Protein chip analysis of pluripotency-associated proteins in NIH3T3 fibroblast

Specific transcription factors regulate the totipotent and pluripotent capability of embryonic stem cells. Amongst these regulatory transcription factors in embryonic stem cells, Oct4 and Nanog are master factors that also have unique characteristic ability of cell-specific pluripotency and self-renewal. The expression of Nanog in fibroblasts confirms increased cell proliferation and transformation of foci-forming phenotype indicative of its oncogenic potential. The expression of Oct4, interestingly, leads to transformation of non-tumorgenic mouse into tumorigenic mouse. Our current investigation ascertains that the resultant increase in DNA synthesis and cell proliferation is the consequence of transforming the phenotype into foci formation. We used a manually curetted ProteoChip to carry out the signaling protein microarray analysis, which revealed up-regulated expression of various proteins including FAK1, MEK1 and Raf1. Some of the proteins explain the mechanism by which Oct4 and Nanog transform the phenotype. In NIH3T3 cells expressed with mouse Oct4 (mOct4), mouse Nanog (mNanog) separately as well as together, the specific knockdown of mFAK1 inhibited morphological transformation of the cells, and their invasion activity. The mFAK1 overexpression leads to morphological transformation as shown with mOct4 and mNanog. Additionally, we showed that the ERK1/2 pathway is involved in the up-regulation of c-myc and cyclin D1 expression mediated by mFAK1. Our results signify that the combinatorial signaling protein-array using biomolecular approach may possibly provide us with a new tool to understand cellular homeostasis.