Incucyte zoom software

To measure apoptosis and caspase activity, THP‐1 cells were seeded in an ImageLock 96‐well plate (#4379, Essen Biosciences) and differentiated as described above. Cells were then primed with 50 IU/ml IFNγ overnight and infected with type I or type II Tg‐expressing tdTomato as previously described. To measure caspase activity, NucView™ 488 caspase‐3 substrate was added to the medium (RPMI without phenol red + 10% FCS) to a final concentration of 5 μM. Following infection, the cell culture plate was placed in an IncuCyte® ZOOM (Essen Biosciences) and phase‐contrast and green fluorescence images recorded every 15 min over a time course of 18 h. Images were analyzed using the IncuCyte® ZOOM software (Essen Biosciences).

The plates were scanned with a 10× objective using an IncuCyte^® ZOOM live cell imaging system (Essen BioScience, Ann Arbor, MI, USA) 48 h post-transfection, after loading the plate into the system incubator, 25 images per well were obtained using the green and phase channels through the IncuCyte^® ZOOM software (2018A, Essen BioScience, Ann Arbor, MI, USA). The images were analyzed by dividing the total green object integrated intensity (green calibrated units (GCU) × μm²/image) values of each image by its corresponding total phase area (μm²/image) to obtain the normalized GFP expression (GCU) values per image. The GFP expression from all the images of each well and any replicate wells was averaged to obtain the group means, which represented the final GFP expression values and could be used for statistical analysis. Detailed protocols for the IncuCyte^® system and software operation have been described by our group [43 (link),44 (link)].

Invasive wound healing assay was performed as previously detailed [21 (link)]. Briefly, 96 well ImageLock™ plates (Essen BioScience) were pre-coated with Basement membrane extract (BME, Trevigen, 50 μg/mL) and maintained at room temperature for at least 1 h. Next, BME solution was removed from the plates and fibroblasts were plated (3.5 × 10⁴/well) and incubated overnight at 37 °C and 10% CO₂. The wells containing cells were scratched using the WoundMaker™ (Essen BioScience), washed with PBS and then layered with 4 mg/ml Matrigel containing vehicle or 500 nM Nu7441. Cells were monitored using an IncuCyte ZOOM live cell imager (Essen Biosciences) at 37 °C, 10% CO₂ for 150 h. Wound closure was measured using the IncuCyte ZOOM Software (Essen Biosciences) as relative wound density [measurement of the spatial cell density in the wounded area relative to the area outside the wounded area over time]. 0% represents the wound when there are no cells in the scratched site at t = 0 and 100% represents a wound density where the cell density in the wound is equivalent to the density outside the wound, i.e. 100% wound healing.

For the wound‐healing assay, U87 cells that were transfected for 24 h were seeded in a 96‐well plate to ~ 90% confluence. Wound Maker generated wounds in the middle of each well after cells were starved for 12 h. Then, the cells were cultured in serum‐free medium in the absence or presence of 18:1 LPA (2 μm) as indicated. The percentage of relative wound density was analyzed with the incucyte zoom software (Essen Bioscience, Irvine, CA, USA).

Cells (4 × 10⁴/well) were seeded in IncuCyte^® ImageLock 96-well plates (Essen BioScience, Ltd., Royston, UK) and incubated for 16 h at 37 °C to reach 90–100% confluency. The cell layer was then scratched with the 96-well Wound-Maker^TM (Essen BioScience) and washed 2 times with PBS 1X (Gibco). For invasion assays, 50 µL of reduced Matrigel (Corning) diluted at 1 mg/mL in medium was added to each well and the plate was incubated 30 min at 37 °C to allow the gelation of the matrix. Afterwards, 100 µL of medium alone or containing CuPRiX₂ (800 µM of DOTAGA(Gd) and 500 µM of uncomplexed DOTAGA) or AGuIX^® (800 µM of DOTAGA(Gd)) was added to the appropriate wells. Finally, the plate was placed in the IncuCyte (objective 10X), and images of each well were taken automatically every 2 h in the CO₂ incubator. Data were analyzed using IncuCyte ZOOM software (v. 2018A, Essen BioScience, Ltd., Royston, UK) and expressed as a percentage of wound confluency (relative wound density).

HeLa cells (40,000 cells/mL) were plated in 96-well plates (0.1 mL/well). The following day the cells were treated with different concentrations of Raphin1, in triplicate, as indicated and incubated at 37°C for 48 hr. To monitor cell death 1:2,000 dilution of the CellTox Green Dye (Promega) was added to the medium. The growth of the cells was monitored over time and pictures taken every 2 hr (two per well) with the IncuCyte ZOOM system (Essen BioScience). The default software parameters for a 96-well plate (Corning) with a 10x objective were used for imaging and the images were analyzed by the IncuCyte ZOOM software (Essen BioScience). The software was used to calculate the confluency from the two non-overlapping images of each well at each time point. The phase confluency refers to the percentage of the image area that is occupied by cells. The green confluency refers to the percentage of the image area that fluoresces green. The CellTox Green Dye (Promega) preferentially stains dead cells’ DNA, but is excluded from viable cells. To assess the effect of Raphin1 on cell viability, the percentage of dead cells was calculated as follows: $\%ofdeadcells=\frac{Greenconfluency(\%)atXhours}{Phaseconfluency(\%)atXhours}\times 100$

Cell lines expressing GFP-H2B were transfected with the re-pooled custom siRNA SMARTpool and non-targeting siRNA according to the protocol used in the primary screen. 48 hr post-transfection cells were treated with DMSO or PARGi and imaged by time-lapse microscopy, taking 9 phase contrast and fluorescence images per well every 1-4 hr for a total of 96 hr. IncuCyte^® ZOOM software (Essen Bioscience) was used in real-time to measure confluence and green fluorescent object count. After imaging for 96 hr, cells were fixed and stained according to the immunofluorescence protocol and imaged using the Operetta® High Content Imaging System to give matching γH2AX fluorescence intensity measurements.