Cells were grown on Nunc Lab-Tek II chambered cover glass for imaging experiment.
To express ATM-SPARK, HEK293 cells were transiently transfected using calcium phosphate transfection reagent (15 (link)) with 300 ng of ATM-SPARK plasmid plus 50 ng of H2B-mIFP-T2A-mCherry plasmid, 100 ng of mCherry-POLQ plasmid, or 300 ng of dnATM (ATM kinase dead) or dnATR (ATR kinase dead). Hela cells were transiently transfected using Lipofectamine 3000 transfection reagent with 300 ng of ATM-SPARK and 100 ng of mApple-53BP1 or BRCA1-mCherry plasmids. HEK293 and Hela cell were imaged 1 day after transfection. U2OS, T98G, CHLA, and U87 cells were infected with medium containing ATM-SPARK lentivirus and imaged 3 days after infection.
All imaging was carried out on Nikon Eclipse Ti inverted microscope equipped with Yokogawa CSU-W1 confocal scanner unit (Andor), digital complementary metal-oxide semiconductor camera ORCA-Flash4.0 (Hamamatsu), and ASI MS-2000 XYZ automated stage (Applied Scientific Instrumentation). Imaging was performed in environmental control unit incubation chamber (In Vivo Scientific) maintained at 37°C and with 5% CO2. Fluorescence images were acquired using Nikon CFI Plan Apochromatic 20× dry [numerical aperture (NA), 0.75] objective or CFI apochromatic TIRF 60× oil objective (NA, 1.49) against GFP, mCherry, or mIFP per experiment settings. Imaging started before adding NCS (500 ng/ml) or etoposide (3 μM) and continued for around 1.3 hours. For experiments with inhibitors (KU-55933, AZD6738, and NU7026), cells were pretreated with inhibitors (20 μM) or DMSO before NCS treatment. To demonstrate sensor reversibility, cells were preincubated with NCS and then treated with KU-55933 or DMSO, and imaging started for several frames before adding KU-55933 or DMSO and continued for around 1 hour.
To express ATM-SPARK, HEK293 cells were transiently transfected using calcium phosphate transfection reagent (15 (link)) with 300 ng of ATM-SPARK plasmid plus 50 ng of H2B-mIFP-T2A-mCherry plasmid, 100 ng of mCherry-POLQ plasmid, or 300 ng of dnATM (ATM kinase dead) or dnATR (ATR kinase dead). Hela cells were transiently transfected using Lipofectamine 3000 transfection reagent with 300 ng of ATM-SPARK and 100 ng of mApple-53BP1 or BRCA1-mCherry plasmids. HEK293 and Hela cell were imaged 1 day after transfection. U2OS, T98G, CHLA, and U87 cells were infected with medium containing ATM-SPARK lentivirus and imaged 3 days after infection.
All imaging was carried out on Nikon Eclipse Ti inverted microscope equipped with Yokogawa CSU-W1 confocal scanner unit (Andor), digital complementary metal-oxide semiconductor camera ORCA-Flash4.0 (Hamamatsu), and ASI MS-2000 XYZ automated stage (Applied Scientific Instrumentation). Imaging was performed in environmental control unit incubation chamber (In Vivo Scientific) maintained at 37°C and with 5% CO2. Fluorescence images were acquired using Nikon CFI Plan Apochromatic 20× dry [numerical aperture (NA), 0.75] objective or CFI apochromatic TIRF 60× oil objective (NA, 1.49) against GFP, mCherry, or mIFP per experiment settings. Imaging started before adding NCS (500 ng/ml) or etoposide (3 μM) and continued for around 1.3 hours. For experiments with inhibitors (KU-55933, AZD6738, and NU7026), cells were pretreated with inhibitors (20 μM) or DMSO before NCS treatment. To demonstrate sensor reversibility, cells were preincubated with NCS and then treated with KU-55933 or DMSO, and imaging started for several frames before adding KU-55933 or DMSO and continued for around 1 hour.
