Sensoplate

Enteroid monolayers were prepared as described previously (Thorne et al., 2018 (link)), in short, single cell suspensions were generated from organoids by mechanical disruption and a digest with TrypLE Express (Thermo Fisher Scientific Cat# 12605-010). Approximately 4000 WT and/or 1000 cancer cells were seeded per well of BME2 coated (0.8mg/ml) 96-well plate in medium supplemented with CHIR-99021 and Y-27632. After 24hrs, cells were washed once and cultured in murine small intestinal organoid medium for the remainder of the experiment. For imaging purposes cells were plated in glass-bottom 96 well SensoPlates (Greiner Bio-One Cat#655892). Small molecule inhibitors were used in the following concentrations: Z-VAD-FMK (50 μM, Bachem Cat# N-1510.0005), CHIR-99021 (3 μM, Tocris Cat#4423), Y-2763 (10 μM, Abmole Cat# M1817).

U4C cells complemented with sfGFP-tagged JAK1 mutants were seeded at 50,000 cell per well on 24-well Sensoplates (662892, Greiner). Two days later, living cells were rinsed twice in 1xPBS and imaged in Live Cell Imaging Solution (A14291DJ, ThermoFisher). Nuclei were stained with NucBlue reagent (R37605, ThermoFisher). Micrographs were acquired at a Leica SP5 confocal microscope.

Human neuroblastoma SH-SY5Y cells containing a stable integration of a doxycycline (dox)-inducible, COPA targeting shRNA⁴⁵ (link) were gifted by Androphy lab, Indiana University, Indianapolis, USA. Cells were cultured in DMEM:F12 (Gibco 11320033) and were selected in media containing final concentrations of 4 μg/mL blasticidin and 0.75 μg/mL puromycin. Cells were routinely cultured in 10% FBS supplemented media but serum was reduced to 1% FBS for 2 passages prior to plating for experiments, to encourage proliferation of neuronal-like cells over endothelial-like cells. For experiments, cells were seeded onto laminin-coated coverslips or glass-bottomed, 96-well Sensoplates (Greiner) at 2.5x10⁴ cells/cm² in DMEM:F12, 1% FBS. After 24 h, cells were treated with a final concentration of 4 μg/mL dox in DMEM:F12, 0.5% FBS for 72 h prior to experimentation. Media was changed to fresh 4 μg/mL dox in DMEM:F12, 0.5% FBS every 24 h.

To study the effect of nisin on pre-formed biofilms and to precisely control the exposure times, biofilms were inoculated and grown in 24 well Sensoplates (Greiner Bio-One, Monroe, NC, USA) as described above using CCS and CFS for 20–22 h at 37°C. Following overnight growth, the biofilms were treated with nisin (10, 50 μg/ml) with short exposure times (1, 5, 10 min). Following the treatment of pre-formed biofilms with or without nisin in CFS, all wells were washed with PBS three times. The same biofilm staining protocol was followed as described above.

Vero cells on glass bottoms of 24-well SensoPlates (Greiner bio-one, 662892) were transfected with CellLight Actin-RFP, BacMam 2.0 (Molecular Probes, C10502) 1 dpi and CellLight Lysosomes-GFP, BacMam 2.0 (Molecular Probes, C10507) the night before imaging at 3 dpi. CellLight reagents were used at concentrations recommended by the manufacturer. Cells were washed to remove CellLight reagents before imaging on a Nikon ECLIPSE Ti spinning disk confocal fluorescence microscope. For imaging of LAMP1⁺ vesicles fusing with CCV membranes, cells were incubated at 37°C with 5% CO₂ and imaged at 1 frame every 2 sec. For LatA (Sigma) treatment, cells were imaged at room temperature at 1 frame per min. Imaging started 15 min before adding LatA followed by 30 min post-treatment.

HeLa, U2OS, and RPE1 cells co-expressing GFP-PCNA and H2B-RFP were seeded into 96-well glass bottom Sensoplates (Greiner) at 10,000 cells/well 16 h before inhibitor addition. Prior to seeding, glass-bottom plates were coated with poly-l-lysine (Sigma). All inhibitors were diluted in DMSO and added to cells in complete growth media (2× desired concentrations were prepared in complete growth medium and added to wells). Movies were acquired on a CV1000 spinning disk confocal system (Yokogawa Electric Corporation) with a 20× U-PlanApo 0.75 NA objective and 512 × 512 EM-CCD camera with 2 × 2 binning. The humidity controlled imaging chamber was maintained at 37°C and 5% CO₂. Three fields per well were imaged, with duplicate wells for each condition. 3 μm × 2 μm z-sections in the GFP (25% power, 100 ms, 20% gain) and RFP (20% power, 100 ms, 20% gain) channels were captured in each field at 12-min intervals for 24 h. Cells were manually tracked from appearance of GFP-PCNA foci to the beginning of the next mitosis (NEBD). GFP-PCNA foci appear in the nucleus during mid to late S-phase, and the first frame in which these foci are no longer visible was defined as the beginning of G2 phase. Results represent combined measurements of 40-100 cells per condition from two independent experiments. Data were fit using a 4-parameter, variable slope fit in Prism (GraphPad).