and connexin-43 mRNA expression in Lines 31E + 30F co-cultures after Wnt-3a stimulation. Northern blots were employed to show the expression of cyclin D1 and connexin-43 polyA+ RNAs after stimulation of the cultures for 12 hours with Wnt-3a or insulin + epidermal growth factor (EGF). Co-cultures, made with either pBabeVector or pBabeSFRP-4-infected Line 31E cells together with Line 30F cells, were maintained in 3% serum-containing medium and compared in their responses to Wnt-3a. With Line 31E-Vector cells (31E-V) in co-culture, either Wnt-3a alone or insulin + EGF (Ins+EGF) treatments elicit stimulation of cyclin D1 (CD1) mRNA expression, whereas connexin-43 (Cx43) expression responds only to Wnt-3a, and is strongly inhibited by EGF+insulin. A response to Wnt-3a was not seen; however, when co-cultures were made using Line 31E cells expressing pBabeSFRP-4 (31E-SFRP4), up-regulation of connexin-43 but not cyclin D1 was prevented. However, cyclin D1 mRNA up-regulation is reduced compared to the co-cultures containing Line 31E-Vector cells. Residual ribosomal RNA (loading control) was stained with acridine orange.