Fig. 2.

Acetaminophen (AAP) inhibits CSC growth, self-renewal and CSC marker expression in vitro.

(A) Dose response curves of AAP for primary spheroid inhibition in selected NSCLC cell lines and primary human tumor-derived cells. Sigma plots software was used to determine IC₅₀ of spheroid formation. H460, CUTO29 and H1975 cells were plated in CSC media with increasing concentrations of AAP. Four days later, the number of spheroids per well 50-150 μM was counted. Results reported as percent of untreated vehicle. (B) In vitro limiting dilution assay (LDA) of H460, CUTO 29 and H1975 NSCLC cells. Cells were treated with vehicle or AAP for 24 h and then plated in fresh CSC media (no treatment) in limiting dilutions (0→128 cells /well). Four days after plating, wells were assessed for spheroid formation (yes/no). (C to E) H460 NSCLC cells were grown in CSC media as spheroids and treated for 24 h with DMSO (veh) or AAP (1 mM) prior to harvesting. (C, D) Immune-blot analyses for CSC markers, and (E) quantitative real-time PCR analyses for self-renewal factors in vehicle and AAP treated samples. (n = 3, 2 independent experiments). * P < 0.05.