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. 2020 Nov 19;11:591275. doi: 10.3389/fphar.2020.591275

FIGURE 2.

FIGURE 2

Endosomal transduction of micellized PTD-BMP-7 (mPTD-BMP-7) and exosomal secretion of bone morphogenetic protein-7 (BMP-7). (A) HEK 293 cells were treated with 200 ng of mPTD-BMP-7 for the indicated period, and the insoluble fraction of whole cell lysates were subjected to immunoblot analysis with anti-HA antibody (left). Arrow and arrowhead denote monomeric and aggregated PTD-BMP-7, respectively. The 293 cells were treated with 200 ng of mPTD-BMP-7, and intracellular transduction was monitored by confocal microscopy (right). Z-stack in blow. Scale bar, 5 μm. (B) The 293A cells were transiently transfected with mCherry-tagged Rab7 and treated with 200 ng of HA-tagged mPTD-BMP-7 for 3 h. Intracellular localization was observed with confocal microscopy and Nuc-blue served as nuclear staining of live cell imaging. (C) The 293 cells were treated with 200 ng mPTD-BMP-7, and the cells were subjected to transmission electron microscopy. Note a membrane-coated mPTD-BMP-7 in endosomes (middle) and Golgi (right). (D) The 293 cells were transfected with flag-tagged N-terminal of fibrillin-1 (FBN-1) and treated with 500 ng HA-tagged mPTD-BMP-7 for 16 h. The culture medium was harvested and subjected for immunoblot analysis (left) and immunoprecipitation with anti-flag (FBN-1). Arrows indicate active BMP-7. (E) Exosomal secretion of active BMP-7 (arrow) and mPTD-BMP-7 (arrow head) with FNB-1. CD63 serves as exosomal marker. (F) Cellular toxicity of mPTD-BMP-7. The 293A cells were incubated in various concentrations of mPTD-BMP-7, and cell growth was monitored by JuLi real-time live cell analyzer (NanoEntek).