Fig. 1.

Combination of erastin and celastrol induced NSCLC cell death in vitro. (A) HCC827, A549 and H1299 cells were treated with erastin at the indicated concentrations for 24 h, and cell growth was assayed by a CCK‐8 assay. (B) HCC827, A549 and H1299 cells were treated with celastrol at the indicated concentrations for 24 h, and cell growth was assessed by a CCK‐8 assay. (C) HCC827, A549 and H1299 cells were treated with indicated combinations of concentrations of erastin and celastrol for 24 h. Cell viability was measured by a CCK‐8 assay. (D) Cell death was measured by a PI assay using flow cytometry. (E) Combination index (CI) analyses were performed to determine synergy using the Calcusyn software. (F) Representative phase‐contrast microscopic images of HCC827 cells treated with either erastin or celastrol or their combination for 24 h. Most of the cells became smaller in size and round in shape after cotreatment with celastrol and erastin for 24 h. Scale bars, 200 µm. (G) Cell morphology was examined by quantitative holographic phase microscopy. The scale bar indicates the cell height. Scale bars, 200 µm. (H) Representative images of colony formation of HCC827 cells after exposure to either erastin or celastrol, or in combination for 24 h. The mean ± SD is shown, n = 3. Statistical significance was determined using two‐tailed unpaired Student’s t‐tests. ***P < 0.001. n.s., not significant.