(A) RT-PCR for Sox2, Fgfr2, Fgf4, Cdx2, Eomes, Tead4, Oct4, Nanog, Sox1, Sox3, Sox14, Sox15, Sox21 and beta-actin (40 cycles) on day 4: incubator-control embryos (lane 1); Sox2-duplex-2-siRNA embryos (lane 2); genomic DNA (lane 3); incubator-control morulae –RT (lane 4); Sox2-duplex-2-siRNA morulae –RT (lane 5). In the absence of Sox2 transcript after siRNA, a clear reduction of Fgfr2, Ffg4, Cdx2, Eomes and Tead4 transcripts in Sox2-siRNA embryos was observed. Oct4 and Nanog transcripts were unaffected in Sox2 knock-down morulae compared to incubator-control morulae. To ensure absence of signal did not reflect low detectable message, we performed PCR at 40 cycles. PCR for all transcripts was determined at 25, 30 and 35 cycles with results similar to those presented, but with weaker band intensity where present. Sox1, Sox3, Sox14, Sox15 and Sox21 transcripts were not detected in incubator-control morulae or Sox2 knock-down morulae. Atp1b1 (ATPase, Na+/K+ transporting, beta 1 polypeptide), Gata3, Gata4 and Gata6 transcripts were detected in comparable levels in Sox2 knock-down morulae compared to incubator-control morulae. Beta-actin transcripts were detected in both Sox2-siRNA and incubator-control embryos. This figure illustrates Sox2-duplex-2-siRNA embryo transcripts, whereas similar patterns of transcript expression for Sox2-siRNA duplexes -1 and -3 are shown in Figure S4. (B) Comparison of Sox2 knock-down embryos (as assessed by absence of fluorescence after Sox2 staining) with incubator (untreated) control embryos for ICM and TE markers. Immunofluorescence for Sox2 (Abcam), Oct4, Nanog, Cdx2 (rabbit polyclonal from Jane Collins), Eomes, Fgfr2, Fgf4, Occludin, ZO1, Desmoplakin, E-cadherin, Gata4 and Gata6 to compare protein expression between incubator control and Sox2-siRNA embryos at day 4 and 5 of preimplantation development. The images are single optical sections from confocal Z-series. Sox2-duplex-1-siRNA embryos stained for Sox2 are shown in this Figure. In all cases, after knock-down with each of the three duplexes, Sox2 siRNA embryos were negative for Sox2. For assessment of the other markers, embryos from all three knock-down construct groups were pooled. A large reduction in Sox2, Cdx2, Eomes, Fgfr2, Fgf4 and Occludin protein was observed, whereas Oct4, Nanog, ZO1, Desmoplakin, E-cadherin, Gata4 and Gata6 remained unaffected. However, Nanog staining appeared more cytoplasmic in Sox2 knock-down embryos. The selected image for the day 4 incubator control embryo stained for Cdx2, is a plane through the top of the embryo and the positively stained Cdx2 cell in the center is located in the outer layer of the embryo. The staining patterns for Sox2 and Cdx2 were confirmed with different Sox2 (Chemicon) and Cdx2 (Biogenex) antibodies (Figure S3). At least 10 embryos were stained for each antigen and representative embryos are shown. Blue indicates DAPI stained nuclei and green staining for the protein of interest. In all cases immunological controls were negative (Figure S6). Where antibodies were compatible, embryos were double-stained for two markers. In all cases the anti-Sox2 staining for Sox2-siRNA embryos was negative. Bar: 50 µm.