Hybrid microscope

An ALDEFLUOR assay was performed using an ALDEFLUOR kit (Stemcell Technologies, Cambridge, MA, USA), according to the manufacturer’s instruction. Briefly, control and NQO1 stable cells were seeded into a six-well plate and cultured for 24 h at 37 °C in an incubator supplied with humidified air containing 5% CO₂. Twenty-four hours later, bL (2 µM) was added to the culture media, and the cells were cultured for an additional 24 h; next, 10 µL of activated ALDEFLUOR was added to the media for 45 min. The cells were washed with PBS three times, and pictures were taken under a hybrid microscope (Echo, San Diego, CA, USA).

Mammosphere formation was performed using MammoCult media (Stemcell Technologies, Cambridge, MA, USA) supplemented with 4 µg/mL heparin and 0.48 µg/mL hydrocortisone, according to the manufacturer’s instructions. In brief, 4 × 10⁴ control and NQO1 stable cells were seeded into ultralow adherent six-well plates (Stemcell Technologies, Cambridge, MA, USA)) and incubated with MammoCult media for one or two weeks. Then, bL (2 µM) was added for 24 h, and the cell morphology was observed under a hybrid microscope (Echo, San Diego, CA, USA).

Cells were fixed in 4% (w/v) paraformaldehyde (PFA)/PBS for 20 min and then permeabilised with 0.1% Triton X-100 for 15 min. After blocking with bovine serum albumin for 30 min, cells were incubated with primary antibody overnight at 4 °C. Cells were then incubated with FITC conjugated secondary antibodies (Proteintech) at room temperature for 1 h; Echo Revolve Hybrid Microscope (San Diego, CA, USA).
Use image j to analyse the quality of co-localisation.