Fig. 4.

Short pulse treatment of doxorubicin at micromolar concentration: its application in the 3D culture system and survey in various cancer cells. (A) Schematic diagram of pulse-treatment experiments. (B) Northern and Western blots, as performed according to the scheme in A. (C) A brief summary illustrating how to generate the 3D culture system, with schematic (Upper) and actual images (Lower). (D and E) Northern and Western blots described in A–C. Lane 1, continuous doxorubicin treatment; lane 2, no doxorubicin control; lanes 3–4, doxorubicin pulse as described in A. (F) A rank distribution plot of IC₅₀ values of 791 cell lines in ascending order (see the text for detailed description and Dataset S2 for full information). We selected two groups of cells: doxorubicin-sensitive (IC₅₀ < 0.6) and -resistant (IC₅₀ > 1.5). In each group, cells were further classified according to the degree of nc886 CpG methylation (from 10 CpG sites in the nc886 genomic region) and depicted in the pie charts on the top. Hypermethylation was defined as >90% methylation on average. Statistical significance of differential methylation between the two groups was calculated by a χ² test.