Fig. 3. Reduced proliferation of Sall4-null ES cells. (A) Northern blot analysis showing the reduction of Sall4 upon Sall4-siRNA treatment. KD (knockdown), treated with Sall4-siRNA; NC (negative control), treated with control siRNA. (B) Transiently reduced proliferation of ES cells upon Sall4-siRNA treatment. Cells were counted in triplicate. (C) Conditional disruption of Sall4 in ES cells. When a Sall4-IRES-Hyg vector was introduced into cells heterozygous for a floxed allele of Sall4 (flox/+), both alleles were targeted with a similar frequency, resulting in two types of cells: flox/- and +/-. Upon infection with adenovirus expressing Cre, flox/-cells became almost Sall4-null by day 3, determined by western blot (lowest panels). White triangle, Frt; black triangle, loxP. (D) Reduced proliferation of Sall4-null ES cells. Cell expansion rate over 16 days is shown, using Sall4-null (-/-) versus flox/-cells obtained upon the same Cre treatment. Analysis was carried out in triplicate. (E) Reduced S phase and increased G1 phase in the Sall4-null ES cells. Consistent data were obtained from two independent experiments using three Sall4-null cells, and the representative data is shown. (F) Normal morphology and positive staining of Oct3/4 of a Sall4-null ES colony. (G) Northern blot analysis of Sall4-deficient ES cells. Two heterozygous (flox/-), and two Sall4-null ES (-/-) clones are shown. (H) Chimeric embryo formation from Sall4-null ES cells transfected with GFP. (left) High contribution of GFP-expressing cells in the E7.5 embryo. (Right) A section of the chimera was stained by an anti-GFP antibody and detected by DAB.