Janelia fluor 549 dye

Cells stably expressing LC3-Halo or Su9-Halo were seeded for 80% confluence on the day of the assay in 12-well tissue culture plates. To pulse-label the reporter, cells were incubated for 20 min in 100 nM cell-permeable Halotag conjugated to Janelia Fluor 549 dye (Promega) before washing twice with PBS. Cells were incubated in a growth medium or in a growth medium supplemented with 10 µM oligomycin and 5 µM antimycin, or in a growth medium supplemented with 1 mM deferiprone (Millipore Sigma), or in Earle’s Balanced Salt Solution before harvesting cells via scraping. Incubation times are listed in the respective figure caption. Cells were lysed and the protein concentration was quantified in a plate reader using a Pierce BCA protein quantification kit (Thermo Fisher Scientific). For each condition, 20 µg of protein was loaded onto a gel for SDS-PAGE and then the gels were imaged using a ChemiDoc fluorescent imager (Biorad).
Images of gels (Fig. S4) were imported into FIJI and analyzed using the Gel Analyzer tool. Briefly, selections were defined that contained the bands of interest, and the integrated signal for each band was recorded. The intensity of the processed HaloTag band as a fraction of the total Halo signal was calculated.

For time-lapse imaging, swarmer cells isolated from a small-scale synchrony were either prepared for imaging immediately or allowed to grow to the appropriate stage of the cell cycle prior to imaging. For HaloTag imaging, cells were incubated in 2 nM Janelia Fluor 549 dye (Promega GA1110) in M2G for 10 minutes at room temperature and washed three times with M2G prior to imaging. Images were taken at 10-minute time intervals. For PleC-HaloTag, cells were labeled immediately after synchrony. For DivJ-HaloTag, swarmer cells were allowed to progress to stalked cells for 40 minutes prior to labeling. For imaging DivJ-eYFP and eYFP-ParB cells, isolated swarmer cells were grown in M2G or M2G with 0.01% xylose for 1 hour to allow cells to progress to the pre-divisional stage. Agarose pads made with M2G or M2G with 0.01% xylose were cut in half in order to image WT and pleC(ΔPAS) cells simultaneously. The microscope was programmed to take images at 5-minute intervals and move the stage to take images of both halves of the agarose pad. The genotypes associated with raw images were blinded, and Δt intervals were acquired manually.