Figure 6. Converted MCF7 RR cells biochemically similar to RR cells.

RU and RR cells derived from MCF7 were exposed to 0.5 mM H₂O₂ for 6 hours in complete culture media and further cultured for 3 days in fresh media. The mRNA expression levels of PROM1 A., GPR49 B. and MUC15 C. were measured by qRT-PCR by using specific primers. The data showed the significant increase in the mRNA expression of all three genes upon H₂O₂ treatment in the RU cells. RR cells did not show any significant change in gene expressions except in case of GPR49. D. Cell lysates derived from the nuclear fraction of H₂O₂ treated RU and RR cells were incubated with the biotinylated SRR2 probe. A similar amount of lysate proteins derived from treated and untreated RU and RR cells was used. The SRR2 probe-protein complexes were then captured by streptavidin beads. By western blots, Sox2 was prominently detectable in H₂O₂ treated RU cells contrary to very low to no detection in untreated RU cells. RR cells did not show any noticeable change in Sox2 binding to SRR2. Mutation of the SRR2 completely abrogated the binding of Sox2 to the probe (top panel). The western blot of input samples were evaluated for the quality of lysates of nuclear fractions by using an anti-Sox2 antibody. HDAC-1 was used as loading control (bottom panel). E. Cell lysates of H₂O₂ treated and untreated cells were analyzed by western blot. Protein level of E-cadherin was prominently increased, and Slug and vimentin were decreased after H₂O₂ treatment in RU cells in comparison to that of untreated RU cells. RR cells showed similar pattern as converted RR cells.