24 well plate

KTR-expressing ES2 or PtD cell lines were established by lentiviral transfection with the ERK KTR mClover and JNK KTR mRuby2 constructs, gifted from M. Covert (Addgene plasmids #59150 and #59154) (42 (link)). For in vitro characterization, reporter cell lines were seeded 40,000 cells per well into a 24-well plate (Ibidi) and then treated the following day with EGF (PeproTech), anisomycin (Sigma-Aldrich), or JNK-IN-7 (MedChemExpress). After a 24-hour incubation period, cells were fixed with warm 4% paraformaldehyde in PBS for 15 min, stained with Hoechst 33342 (1 μg/ml; Invitrogen) for 10 min, and imaged with a Leica DMI 6000 microscope and Oasis Surveyor software. ImageJ [National Institutes of Health (NIH)] was used to quantify the C/N intensity ratios of mClover and mRuby2 fluorescence. Similar experiments were also performed ±trametinib and ±transwell coculture with M2-MΦ.

RKO FKBP-WRN triple reporter cells were seeded in a 24-well plate (Ibidi, #89626) approximately 18 hours prior to imaging. Cells were plated such that cell density remained sub-confluent until the end of the imaging period (3–6 days). Depending on the goal of the experiment, different drug treatment and imaging schedules were used. To examine 53BP1 induction, cells were first imaged for approximately 20 h in drug-free media to establish baseline 53BP1 level and behavior, after which 0.5 μM dTAG-13 or vehicle (DMSO) was quickly added and imaging resumed for an additional three days. To conduct long-term cell fate mapping, cells were treated with DMSO or 0.5 μM dTAG-13 and imaged over a span of six days. To explore the effect of WRN/ATR single or co-inhibition, cells were first treated with vehicle or 10 nM dTAG-13, followed by the addition of vehicle or 0.25 μM AZ-20 two hours later before commencing image acquisition (6 days). In all cases, time-lapse images were taken in CFP, YFP, and RFP channels every 12 min on a Nikon Ti2-E inverted microscope (Nikon) with a 20X 0.45NA objective. NIS Elements (Nikon, v5.11.00) software was used for controlling image acquisition. Total light exposure time was kept under 600 msec for each time point. The microscope was housed within an environmental chamber to maintain cells at 37 °C with 5% humidified CO₂.

Pairs of VFF and oral mucosa fibroblasts (OMF) with similar passage numbers were seeded into 2-chamber silicone inserts which were placed in a 24 well plate (ibidi, Martinsried, Germany). Depending on preliminary experiments, 20,000–30,000 cells resuspended in 70 μL GM were pipetted into each chamber and incubated overnight, which resulted in confluent cell layers. Subsequently, silicone inserts were removed with sterile forceps, cells were washed 3 times with PBS and 1 mL of standard medium (SM), consisting of DMEM/F12 Nutrient mix (3:1) supplemented with 5% FBS and 100 μg/mL Normocin, was added per well. Closure of the 500 μm gap between the 2 regions of cells was monitored using a Cell-IQ system (Chipman Technologies, Tampere, Finland) with automated phase contrast imaging (4–6 regions per well) performed every 30 min over a period of 72 h. Semi-automatic segmentation of the image series and quantitative analysis was performed using the Cell-IQ software. For each cell type, at least 3 wells (assayed in parallel) were averaged as technical replicates.