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. 2016 Jul 8;7(32):51503–51514. doi: 10.18632/oncotarget.10491

Figure 3. Hypomethylation of the HOXA9 promoter in lrECM 3D culture.

Figure 3

(A) Total cell DNA was extracted from MDA-MB-231 cells in 2D and lrECM 3D cultures on day 6. CpG methylation of the HOXA9 promoter was compared between two culture conditions using bisulfite treatment coupled with methylation-specific PCR. The intensity of each PCR product was quantified by densitometry using NIH Image J. (B) Total cell RNA was extracted from 2D culture of MDA-MB-231 cells with exposure to either a DNMT inhibitor, 5-Aza-2dC (5 & 10 μM) or DMSO for 72 hrs. The mRNA levels of HOXA9 were measured using qRT-PCR. A fold change was obtained by normalizing to the housekeeping gene RPLP0 and setting thevalues from the DMSO group to one. (C) Similar to part B except that the mRNA levels of HOXA9 were compared between the TSA (500 nM, 72 hrs) treated group and the DMSO treated group. (D) Similar to part C except that the mRNA levels of HOXA9 were compared between the TSA (500 nM, 72 hrs) treated group and the DMSO treated group in Hs578T cells. When presented, means and standard deviations were obtained from three independent experiments. *, **, *** indicate a P value < 0.05, 0.01, 0.001, respectively.