Dhe dye

DCHF-DA was used to assess the presence of a broad array of cellular ROS levels, as previously described.^{53 (link)} DHE preferentially detects superoxide moieties and was used to assess superoxide levels.^{47 (link)} Cells were collected and re-suspended in 1 × PBS at 37 °C containing 50 μM DCHF-DA dye (Sigma) for 20 min or for 30 min in RPMI media containing 20 μM DHE dye (Life Technologies, Carlsbad, CA, USA). For the DCHF-DA dye, samples were treated with 10 Gy or sham treated and immediately placed on ice and analyzed within 8 min by FACS (FL-1). Samples incubated with DHE were also sham treated or irradiated with 10 Gy and analyzed by FACS (FL-2) within 8 min. Annexin V has been previously described to label apoptotic cells for rapid analysis by FACS.^{54 (link)} The flow cytometry-based Annexin V Apoptosis kit was used per manufacturer's protocol (BD Biosciences, San Jose, CA, USA). Cells were collected 48 h post-10 Gy and re-suspended in 1 × binding buffer containing Annexin V and propidium iodide. Cells positive for Annexin V staining were scored as positive for apoptosis.

The level of intracellular reactive oxygen species (ROS) in HCT116 and HT29 was measured using the fluorescent probe dihydroethidium (DHE). For DHE staining, cells were seeded at a density of 50000 cells on coverslips in 24-well cell culture plates and allowed to become 40%-50% confluent. Following 48 h incubation with DIQ treatment at the IC₅₀ dose, CRC cells were fixed in 4% formaldehyde for 20 min. After fixation, CRC cells were washed twice with 1 × PBS, then incubated with 20 μmol/L DHE dye (Invitrogen, Carlsbad, CA, United States). After 45 min staining, the DHE stain was removed, and the cells were washed with 1 × PBS. Mounting media with 4’,6-diamidino-2-phenylindole dye was added. Fluorescence images were taken immediately under a Zeiss LSM710 Laser confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with Zen software to process the images.

Flies were starved for 8 hr and then fed a solution containing 5% sucrose alone or 5% sucrose supplemented with 5 mM paraquat (Sigma-Aldrich) for 20 hr. Flies were then transferred to standard food for 20 hr to allow for recovery (referred to as a 2-day treatment). For time-course experiments, flies were treated with 5 mM paraquat for 6 days after starvation, followed by a 20 hr-recovery (referred to as a 7-day treatment). Testes were dissected into 1 mL of Schneider medium (SM) with 10% fetal bovine serum. One microliter of reconstituted DHE dye (Thermo Fisher) was added and rocked for 5 min in the dark. Testes were washed with SM, followed by fixation with 4% paraformaldehyde for 5–10 min. For the quantification of ROS, dissected testes were dissociated in a cocktail comprising 0.25% collagenase (Thermo Fisher) and 0.5% trypsin (Thermo Fisher) in 1× PBS for 15 min, filtered through 40-μm mesh; the reaction was stopped with SM and centrifuged for 5 min at 450 × g. The cell pellets were resuspended and stained with 10 μM CM-H₂DCFDA dye (Thermo Fisher) for 30 min at 37°C in the dark. The cells were centrifuged again and resuspended in 200 μL of 1× PBS for measurement.