Fig. 4. Tetracycline targets the cytoplasmic membrane.

a Fluorescence microscopy images of cells treated with either tetracycline or anhydrotetracycline for 30 min. Both antibiotics display green autofluorescence allowing direct localization of the compound. Cell membranes were stained with Nile red. Arrows indicate fluorescent membrane patches coinciding with accumulation of the respective antibiotic. b SIM microscopy images of cells treated with either tetracycline or anhydrotetracycline for 30 min. Membranes were stained with Nile red. Arrows indicate membrane staples or invaginations. Scale bars 2 µm. c Quantification of cells showing membrane patches from widefield (a) and SIM (b) images. Images were quantified manually. Sample size was ≥100 individual cells per condition for widefield and ≥50 individual cells per condition for SIM. Error bars show standard deviation of the mean of three replicate experiments. Circles indicate individual datapoints.