Figure 4. Butein unlocks the repression of DDIT3mRNA in the chemoresistant ALDH^bright cells.

A. Heat map. DDIT3 mRNA levels in ALDH^bright and ALDH^low cells purified from the 6M PM cell lines and treated with vehicle (V: DMSO 0.05%) and butein (B: 18 μM), alone or in combination with pemetrexed + cisplatin (P+C: 10 μM + 5 μM, respectively) for 16hrs. B. Upper. Western blotting with DDIT3 antibodies of whole cell lysates from purified ALDH^bright and ALDH^low MSTO-211H cells treated as in 4A. Coomassie staining used as a loading control. Lower. Histograms showing the changes in intensity signal of DDIT3 from duplicate experiments (normalized to two reference protein bands in the coomassie stained gel with Image J software). C. Clonogenic assays. Number of formed colonies from purified cell subpopulations of MSTO-211H and HP-1 cells treated with butein for 16hrs before seeding at clonal density. Histogram bars represent the mean ± s.e.m of triplicate experiments. Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group).