8 chamber glass bottom vessel

For laser micro-irradiation, 5 × 10⁴ cells were seeded into each well of an 8-chamber glass bottom vessel (Thermo Fisher Scientific; Waltham, MA, USA; Cat# 155409). After 24 h, laser micro-irradiation and subsequent time-lapse imaging were performed using a Nikon A1rsi laser scanning confocal microscope equipped with 6 visible wavelength lasers (405, 441, 514, 561, 647 nm, Coherent), customized to add a UVA 355 nm laser (PicoQuant; Berlin, Germany) controlled by a Bruker XY Galvanometer, and equipped with a live-cell incubation chamber (Tokai Hit; Shizuoka, Japan) maintained at 5% CO₂ and 37 °C, using a 20× (NA = 0.8) non-immersion objective for 405 nm laser micro-irradiation. For experiments using PARPi (ABT-888; 10 μM) or PARGi (PDD00017273; 10 μM), cells were treated with the compounds for 1 h prior to micro-irradiation. Micro-irradiation was performed at 100% laser power with a stimulation time of 0.125 s per site. For parallel irradiation, time lapse images were collected every 15 s during a 20 min interval. Images of focal recruitment were quantified using MIDAS for quantitation of and statistical analysis of focal recruitment [5 (link)]. Forty individual cells (2 sets of 10 cells were performed on 2 separate days) were analyzed and used to generate recruitment profiles and kinetic parameters.

A549/LivePAR cells were seeded into each well of an 8-chamber glass bottom vessel (Thermo Fisher Scientific; Waltham, MA, USA; Cat# 155409) at a density of 5 × 10⁴ cells per well. After 24 h, cells were washed twice with 1XPBS, then fixed with 4% formaldehyde while simultaneously staining with Alexa Fluor 647 phalloidin (Invitrogen; Waltham, MA, USA; Cat# A22287) and NucBlue Fixed ReadyProbes reagent (Invitrogen; Waltham, MA, USA; Cat# R37606) for 30 min at 4 °C according to the manufacturer protocols. Cells were washed three times with 1XPBS, and cells were imaged using a Nikon A1rsi laser scanning confocal microscope.