Softworx software

Time-lapse microscopy of E. coli MG1655 (wild-type) and ΔaceK strains expressing YFP (Supplementary Table 4) was performed with minor modifications51 (link). In brief, bacteria were grown to mid-log phase (OD₆₀₀ ∼0.3) in M9 liquid medium containing 2 g l⁻¹ glucose and 2 μl of culture were spread on a semipermeable membrane, which was then inverted onto a glass coverslip micro-patterned with rings of SU-8 (65 μm diameter × 1.5 μm height) containing a central post (10 μm diameter × 1.5 μm height). The bacteria-free side of the membrane was overlaid with a PDMS-based microfluidic device and the assembly was clamped between a transparent acrylic cover and base adapter, as described51 (link). The inlet and outlet ports of the microfluidic device were connected to plastic tubing and bacteria were fed by continuous flow of M9 minimal medium containing 2 g l⁻¹ glucose or acetate (flow rate, 25 μl min⁻¹). Bacteria were maintained at 37 °C in a microscope-fitted environmental chamber and imaged using a DeltaVision personalDV microscope (Applied Precision) equipped with a 100 × oil-immersion objective. Images were recorded at 5-min intervals on phase-contrast and fluorescence channels (GFP, excitation filter 490/20/, emission filter 528/38) using a CoolSnap HQ2 camera. Images were processed using Softworx software (Applied Precision) and custom-made macros in ImageJ.

Cells were plated at 8 × 10⁴ cells/mL on 19 mm (VWR International) coverslips at 500 μL. After overnight incubation, the Cenp-E inhibitor was added for various time periods. Cells were fixed with 1% formaldehyde, quenched with glycine, and then permeabilised with PBST (PBS and 0.1% Triton X-100). For microtubule staining the PEM buffer was used. Cells were pre-extracted with 100 mM Pipes, 1 mM MgCl₂, 0.1 mM CaCl₂, and 0.1% Triton X-100 for 90 seconds, followed by fixation with 4% formaldehyde in PEM buffer for 10 minutes. Cells were then incubated with sheep anti-Bub1 SB1.3 [49 (link)] and with mouse anti-tubulin TAT1 [63 (link)], for 30 minutes, and then washed and incubated with secondary antibodies Cy2-, and Cy3-, antisheep/mouse (Millipore) for 30 minutes. Hoechst 33358 (Sigma) at 1 μg/mL was then added to the cells, followed by mounting onto slides with 90% glycerol and 20 mM Tris-HCl, pH 8.0. Images were taken at room temperature with a restoration microscope (DeltaVision RT; Applied Precision) using a 100x 1.40 NA Plan Apo objective and a filter set (Sedat Quad; Chroma Technology Corp.). Images were captured with a charge-coupled device camera (CoolSNAP HQ; Photometrics) with a z-optical spacing of 0.2 μm. Raw images were then deconvolved with the SoftWorx software (Applied Precision), and these were then processed, and PhotoShop (Adobe) was used to analyse the images.

Fluorescence signals were visualized with a DeltaVision RT fluorescence microscope (Applied Precision) equipped with a CoolSNAP HQ camera (Photometrix). Images were generated by collecting a stack of 20 pictures with focal planes 0.2 μm apart in order to cover the entire volume of a yeast cell and by successively deconvolving them using the SoftWoRx software (Applied Precision). A single-focal plane is shown at each time point. The FM 4–64 dye (Invitrogen) was used to specifically stain the vacuolar rim^{41 (link)}. The GFP intensity in vacuolar rim or lumen was determine as an average of the intensity of several points in each region by using the multi-point tool in ImageJ.
For time-lapse imaging experiments, cells nitrogen starved in SD-N medium and stained with the CellTracker Blue 7-amino-4-chloromethylcoumarin (CMAC) dye (Invitrogen) for 30 min, were imaged every 30 s, collecting a Z-stack of six pictures with focal planes 0.30 μm apart. Images were deconvolved and mounted into movies before measuring the life of GFP-Atg8∆R using the SoftWoRx software. The time point at which GFP-Atg8∆R appeared as a punctuate structure was considered as time 0 min. 2D projections of Z-stack images were employ to quantify the relative intensity of GFP-Atg8∆R-positive structures as previously described^{23 (link)} and setting to 1 the relative intensity of cells expressing WT Atg4.

Fibroblasts were grown on coverslips for at least 24 h. Prior to fixation, cells were incubated with MitoTracker® Orange CMTMRos (Life Technologies) for 15 min and then fixed in 4% paraformaldehyde for 20 min at 37°C. Cells were washed in PBS and mounted in histology mounting medium containing DAPI (Fluoroshield™; Sigma-Aldrich, St.Louis, Missouri, USA). Images were taken using a DeltaVision Spectris epifluorescence microscope (Applied Precision, Issaquah, Washington, USA) at 60× magnification, equipped with a TRITC (excitation 542/27, emission 594/45) and DAPI (excitation 390/18, emission 435/48) filter set. Series of 10–15 sections with 0.5 μm spacing along the Z-axis were taken. Images were deconvoluted and a maximum projection of the stacks was generated by merging the individual slices using the softWORx software (Applied Precision).

Images of ced-1::gfp were analyzed using softWoRx software (Applied Precision), and engulfed nuclei were scored only if there was complete engulfment.

BMDMs (2 × 10⁵) were plated in glass chamber slides (LabTek) and starved overnight. Cells were stained for 20 min with 250 nM MitoSpyTM Orange CMTMRos (BioLegend). Imaging was started immediately after addition of 500 ng/ml LPS on a DeltaVision RT system with SoftWorx software (Applied Precision) at 37 °C and 10% CO₂. Images were acquired every 10 min for 2 h. The data were exported as uncompressed AVI files and processed with Premiere Pro.

Injectoporation was performed as described (Xiong et al., 2014 (link); Liu et al., 2018 (link)). In brief, the organ of Corti was isolated and cultured in DMEM/F12 medium (11330057; Life Technologies) for 2–4 h. Glass electrodes (2-µm diameter) were used to deliver the plasmid (500 ng/µl in 1× HBSS) to the sensory epithelium. A series of three pulses were applied at 1-s intervals with a magnitude of 60 V and duration of 15 ms (BTX ECM 830 square wave electroporator). Cochlear explants were cultured in a 37°C incubator for 2–3 d. Samples were then fixed in 4% formaldehyde for 20 min at room temperature and processed for immunostaining using 6E2 anti-HA antibody (2367, RRID:AB_10691311; Cell Signaling Technology). Whole-mount preparations were imaged on a Deltavision Deconvolution Microscope and processed with SoftWoRx software (Applied Precision).