Cell culture and electroporation
U-2 OS (ATCC) cells were cultured in phenol-red free DMEM supplemented with 10% fetal bovine serum (Corning), 2 mM L-glutamine (Corning), and 100 IU penicillin and 100 µg ml− 1 streptomycin (CellGro) at 37 °C and 5% CO 2 . For the tet-inducible system, FBS that was tested free of tetracycline (Cytvia) was used. Cells were grown on either diluted fibronectin (1:100, Millipore) or Matrigel (1:100, Corning)-coated coverslip chambers or sterilized cover glasses (no. 1.5 Round (EMS)). Lonza Electroporation kit with the U-2 OS specific set-up suggested by the company was used for transient transfection, which was performed 16–20 h prior to imaging. In addition, HeLa (ATCC), COS-7 (ATCC), HEK293T (ATCC) and HT1080 (ATCC) cells were cultured and electroporated according to the manufacturer’s instructions. Cells were routinely tested for mycoplasma contamination.
Plasmids and cloning
A list of plasmids used in this study is provided in Supplementary Table 1.
Generation of stable cell line and doxycycline induction
To generate lentivirus particles, HEK293T cells were transfected with MCP-GFP, MCP-HaloTag or scFv-sfGFP plasmids, along with viral packaging plasmids. The supernatant was collected 48–72 h after transfection, filtered through a 0.22-µm syringe filter, and concentrated using Lenti-X Concentrator (Takara). U-2 OS cells were exposed to lentiviral particles, and MCP-HaloTag and scFv-sfGFP double positive U-2 OS cells were generated via sequential viral transduction and sorted via fluorescence-activated cell sorting.
To generate cells stably expressing the protein of interest with only MS2 binding sites mRNAs, lentiviral transduction was used on pre-sorted MCP-GFP or MCP-HaloTag positive U-2 OS cells. Cells stably expressing a tet-inducible cytERM–SunTag were generated using the PiggyBac system. MCP-HaloTag/scFv-sfGFP double positive U-2 OS cells were then electroporated with cytERM-SunTag plasmid with hyperactive piggyBac transposase (hyPBase). After 4 days post-electroporation, the cells were selected under 10 µg ml−1 Blasticidin S for two weeks. The stable cells expressing MCP-HaloTag/scFv-sfGFP/cytERM–SunTag were induced with 100 ng ml− 1 doxycycline 3–5 h prior to imaging.
Labelling JF dyes with HaloTag ligand and SNAPTag ligand
For cells expressing HaloTag, they were incubated in complete media with 100 nM JF646 HaloTag ligand (JF646-HTL) at 37 °C for 30 min and then washed twice. The rinsed cells were then equilibrated in the media at 37 °C for 30 min prior to imaging. Similar methods were used for PA-JF646-HTL, PA-JF549-HTL, JF549-HTL, JF646-HTL and JF635-HTL.
Cells expressing SNAPTag were incubated in complete media with 250 nM JF549 with chloropyrimidine SNAPTag ligand (JF549-cpSNAP) for 1 h at 37 °C and then washed twice. The rinsed cells were then washed twice again after 30 min to remove residual dyes, and then equilibrated in fresh media at 37 °C for 30 min prior to imaging.
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