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. 2016 Dec 22;8(1):95–111. doi: 10.1016/j.stemcr.2016.11.009

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© 2017 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Functional Analysis of STAT3(Y705) Activity in HPCA-Mediated Neuronal Differentiation

(A) NSCs were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and induced to differentiate for 1 day. Cell lysates were analyzed by western blotting with anti-p-STAT3(Y705), anti-p-STAT3 (S727), anti-STAT3, anti-MYC, and anti-GAPDH. Data are shown as mean ± SD (n = 3). ^∗p < 0.05 compared with the +bFGF/vector control. ^∗∗p < 0.05 compared with the −bFGF/vector control.

(B) Cells were transfected with control siRNA or Hpca siRNA for 48 hr and allowed to differentiate for 1 day. Proteins were analyzed by western blotting with anti-p-STAT3(Y705), anti-STAT3, anti-TUJ1, anti-GFAP, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3).

(C) Diagrams of Stat3WT (wild-type) and Stat3YF (mutant). CC, coiled-coil domain; DBD, DNA-binding domain; LK, linker domain; SH2, Src homology 2; TA, transcriptional activation domain.

(D) Vector (pBOS-FLAG), Stat3WT, or Stat3YF were transfected into NSCs for 48 hr, and the cells were induced to differentiate for 2 days. Cells were lysed and analyzed by western blotting with anti-p-STAT3(Y705), anti-STAT3, anti-TUJ1, anti-GFAP, and anti-GAPDH.

(E and F) Cells were transfected with Stat3WT or Stat3YF for 48 hr. After differentiation for 3 days, cells were stained for neuronal (TUJ1, green) and astrocytic (GFAP, red) markers. Scale bar, 10 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 3). ^∗Significantly different from −bFGF/Stat3WT at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/Stat3WT at p < 0.05 (for GFAP).

(G) Neurite lengths were measured in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with −bFGF/Stat3WT (mean ± SD; n = 3).

(H) Cells infected with retroviruses expressing pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc were transiently transfected with Stat3WT or Stat3YF for 48 hr. The cells were induced to differentiate for 2 days. Levels of TUJ1 and GFAP were determined by western blotting. Data are shown as mean ± SD (n = 3). ^∗p < 0.05, ^∗∗p < 0.01.

(I and J) Cells were treated as in (H) and induced to differentiate for 3 days. Cells were stained for immunocytochemical analysis of neuronal (TUJ1, green) and astrocytic (GFAP, red) markers. Scale bar, 10 μm. Graphs show the percentages of TUJ1- and GFAP-positive cells. Data are shown as mean ± SD (n = 3). ^∗Significantly different from −bFGF/Stat3WT/Hpca at p < 0.05 (for TUJ1). ^†Significantly different from −bFGF/Stat3WT/Hpca at p < 0.05 (for GFAP).

(K) Neurite lengths were measured in randomly selected areas from at least three slides of each condition. ^∗p < 0.05 compared with −bFGF/Stat3WT/Hpca (mean ± SD; n = 3).