Streptomycin

In vitro reconstituted E. coli 70S ribosomes were generated from the E. coli K12 strain BW25113, as described previously^{56 (link)}. Antibiotic–ribosome samples were generated by incubating antibiotic cocktails 1–5 with E. coli 70S ribosomes in buffer A (50 mM HEPES-KOH, pH 7.5, 25 mM Mg(OAc)₂, 80 mM NH₄Cl, 100 mM KOAc, 1 mM DTT, 0.05% DDM) at 37 °C for 15 min, before being frozen at −80 °C until use. Final antibiotic concentrations for complexes formed with each cocktail was: cocktail 1 contained 200 μM omadacycline (MedChemExpress), 200 μM spectinomycin (Santa Cruz Biotechnology), 200 μM streptomycin (Santa Cruz Biotechnology), 200 μM evernimicin, 200 μM hygromycin B (Cayman Chemical); cocktail 2 contained 100 μM capreomycin (Sigma Aldrich), 100 μM kasugamycin (Sigma Aldrich) and 100 μM retapamulin (Sigma Aldrich); cocktail 3 contained 100 μM tetracycline (Sigma Aldrich), 100 μM viomycin (Sigma Aldrich), 100 μM streptomycin (Santa Cruz Biotechnology), 100 μM lincomyin (Sigma Aldrich) and 100 μM avilamycin (Cayman Chemical); cocktail 4 contained 10 μM apramycin (Sigma Aldrich), 10 μM eravacycline (MedChemExpress) and 100 μM clindamycin (Santa Cruz Biotechnology); cocktail 5 contained 100 μM pentacycline (Tetraphase), 10 μM gentamicin (Carl Roth) and 100 μM tiamulin (Sigma Aldrich).

Purified B cell subsets from HD or SLE patients were co-cultured in B cell media in the presence of IL-2 (50 U/ml)±IL-21 (10 ng/ml) with allogeneic in vitro generated Th1 or Th2 effectors (0.6 × 10⁶ cells/ml, ratio 5B:1T) for 5–6 days, as indicated. B cell media contained Iscove’s DMEM supplemented with penicillin (200 μg/ml), streptomycin (200 μg/ml), gentamicin (40 μg/ml), 10% FBS, and insulin (5 μg/ml; Santa Cruz Biotechnology).

HeLa-Kyoto BAC lines stably expressing PRC1-GFP were courtesy of Ina Poser and Tony Hyman (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany). HeLa cells stably expressing EGFP-CENP-A and EGFP-centrin1 were a courtesy of Emanuele Roscioli and Andrew McAinsh (University of Warwick). Human U2OS cells, both unlabeled and permanently transfected with CENP-A-GFP, mCherry-α-tubulin, and photoactivatable (PA)-GFP-tubulin, were courtesy of Marin Barišić and Helder Maiato (University of Porto). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with Ultraglutamine (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA), penicillin, streptomycin, and geneticin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). The cells were kept at 37 °C and 5% CO₂ in a Galaxy 170S CO₂ humidified incubator (Eppendorf, Hamburg, Germany). All used cell lines were confirmed to be mycoplasma free by using MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).