Generation of Fluorescent HeLa Cell Lines

HeLa cell lines (ATCC CCL2) used were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; 10270106; Thermo Fisher Scientific), 1% Penicillin/Streptomycin (15140122; Thermo Fisher Scientific), and 0.1% Amphotericin B (Fungizone; 11510496; Thermo Fisher Scientific). Cell lines were cultured as a monolayer at 37°C and 5% CO₂. HeLa H2B-GFP, mCherry-Tubulin cell line was generated by transfecting mCherry-Tubulin expressing eukaryotic plasmid vector into HeLa H2B-GFP cells (Draviam et al., 2006 (link)). The HeLa mKate2-EB3 cell line was generated by transfecting a pmKate2-EB3 plasmid vector (#FP316; Evrogen) into HeLa cells. Plasmid transfection was carried out using DharmaFECT duo (T-2010; Dharmacon). The HeLa H2B-GFP, SiR-Tubulin cell line was generated by adding SiR-Tubulin dye, a paclitaxel-based fluorescent compound (Lukinavičius et al., 2014 (link); Spirochrome SC002; 100 nM) just 1 h before imaging. The HeLa FRT/TO cell line expressing siRNA-resistant MARK2-YFP-WT or KD mutant was generated by transfecting a Tet-inducible expression vector encoding siRNA-resistant MARK2-YFP-WT or KD, followed by colony picking (Zulkipli et al., 2018 (link)). Vectors bearing point mutants of MARK2 were generated by polymerase chain reaction–based point mutagenesis and confirmed by DNA sequencing (Hart et al., 2019 (link)). Fluorescent cells were enriched using a BD FACSAria III Cell Sorter for fluorescence-activated cell sorting (FACS).