35 mm glass bottom imaging dishes

HaCaT cells were seeded onto either sterilized glass coverslips in 6-well tissue culture-treated plates (Corning) for IF microscopy experiments, 35 mm glass bottom imaging dishes (MatTek) for live imaging experiments, or 24-well tissue culture-treated plates (Eppendorf) for cytotoxicity assays and other keratinocyte treatments. Cells were grown to 80-90% confluency, and were washed with sterile 1x PBS (Gibco) prior to the addition of fresh DMEM + 10% FBS. For infections that were performed with chemical inhibitors, this fresh media contained either 100 µM of the compound, or DMSO as a vehicle control. The final DMSO concentration in all wells was 0.8%. Cells were pre-treated for 1 hour at 37° C with 5% CO₂. Meanwhile, overnight GAS cultures were centrifuged and re-suspended in fresh TH, and their optical densities were normalized. Following the pretreatment step, HaCaT cells were infected at a multiplicity of infection (MOI) of 10 for cytotoxicity and IF microscopy experiments, and MOI 0.01 for live imaging experiments. Sterile TH was used as an uninfected control. Infections were carried out at 37° C with 5% CO₂ for the specified times. A summary of the general infection protocol is shown in Figure S1.

HeLa cells were incubated at 37 °C and 5% CO₂ in a humidified atmosphere and maintained in DMEM complete medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% L-Glutamine (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were transiently transfected using LipofectamineTM 2000 transfection reagent (ThermoFisher Scientific). Briefly, cells were seeded in a six-well plate the day prior to transfection in complete medium. At 70–80% confluency, cells were co-transfected with 1 µg of the mCherry-Lifeact-7 plasmid (Addgene) together with wild-type or mutant pEGFP_YARS1 plasmid (1:1 ratio). The next day, cells were reseeded in 35 mm glass bottom imaging dishes (MatTek Corporation). Fixation (15 min 4% PFA, Laborimpex) or live cell imaging was performed the next day (48 h after transfection) on a Zeiss LSM700 laser scanning confocal microscopy. Fixed cells were imaged with Plan-Neofluar 40×/1.30 Oil objective. Time lapse imaging of living cells was done at 2fps with a Plan-Apochromat 63×/1.40 Oil objective and using the line switching mode to avoid any fluorescence channel crosstalk and to minimize acquisitional delay between channels. Images in Supplementary Fig. 6 and Supplementary Movie 2 are 332 × 268 with 85 nm pixel dimension. Channels were combined and movies were annotated with the Fiji distribution of ImageJ.

U20S and HEK293T cells (ATCC) were grown in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human fibroblasts were cultured in RPMI medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. HCT116 cells (ATCC) were grown in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For imaging, U2OS cells were seeded at ~0.5 × 10⁵ cells per ml 24 h prior transient transfection in 35-mm glass-bottom imaging dishes (MatTek). Plasmid transfections were performed for 5 h in reduced serum medium (Opti-MEM^TM, ThermoFisher) with Lipofectamine 2000 transfection reagent (ThermoFisher). Cells were imaged 18 h post transfection in DMEM supplemented with 10% FBS. All DNA plasmids used in this work are listed in Supplementary Table 1. DNA primers used for generating constructs reported in this work are listed in Supplementary Table 2.