Dlx-2 shRNA prevents metabolic stress-induced necrosis in MTS. (A) Formation, growth, and morphology of MTSs derived from MCF-7 cells, which were cultured for up to 13 days. (B-C) MCF-7 spheroids cultured on agarose for the indicated times were analyzed by real-time PCR for Dlx-2 expression (B). The values are expressed as mean ± SE (n = 3). *P < 0.05, **P < 0.01 versus two-dimensional cultured cells. The MCF-7 spheroids were also analyzed using Western blotting with antibodies against Dlx-2 and α-tubulin (C). (D-E) After 7 days of MCF-7 MTS culture, the MTSs were dissociated into subpopulations of cells from different locations in the spheroids, as described in Materials and Methods. The cells isolated from different locations within the MCF-7 spheroids were analyzed by RT-PCR using primers for Dlx-2 and β-actin (D). The cells were also analyzed using Western blotting with antibodies against Dlx-2 and α-tubulin (E). Arrow in panels C and E, a putative modified form of Dlx-2.