munich, Germany), fixed with 4?% ice-cold paraformaldehyde, and nuclei were stained with DAPI. Fluorescence images were captured with the use of a Leica TCS SP2 confocal microscope (Leica, Germany). activity of KST peptide in complex with Avidin (KSTCAv complex) with that of similarly linked canonical TAT peptide, we found that TAT peptide penetrated SK-N-SH human neuroblastoma cells at a similar rate and efficiency as the KST peptide. Furthermore, KST peptide can carry protein complexes consisting of a specific antibody coupled to the peptide through the Avidin bridge. An antibody to Hsp70 delivered to SK-N-SH cells with high expression level of Hsp70 reduced the protective power of the chaperone and sensitized the cells to the pro-apoptotic effect of staurosporine. We studied the mechanisms of penetration of KSTCAv and full-length Hsp70 inside human neuroblastoma SK-N-SH and human erythroleukemia K-562 cells and found that both used an active intracellular transport mechanism that included vesicular structures and negatively charged lipid membrane domains. Competition analysis of intracellular transport showed that this chaperone reduced intracellular penetration of KST peptide and conversely KST peptide prevented Hsp70 transport in a dose-dependent manner. Electronic supplementary material The online version of this article (doi:10.1007/s12192-014-0554-z) contains supplementary material, which is available to authorized users. peptides around the Hsp70 molecule (SWISSMODEL based on template: 1yuwA) (Kiefer et al. 2009; Kopp and Schwede 2004). KST peptide, 493C512 aa, KSTGKANKITITNDKGRLSK (fusion genes (Addgene, USA) and then incubated them with 1?M Hsp70 labeled with Alexa488 or 1?M KSTCAvCFITC complex. In other experiments, SK-N-SH cells were incubated with 1?M of Hsp70 labeled with Alexa488 or 1?M KSTCAvCFITC complex and then stained with CytoPainter Lysosomal Staining Kit (Abcam, UK) according to the manufacturers protocol. To reveal the possible contribution of cell surface Hsp70 to endocytosis of full-sized Hsp70 and KST peptide, we transfected K-562 and SK-N-SH cells with pFusionRed-f-mem plasmid (Evrogen, Russia). Cells were heat shocked at 43?C for 30?min and then were cultured overnight. Next morning, cells were washed with Mouse monoclonal to MPS1 ice-cold PBS incubated with monoclonal cmHsp70.1-FITC antibody on ice (kindly provided by Prof. Gabriele Multhoff, Technical University, Munich, Germany), fixed with 4?% ice-cold paraformaldehyde, and nuclei were stained with DAPI. Fluorescence Nutlin carboxylic acid images were captured with the use of a Leica TCS SP2 confocal microscope (Leica, Germany). To avoid possible cross interference among the various fluorochromes, images for DAPI, FITC, Alexa488, or deep red were acquired using the sequential image recording method. Western blotting K-562 and SK-N-SH cells were heat shocked at 43?C for 30?min and after overnight incubation were collected and lysed, and the lysates were analyzed with the aid of Western blotting using 3B5 anti-Hsp70 monoclonal antibody; 6C5 anti-GAPDH antibody (Abcam, UK) was employed to represent loading control. Flow cytometry Flow cytometry was used to analyze the time- and dose-dependent Nutlin carboxylic acid penetration of the KSTCAvCFITC complex. SK-N-SH or K-562 cells were incubated with 5?M KSTCAvCFITC for 30?min, 1?h, 3?h, or 6?h, washed in PBS, and analyzed with the aid of a Coulter Epics XL (Beckman Coulter, USA) flow cytometer using laser with assessments were used to evaluate differences between the control and treatment groups; differences were considered to be statistically significant when 5?m. b K562 and SK-N-SH cells were incubated with studied peptides as described above and than were applied for flow cytometry. Data of Nutlin carboxylic acid five impartial experiments are summarized. Differences between control Nutlin carboxylic acid and treatment groups were considered to be statistically significant when test. c We used sequential image recording with a step of 0.5?m from top to bottom to demonstrate that KST peptide can penetrate SK-N-SH cells and reside within the cytosol Notably, KST peptide demonstrated no or minimal toxicity. After 72?h of incubation with KST peptide, we observed 6C7?% dead cells in populations of K-562 and SK-N-SH cells (data not shown). One of the most important properties of CPPs is usually their ability to deliver certain cargo molecules, including drugs, DNA, RNA, and proteins. To check whether Hsp70-based peptides were able to carry high molecular weight cargoes into living cells, we used a biotin tag to construct a complex with Avidin, a protein with a Nutlin carboxylic acid molecular mass more than 30-fold that of the Hsp70 peptides. The complex containing AvCFITC and the peptides to be investigated, KSTCbiotin, YFNCbiotin, or TATCbiotin, was formed at room temperature, and then, the solution was dialyzed to remove.
Gabriele Multhoff, Technical University, Munich, Germany), fixed with 4?% ice-cold paraformaldehyde, and nuclei were stained with DAPI