(A Immunofluorescence show specific expression of TWIST-1 only in cells undergoing EMT; (i) CD44+/MyD88+ EOC stem cell monolayer is negative for TWIST-1; (ii) TWIST-1 is expressed in cells with fibroblast/mesenchymal morphology; (iii) TWIST-1 is highly expressed in cells forming spheroids; (iv) Western blot analysis showing TWIST-1 expression is mainly nuclear. EOCSC - CD44+/MyD88+ EOC stem cells; MSFC - mesenchymal-like spheroid forming cells.
(B (i) Transfection of pEMSV-TWIST1 into CD44+/MyD88+ EOC stem cells is not able to induce expression of TWIST-1 protein; (ii-iii) the proteasome inhibitor, MG132 is able to increase endogenous and ectopically expressed TWIST-1.
(C (i) Co-transfection of E12 and TWIST-1 significantly increased TWIST-1 expression in the CD44+/MyD88+ EOC stem cells; ii) parallel increase in E12 and TWIST-1 was observed in the MSFCs and in mouse xenograft. EOCSC - CD44+/MyD88+ EOC stem cells; MSFCs - mesenchymal spheroids forming cells.