Fig 2: Comparison of Subcellular localization for SALL2 Alternative Isoforms. Representative model of alternative SALL2 isoforms displaying main structural characteristics; green ovals represent C2H2 zinc finger motifs, and the pink rectangle represents the Q-rich sequence present in both E1 and E1A. E1 contains the MSRKKQ motif, found in other SALL members but not in E1A or short E1A isoforms. The number of residues, “NNCN” score, and immunocytochemistry data are indicated.

Fig 3: SALL2 binds and represses CCND1 and CCNE1 promoters. Bioinformatic analyses of cyclin promoters to identify putative SALL2 sites were performed using a previously reported binding site matrix (consensus sequence GGG(T/C)GGG) (Gu et al., 2011) in Transcriptional Regulatory Element Database (TRED) (https://cb.utdallas.edu/cgi-bin/TRED/tred.cgi?process=home) (Jiang et al., 2007). Sequences analyzed [-2000 bp from transcription start site (+1)] were obtained from Eukaryotic Promoter Database (EPD) and included mouse Ccnd1 (NM 007631), mouse Ccne1 (NM 007633), human CCND1 (NM 053056), and human CCNE1 (NM 001238). A. Schematic representation of human CCND1 (NM 053056) and CCNE1 (NM 001238) gene promoters. SALL2 putative binding sites are represented by square symbols. Putative SALL2 binding sites are classified as high, middle, and low scores according to their identity with the SALL2 consensus matrix (Gu et al., 2011) The transcription start site (+1) is represented by a flag. (B,C) Repression of CCND1 (B) and CCNE1 (C) promoters’ activities by SALL2. Transient co-transfections of pGL3-CCND1 or pGL3-CCNE1 reporter with or without SALL2 E1 (blue bars) (or E1A, green bars) into HEK293 cells were performed as described under ‘Materials and Methods’. Luciferase activity was measured from cell lysates and normalized to ß-galactosidase activity, and promoter activity was expressed as relative luciferase units (R.L.U). pGL3 vector served as control. Data are expressed as mean ± SD from three independent experiments performed in triplicate. ***P = 0.001, one-way ANOVA. (D–F) HEK293 cells were transfected with pcDNA3-SALL2 (E1, blue) or pcDNA3-SALL2 (E1A, green) vector. Chromatin was immunoprecipitated 24 h after transfection using SALL2 antibody or normal rabbit IgG (control antibody), and specific genomic regions in the human CCND1 (D), CCNE1 (E), proximal promoters, and a nonrelated promoter region (NRR) were analyzed by real-time PCR. Graphs show quantification of the amplified DNA for each immunoprecipitation relative to IgG. Results are representative of two assays performed in triplicate.

Fig 4: 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. ß-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 ß-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.

Fig 5: Immortalized Sall2-deficient cells possess transforming ability. (A,B) Foci formation assay. iMEFs were grown in regular culture medium for 12–18 days prior to staining with crystal violet. (A) Microscopic visualization of three individual focus from Sall2 -/- iMEFs photographed at 4× magnification. The appearance of the Sall2 +/+ iMEFs culture (left) is shown for comparison.(B) Top, crystal violet staining of Sall2 +/+ and Sall2 -/- iMEFs. Bottom, quantification of number of foci per plate. Results are representative of three independent experiments performed in triplicate (**P = 0.01, Student's t-test). (C) Sall2 -/- MEFs showed increased anchorage-independent growth. Sall2 +/+ and Sall2 -/- iMEFs were grown in soft agar for 3–4 weeks. Top, colonies were photographed at 4× magnification. Primary Sall2 +/+ MEFs were used as negative control. Bottom, quantification of number of colonies per plate. Results are representative of three independent experiments performed in triplicate (**P = 0.01, Student's t-test).