Nile red

Nile Red (Santa Cruz Biotechnology Inc., Dallas, TX) as a lipophilic stain was used to stain untreated and P1-treated cells according to the literature.^{77 ,78 (link)} Briefly, cells (OD600 ~ 0.2) were treated with 0–100 μM of P1 in a shaker-incubator (1h, 37 °C, 250 rpm). The cell suspension was centrifuged (4700 rpm, 15 min) and the pellet was resuspended in 1 mL of 5 mM PBS buffer. A stock solution of 25 mM Nile Red in dimethylsulfoxide (DMSO; Alfa Aesar, Haverhill, MA) was prepared first, and 2 μL of this stock dye solution were added to the bacterial suspensions (untreated and P1-treated) to yield the final concentration of Nile Red as 50 μM in the suspension. The cells were incubated in the dye for 3 h, after which the cells were centrifuged, washed twice with 5 mM PBS and resuspended in the same buffer. For CLSM imaging, 10 μL of these cells were added to glass slides (Home Science Tools, Billings, MT) and covered with coverslips. A Nikon A1R-Ti2 confocal laser scanning microscope (Center for Biotechnology, Beadle center, UNL) with a 60x objective lens was used to image the samples. The Nile Red was excited with a 560 nm laser line and emission was collected from 570–620 nm.