Characterization of Itgβ1-, ItgαV- and Itgα6-KD ES-D3 cells and their adhesive properties. A) ES-D3 cells expressing the indicated shRNAs were generated using lentiviral vectors and the efficiency of the target mRNA depletion was determined by qPCR. Data shows means +/− STDs from 3 independent experiments. B) Control (Ctrl), Itgα6-knockdown (KD), ItgαV-KD and Itgβ1-KD ES-D3 cells were seeded onto LN-511-coated cells and grown for 3 days in the presence of 10 ng/ml LIF. Cells were lysed and lysates were subjected to SDS-PAGE and western blotting analysis using the indicated integrin antibodies. β-tubulin antibody was used as a loading control. Data shown is representative from 2–3 independent experiments. C) Expression of α6-, D) αV- and E) β1-integrins in ES-D3 cells. ES-D3 cells were seeded onto indicated substrates in the presence or absence of LIF (10 ng/ml). Cells were harvested at day 5 or passage 5, RNA isolated and the expression levels of the mRNAs for the three integrin subunits was determined by qPCR. Data shown is representative of two independent experiments performed in duplicates. F) Control (Ctrl), Itgα6-, ItgαV- and Itgβ1-KD ES-D3 cells were trypsinized, counted and seeded (1000 cells/mm2) onto tissue culture wells coated with LN-511 (+/− LIF), Col-I or FN as indicated. Adhesion onto different substrates was determined as described in Figure 1C,D.