Sall2 interacts with p75NTR in cells. HEK 293 cells were transfected with p75NTR and FLAG–Sall2. (A) FLAG–Sall2 was immunoprecipitated from a cell lysate and a western blot was probed for p75NTR or FLAG (Sall2). (B) p75NTR was immunoprecipitated from a cell lysate and a western blot was probed for FLAG (Sall2) or p75NTR. (C) Overexpression of p75NTR and Sall2 in the lysates was confirmed by western blotting.

NGF induces translocation of Sall2 into the nucleus. (A) NGF diminishes interaction of Sall2 with p75NTR. HEK 293 cells were transfected with p75NTR and FLAG–Sall2. After 24 h, cells were cultured in serum-free medium for 24 h and then NGF was added. Cells were incubated in the absence or presence of NGF (100 ng/ml) for 15 min. FLAG–Sall2 was immunoprecipitated from cell lysates and western blots were probed with antibodies to p75NTR or FLAG. The percentage of p75NTR in the Sall2 complex (immunoprecipitate) is given above the blot and is the average of results from three independent experiments. (B) Overexpression of p75NTR and Sall2 in the lysates was confirmed by immunoblotting (right). (C) Localization of Sall2 in PC12 cells. Cells were incubated in the absence or presence of NGF for 1 h, after which cell fractions were isolated using NE-PER nuclear and cytoplasmic extraction reagents from Pierce Inc. Fractions were characterized by immunoblotting for Sall2, GAPDH and PARP, and a representative figure is shown. The percentage of Sall2 in fractions is the average of results from three independent experiments.

TrkA is necessary for entry of Sall2 into the nucleus. (A) PC12 cells were serum-starved for 24 h, incubated in the absence or presence of K252a for 30 min (10 nM) and then treated with NGF for 1 h. The cells were fixed, stained with anti-Sall2 and then with a secondary antibody coupled to Alexa Fluor 488. DAPI was used to visualize the cell nucleus. (B) Nuclear Sall2 increases after NGF treatment. The fluorescence intensity of Sall2 staining was quantified by calculating the ratio of nuclear pixel intensity × nuclear area/total pixel intensity × total cell area. Data are the mean±s.e.m. (n=35–40) from three independent experiments; ^*Student's t-test (P<0.005) for NGF versus control and for NGF versus K252a+NGF treatments. (C) PC12nnr5 cells were serum-starved for 24 h, incubated in the absence or presence of NGF (100 ng/ml) for 1 h, fixed, stained with anti-Sall2 and then with a secondary antibody coupled to Alexa Fluor 488. DAPI was used to visualize the cell nucleus. (D) Sall2 nuclear localization was quantified as in (B). Data are the mean±s.e.m. (n=35) from three independent experiments.

Sall2 is required for NGF-dependent p21^WAF1/CIP1 expression and cell cycle arrest. (A) PC12 cells were cultured in differentiation medium (DM) containing low serum for several days. Immunoblotting assayed expression of Sall2, p21^WAF1/CIP1 and actin. (B) PC12 cells were transfected with control or Sall2-specific siRNA and then incubated in differentiation medium for 4 days. Immunoblotting assayed expression of Sall2, p21^WAF1/CIP1 and actin. (C) Sall2 regulates p21^WAF1/CIP1 promoter activity. The effect of Sall2 on the p21 promoter reporter was assayed using a firefly luciferase assay in which PC12 cells were transfected with control or Sall2-specific siRNAs (50 nM). After 48 h in the absence or presence of differentiation medium, luciferase activities were determined and normalized to β-gal. Results are the average fold induction±s.e.m. for three independent experiments performed in triplicate, ^*Student's t-test (P<0.005). (D) Cells were co-transfected with empty vector or HA–Sall2 together with a p21 gene reporter and β-gal and then cultured in same medium as in (C), but without NGF. After 2 days, p21 reporter activity was assayed and is presented as a bar graph as described under (C). Results are the average of fold induction calculated from three independent experiments, each performed in triplicate. (E) PC12 cells were transfected with control or Sall2-specific siRNA and then grown in the absence or presence of differentiation medium for 48 h. BrdU incorporation (in RLUs, relative luciferase units) into DNA was then assayed using a kit from Roche Inc. Results are the average±s.e.m. from three independent experiments performed in sextuplicate. ^*Student's t-test (P<0.001). (F) PC12 cells transfected with empty vector or HA–Sall2 were grown for 2 days in the same medium as in (E) but without NGF. BrdU incorporation was assayed and is presented as described in (D). Results are the average±s.e.m. from three independent experiments performed in sextuplicate. ^*Student's t-test (P<0.001).

