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Figure 6 | BMC Cancer

Figure 6

From: P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells

Figure 6

Nuclear p27 Kip1 expression modulated by GSK-3β. A& B, SGC7901 cells were treated with HMBA (10 mM) over a time course (A) or with various concentrations for 24 h. Cytosolic and nuclear protein fractions were extracted and western blotting was performed with antibodies to p27Kip1, α-tubulin or Topo IIβ. C& D, SGC7901 cells were pre-treated with (+) or without (-) 20 mM LiCl (C) or 10 μM SB-415286 (D) for 30 min followed by combination treatment with 10 mM HMBA for 24 h. Cytosol and nuclear proteins were extracted for analysis of p27Kip1 protein expression. E, SGC7901 cells were transfected with siRNA directed to GSK-3β or control siRNA. Twenty-four hours after transfection, cells were treated with HMBA for an additional 24 h. Cytosol and nuclear proteins were extracted for analysis of p27Kip1 protein expression. Knockdown of GSK-3β expression was confirmed by western blotting using anti-GSK-3β antibody. F, SGC7901 cells were infected with Ad-HA-GSK-3βS9A or Ad-β-gal at an MOI of 10 pfu/cell. After 48 h incubation, cytosol and nuclear protein were extracted and western blotting performed using anti-p27Kip1, anti-HA, and anti-GSK-3 antibodies, respectively using anti-α-tubulin or Topo IIβ as loading control. GSK-3β activities were assayed by in vitro kinase assay using Snail protein as substrate (bottom panel). p27Kip1 signals were quantitated densitometrically and expressed as fold-change with respect to α-tubulin or TopIIβ.

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