Figure 2.

Sall2 deficiency accelerates G1‐/S‐phase transition and correlates with increased levels of cyclins D1 and E1. Sall2 ^+/+ and Sall2 ^−/− iMEFs were synchronized at G2‐M by nocodazole as described in materials and methods, and then released into the cell cycle. (A) Cell cycle analysis of Sall2 ^+/+ and Sall2 ^−/− iMEFs by flow cytometry. The graphs show representative cell cycle profiles of each iMEFs over time (hours) after release from nocodazole. (B) Progression of Sall2 ^+/+ iMEFs through the cell cycle was compared with that of Sall2 ^−/− iMEFs every 2 h for a total of 12 h. Data are presented as the percentage of cells at G2‐M, G1, and S phases over time after nocodazole release and are representative of four independent experiments. (C) Sall2 ^+/+ and Sall2 ^−/− iMEFs were harvested at various times after release to evaluate SALL2 protein expression and compare the expression of the indicated cyclins by western blot. β‐actin was used as normalizer. Figure is representative of four independent experiments. (D) Graph shows relative SALL2 protein level in Sall2 ^+/+ iMEFs after various hours from nocodazole release. SALL2 protein level was determined by densitometric analysis and normalized to the corresponding β‐actin level. Data are expressed as mean ± SD from four independent experiments. *P ≤ 0.05, one‐way ANOVA; n.s, not significant. (E) Sall2 mRNA expression was evaluated by qPCR analysis using Ppib as normalizer. Data are expressed as mean ± SD from three independent experiments performed in triplicate. ***P ≤ 0.001, one‐way ANOVA. (F,G) RNA was isolated from Sall2 ^+/+ and Sall2 ^−/− iMEFs and mRNA levels of Ccnd1 (F) and Ccne1 (G) were measured by qPCR. Numbers are relative to Ppib. Data are expressed as mean ± SD from three independent experiments performed in triplicate. *P ≤ 0.05; **P ≤ 0.01, Student's t‐test; n.s, not significant.