Studies with Site-Specific Labeled HAMLET Figure 8. Microscopy studies using site-specific-fluoroprobe-labeled protein. (A) Tumoricidal activity of the labeled protein hLA-ext in molten globule state was tested on A549 cells. MCF-7 cells were incubated for 4 h, with 30 mM Alexa Fluor 488-labeled mg-hLA-ext protein alone (B) or complexed with OA (C). After treatment, cells were fixed and nuclei were stained with vital stain Hoechst 33342 (blue). When complexed with OA, Alexa Fluor 488 labeled mg-hLA-ext (green) internalizes to cytoplasm and cell nuclei, while molten globule protein alone did not enter the cells. (D) Confocal images of SK-BR-3 cells treated with Alexa Fluor 4888-labeled mghLA-ext complexed with OA at the indicated times. doi:10.1371/journal.pone.0026093.g008 Figure 9. Aggregates in the nuclei co-localize with a nucleolus marker. SK-BR-3 cells were treated with Alexa Fluor 488 labeled mg-hLA-ext complexed with OA or without OA (control) for 4 h, fixed, permeabilized, incubated with a-fibrillarin monoclonal antibody, and detected with anti- mouse-Alexa Fluor 555–labeled antibody (red). In cells treated with OA complex, co-localization of green fluorescent signal and fibrillarin protein was found at the cell nuclei in SK-BR-3 and MDA-MD-468 (data not shown) cell lines. doi:10.1371/journal.pone.0026093.g009 PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26093