μ slide 8 well chambered coverslip slide

PANC-1-ABE8e-GFP sorted cells expressing the base editor were infected with pLenti.sgG12D-1-TdTomato, at MOI = 1, resulting in ∼50% infection rate to create an internal competition between cells bearing the base editing gRNA and cells expressing only the base editor. Four days after infection, cells were seeded into a μ‐Slide 8 well‐chambered coverslip slide (Ibidi) containing 300 μL of FluoroBrite DMEM (Gibco) supplemented with 10% FBS, 4 mmol/L GlutaMAX, and 1% Pen/Strep. Time‐lapse microscopy‐based imaging was performed over 5 days using the Deltavision Elite deconvolution microscope. Images were acquired on FITC, TRITC, and Cy5 channels every 30 minutes using a 20×/1.42 plan‐Apochromat objective, at 37°C with 5% CO₂. Subsequently, images were deconvolved and z‐projected using softWoRx analysis software (SoftWoRx, RRID:SCR_019157).

U2OS wild‐type and MLLT6 knockout cells were transfected with pEGFP‐C1 EGFP‐3XNLS (Addgene, 58468) and pmCherry‐C1 mCherry‐NLS (Addgene, 58476) plasmids, respectively, to generate eGFP and mCherry expressing stable lines. A 50:50 mixture of 30,000 WT‐eGFP and MLLT6‐KO‐mCherry cells were seeded into a μ‐Slide 8 well‐chambered coverslip slide (ibidi) containing 100 μl of DMEM supplemented with 10% FBS and Pen/Strep. The media was replaced with 300 μl of FluoroBrite DMEM (Gibco) containing T cells in the ratio of 3:1 to cancer cells and 0.2 μg of anti‐EpCAM‐CD3 bi‐specific antibodies (Creative Biolabs). Time‐lapse microscopy‐based imaging was performed employing the Deltavision Elite deconvolution microscope. Images were acquired on FITC and Alexa 594 channels every 10 min for 10 hours using a 20×/1.00 plan‐Apochromat objective at 37°C with 5% CO₂. Subsequently, images were deconvolved and z‐projected using image processing and analysis software, Fiji (Schindelin et al, 2012). Wild‐type and MLLT6 KO cell numbers were determined by manually counting cells on every image captured at 30‐min intervals to generate a Kaplan–Meier curve.