Cancer cell efflux assay

Cancer cells were seeded in 6-well plates (2 × 10⁵ cells/well) in 1 mL of complete media and allowed to adhere by incubating at 37 °C for 12 h (~70% confluency). Cells were then loaded with 1 μM rhodamine 123 (Rh123) followed by incubation at 37 °C for 1 h. Next, cells were loaded with RSQ (1 or 100 μM), 1 μM of P-gp inhibitor PSC 833 (positive control), or complete media (negative control). Cells were then incubated (30 min at 37 °C) before washing with PBS (1 mL), trypsinizing with Trypsin-EDTA solution, and neutralizing using Trypsin neutralizing solution (5% HI-FBS in PBS). The resulting cell suspensions were centrifuged (300 × g for 5 min), and cell pellets were suspended in 300 μL FACS solution (PBS, 2% HI-FBS, 1 mM EDTA, 0.1% NaN₃) before analysis by flow cytometry to measure intracellular Rh123. Gates for each cell population were set (Supplementary Fig. S4), and 5000 events were collected inside the gate (Fig. 4d). This experiment was performed in hexaplet for all three cancer cell lines. Mean fluorescence intensity from each experiment was normalized relative to cells treated with Rh123 alone (Fig. 4e).