HEK293T (American Tissue Culture Collection), SV589 (SV40 immortalized human fibroblasts; Yamamoto et al., 1984 (link)), RC55, RC65, and RC65+55 cells were cultured at 37°C and 5% CO2 in DMEM (Mediatech) supplemented with 10% cosmic calf serum (HyClone), 100 U/ml penicillin (GoldBio), and 100 µg/ml streptomycin (GoldBio; PenStrep). Expression of OsTIR1-2xMyc was induced with 0.5 µg/ml doxycycline (Sigma) for 6 h or 20 h before inducing degradation of GRASP55 and/or 65 by the addition of 500 µM IAA (Abcam). The Golgi ribbon was disassembled into single stacks by treatment with 3 µg/ml nocodazole (EMD Millipore) for 2 h, while complete disassembly into vesicles was triggered with 5 µg/ml BFA (LC Laboratories) for 30 min. MG132 (Boston Biotech) was added at 10 µM to inhibit protein degradation by the proteasome.
To enrich cells in late M-phase, RC55, RC65, and RC65+55 cells were first synchronized at the G1/S phase transition by double thymidine block. Cells were treated with 2 mM thymidine (Chem-Impex) for 16 h; thymidine was then washed out for 8 h and added again for an additional 16 h. 10 h after the last release from thymidine block, we added 20 µM STLC (Acros) for 4 h to arrest cells in prometaphase. STLC was then removed to allow cells to progress into telophase/cytokinesis, and cells were fixed and processed for immunofluorescence or EM analysis.
To enrich cells in late M-phase, RC55, RC65, and RC65+55 cells were first synchronized at the G1/S phase transition by double thymidine block. Cells were treated with 2 mM thymidine (Chem-Impex) for 16 h; thymidine was then washed out for 8 h and added again for an additional 16 h. 10 h after the last release from thymidine block, we added 20 µM STLC (Acros) for 4 h to arrest cells in prometaphase. STLC was then removed to allow cells to progress into telophase/cytokinesis, and cells were fixed and processed for immunofluorescence or EM analysis.