Figure 1.
EGF and FGF2 have opposite effects on STAT3 phosphorylation in hNSCs.
(A) Lysates from neurospheres as well as hNSCs primed with either EGF (ELL) or FGF2 (FHL) for 24 hrs were analyzed by immunoblotting using antibodies against phosphorylated STAT3-Tyr705 (pY705) or STAT3-Ser727 (pS727). The blots were stripped and re-probed for total STAT3 (tSTAT3) and GAPDH (loading control). Densitometric analysis was performed and ratios of phosphorylated STAT3 to total STAT3 (pSTAT3/tSTAT3) are graphically represented as Mean ± SEM (n = 3). *p<0.05 and ***p<0.001 by One-way ANOVA with Tukey post-hoc test. pSTAT3-Y705 is increased in EGF-primed cells, but reduced in FGF2-primed cells when compared to unprimed neurospheres. pSTAT3-S727 levels, however, remain unchanged after either priming. Representative phase contrast images of primary culture hNSCs are shown as neurospheres (B), one day after ELL- (C) or FHL-priming (D). Scale bars, 20 µm. hNSCs: human neural stem cells; ELL: EGF plus LIF and laminin; FHL: FGF2 plus heparin and laminin.
Figure 2.
Stattic and Niclosamide exhibit dose-dependent effects on hNSCs.
Cell viability is determined by WST-1 assay in FHL- or ELL-primed hNSCs treated with varying doses of Stattic and Niclosamide. (A) Low doses of Stattic (up to 2.5 µM) did not affect viability in either FHL or ELL conditions. Higher concentrations of Stattic, such as 5 µM, are toxic to cells. (B) Low doses of Niclosamide reduced the number of viable cells in ELL-primed group whereas higher concentrations of Niclosamide (5 µM) were toxic to both FHL- and ELL-primed cells. Data are presented as mean ± SEM (n = 6), ***p<0.0001; **p<0.01 by One-way ANOVA with Tukey post-hoc tests. hNSCs: human neural stem cells; ELL: EGF plus LIF and laminin; FHL: FGF2 plus heparin and laminin.
Figure 3.
Increased motor neuron differentiation in hNSCs by STAT3 inhibition.
(A–C) Semi-quantitative RT-PCR analyses of the expression level of HB9 mRNA, a marker for spinal motor neurons, after a 4-day priming. GAPDH mRNA expression serves as internal control. In general, FHL-primed hNSCs (A,B) express more Hb9 mRNA than the corresponding ELL-primed cells (C). Hb9 mRNA levels are significantly increased in FHL-primed hNSCs treated with 0.5 µM Stattic (A) or 0.25 µM Niclosamide (B). In contrast, STAT3 inhibition does not alter HB9 mRNA expression in ELL-primed cells (C). Values are mean ± SEM (n = 3), *p<0.05, One-way ANOVA plus Tukey post- hoc tests (A and C) or Student’s t-test (B). (D–G) Immunofluorescent staining of Hb9/MAP2-labeled motor neurons in hNSCs primed and inhibitor-treated for 4 days and differentiated in B27 for 9 days. Hb9 immunoreactivity is in general higher in FHL-primed than ELL-primed cells. Representative epifluorescent microscopic images show increased Hb9 and MAP2 immunoreactivity in FHL-primed hNSCs after treatment with Stattic (0.5 µM) and Niclosamide (0.25 µM) (D). Inhibition of STAT3, on the other hand, does not affect HB9/MAP2 labeling in ELL-treated cells (E). Arrowheads point to cells that are co-labeled with Hb9 and MAP2, and contain a DAPI-stained nucleus with cytoplasm extended into neurites. Scale bar: 10 µM (F–G) Quantitative analyses show that 0.5 µM Stattic significantly increase the percentage of Hb9+/MAP2+ cells in FHL-primed cells. *p<0.05 by One-way ANOVA with Tukey post-hoc tests. hNSCs: human neural stem cells; ELL: EGF plus LIF and laminin; FHL: FGF2 plus heparin and laminin; Statt: Stattic; Nicl: Niclosamide.
Figure 4.
STAT3 phosphorylation in hNSCs treated with STAT3 inhibitors.
(A–B): Western blot analyses of pSTAT3-Tyr705 (pY705) and total STAT3 (tSTAT3) in FHL- or ELL-primed hNSCs, with or without treatment of Stattic and Niclosamide for 24 hrs. GAPDH was used as loading control. Densitometric analysis was performed and normalized values of pSTAT3-Y705/tSTAT3 calculated from 3 independent experiments are graphically represented (Mean ± SEM). In general, FHL-primed hNSCs exhibit a much lower level of pY705 (A) than that in ELL-primed cells (B). STAT3 inhibitors, Stattic and Niclosamide at the doses showing no cytotoxicity, do not affect the phosphorylation of STAT3 at Y705 (pY705); whereas the cytotoxic dose of niclosamide resulted in a significant reduction of pY705 (n = 3, *p<0.05 by One-way ANOVA plus Tukey post-hoc tests). (C–D) Representative confocal images show the immunofluorescent staining of pY705 in hNSCs. Blue are nuclei counterstained with DAPI. pY705 (red) is localized mainly in nuclei of the FHL- or ELL-primed cells without STAT3 inhibitor treatment (Controls in C or D, respectively). Treatment with Stattic (0.5 µM) or Niclosamide (0.25 µM) decreases the nuclear translocation of activated STAT3 (pY705) in FHL- primed hNSCs (C), but not in ELL-primed cells (D). Scale bar, 10 µm. (E–F) Quantitative localization of pY705 STAT3 immunereactivity in the nucleus vs. in a whole cell. **p<0.01. One-way ANOVA with Dunnett post hoc tests. hNSCs: human neural stem cells; ELL: EGF plus LIF and laminin; FHL: FGF2 plus heparin and laminin; Statt: Stattic; Niclo: Niclosamide.
Figure 5.
Astroglial differentiation in hNSCs treated with STAT3 inhibitors.
Immunofluorescent staining is used to determine GFAP-labeled astrocytic differentiation in human neural stem cells (hNSCs) that were FHL-primed and inhibitor-treated for 4 days, and then differentiated in B27 for 9 days. (A–C) Representative epifluorescent microscopic images show GFAP immunoreactivity in FHL-primed hNSCs (A) or after treatment with Stattic (0.5 µM) (B) and Niclosamide (0.25 µM) (C). Scale bar, 10 µm. (D) Quantitative analyses show that 0.5 µM Stattic and 0.25 µM Niclosamide significantly decrease the percentage of GFAP+ cells. *p<0.05 and ***p<0.001 by One-way ANOVA with Tukey post-hoc tests.