Sall2 regulates neurite outgrowth from primary hippocampal cells. (A) Expression of the Sall2 increases during culture of primary hippocampal cells. Sall2 expression was evaluated by confocal microscopy 1 and 4 days after plating. Cells were fixed and stained with anti-Sall2 (green) and anti-β-tubulin III (red). DAPI-stained nuclei are in blue. β-Tubulin was a neuronal marker. (B) Fluorescence intensity (mean pixel × total area) of Sall2 staining was measured by digital image analysis. Data are the mean±s.e.m. (n=30 cells) from three independent experiments, ^*Student's t-test (P<0.001). (C) NGF induces nuclear translocation of Sall2 in primary hippocampal cells. At 1 day after plating, cells were cultured for 16 h in the absence or presence of NGF, fixed, probed with antibodies to Sall2 and β-tubulin III, and then evaluated by confocal microscopy. DAPI-stained nuclei are in blue. (D) Fluorescence intensity of nuclear and total Sall2 staining was measured by digital image analysis. Fluorescence intensity was quantified by calculating the ratio of nuclear pixel intensity × nuclear area/total pixel intensity × total area. Data are the mean±s.e.m. (n=25 cells) from three independent experiments, ^*Student's t-test (P<0.005). (E) Silencing of Sall2 inhibits neurite outgrowth. At 1 day after plating, primary hippocampal cells were co-transfected with control or Sall2-specific siRNA and dsRed. After 2 days, cells were cultured with NGF for 16 h, fixed and evaluated by confocal microscopy. (F) A bar graph shows the percentage of cells that stained positive for β-tubulin III (green) and dsRed positive extending neurites in cells transfected with control or Sall2 siRNA. Data are the mean±s.e.m. (n=20–25 cells) of results from two independent experiments performed in triplicate, ^*Student's t-test (P<0.001).

Sall2 binds the death domain of p75NTR. (A) The death domain (amino acids 324–399) of p75NTR was used to screen a human fetal brain library. Four interacting clones encompass sequences in a 150-amino-acid region in the N-terminal domain of Sall2. The relationship of the clones to full-length Sall2 (shown as a bar) and the death domain (DD) is illustrated. (B) Alignment of primary amino-acid sequences of mouse, rat and human Sall2 using ClustalW. The region of human Sall2 identified by the two-hybrid screen is compared with the N-terminal regions of mouse and rat Sall2.

Relationship of Sall2 and p75NTR in the brain. (A) Immunofluorescence of coronal mouse brain sections probed with antibody to Sall2 at 10 × and 40 × (inset) magnification. C, cortex; CC, corpus callosum; HC, hippocampus; PT, protectum; TH, thalamus. DAPI-stained nuclei appear blue. (B) Confocal microscopy of coronal mouse brain sections probed with antibodies to: β-tubulin III (red) and Sall2 (green); GFAP (red) and Sall2 (green); p75NTR (red) and Sall2 (green); the negative control shows a confocal image without primary antibodies. (C) Endogenous Sall2 was immunoprecipitated from a mouse brain lysate (L) using a Sall2 polyclonal antibody. Rabbit IgG was a control for the immunoprecipitation. A western blot was probed for Sall2 or endogenous p75NTR.

Localization of Sall2 is regulated by NGF, but not by other neurotrophins. PC12 cells were serum-starved and then incubated in the absence or presence of neurotrophins (100 ng/ml, 1 h). (A) Confocal microscopy characterized Sall2 localization after cells were fixed and stained with anti-Sall2 followed by an Alexa Fluor-conjugated secondary antibody. DAPI stained the nuclei, which appear blue. Inserts show magnified cells for each condition. (B) Confocal microscopy of a negative control in which rabbit IgG instead of Sall2 antibody was used for staining.

Regulation of Sall2 expression during neuronal differentiation. (A) PC12 cells were serum-starved for 24 h and then incubated with NGF in 1.5% serum for up to 6 days. Expression of Sall2 was evaluated by immunoblotting. GAPDH expression served as a loading control. The ratio of Sall2 to GAPDH and the fold increase of Sall2 over that at day 0 were calculated after densitometry and are in the upper box. (B) RT–PCR on total RNA from cells treated as described in (A) was assayed for the expression of Sall2, GAPDH and Dnm3b RNA. (C) PC12nnr5 cells were serum-starved for 24 h and then treated with NGF for 1 day. Expression of Sall2 and GAPDH was assayed by immunoblotting. The ratio of Sall2 to GAPDH and the fold increase of Sall2 expression were calculated as in (A) and is in the upper box.

Sall2 regulates neurite outgrowth from PC12 cells. (A) Equal numbers of PC12 cells transfected with control or Sall2-specific siRNA were seeded into cell culture inserts coated with collagen and then cultured in differentiation medium (DM) consisting of 1.5% serum and 100 ng/ml NGF for 2 days. Neurite outgrowth was quantified by measuring cresyl violet (A_{562 nm}) extracted from stained neurites. Data are the mean±s.e.m. of results from three independent experiments performed in triplicate. ^*Student's t-test (P<0.001). The percentage of neurite outgrowth is obtained by dividing neurite formation from cells transfected with control siRNA, or Sall2 siRNA, by that from untransfected cells, and multiplying by 100. (B) PC12 cells co-transfected with control or Sall2-specific siRNA and the transfection marker dsRed were serum-starved for 24 h and then cultured with differentiation medium for 2 days. Confocal microscopy shows the phenotype of the transfected cells after 2 days in differentiation medium. (C) The bar graph shows the percentage of cells in panel B extending neurites at least one and a half cell bodies in length. About 100 cells were counted for each condition. Data are the mean±s.e.m. of results from three independent experiments, ^*Student's t-test (P<0.001). A representative picture of a cell transfected with control or Sall2-specific siRNA and pEGFP is shown under the bar graph. (D) PC12 cells transfected with empty vector or HA–Sall2 were cultured in the medium described under (A) but without NGF. The cells were stained with an antibody to β-tubulin III, and with DAPI, and then examined by fluorescence microscopy at × 40 magnification.