Pacgfp1 tubulin

The expression vectors in mammalian cells, pAcGFP1-Actin, pAcGFP1-Tubulin, and pDsRed-Monomer-C1, were obtained from Clontech. Oligonucleotides used as PCR primers in the construction of expression plasmids for fusion proteins were custom-synthesized by Gene Design (Osaka, Japan). The WST-1 cell proliferation assay system was purchased from Takara Bio (Kyoto, Japan). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Glass-bottom dishes used for cell culture and imaging experiments were purchased from AGC Techno Glass (Shizuoka, Japan).

HeLa cells were cultured in MEMα Glutamax (Gibco) supplemented with 10% fetal calf serum (Gibco), and incubated at 37 ºC and 5 % CO2 in a humidified atmosphere. For LSCM imaging, cells were grown on MatTek culture dishes (MatTek Corporation). The nuclei were labeled with 5 μg/mL Hoechst 33342 (Molecular Probes), lysosomes were labeled with 200 nM LysoTracker® Red (Molecular Probes), and the plasma membranes with 5μg/mL (Thermo Scientific) ahead of transfection, which resulted in mild labeling of the nuclei. In order to visualize microtubules, HeLa cells were prepared as described and transfected 24 hours before microscopy imaging with pAcGFP1-Tubulin (Clontech) using Lipofectamine LTX (Invitrogen). pDNA:protein complexes were prepared as described above and added to complete-media-containing cell culture, and images were then taken 30 to 60 minutes after transfection.