Distinct doses of NaB differently affect cell proliferation and AlkP and GSTA1 enzyme activities.

<p>Preconfluent Caco-2 cells were treated with NaB (1 mM and 10 mM) in serum-free media. (A) Cellular proliferation was assessed from 24–96 h. Asterisks depict significant differences between control and NaB treatments (*, p≤0.05; **, p≤0.01 and ***, p≤0.001). (B) Cytotoxicity was determined in preconfluent and postconfluent Caco-2 cells treated with 1 mM and 10 mM NaB at 48 h. Cytotoxicity measured LDH release and presented as % cytotoxicity. (C) AlkP activity (µmol/mg/min) and (D) GSTA1 activity (nmol/mg/min) was determined. Values represent the mean ± S.E. of three independent experiments with six replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

GSTA1 down-regulation does not affect the sensitivity of Caco-2 cells to NaB-induced apoptosis.

<p>(A) Representative western blots of GSTA1 (∼25 KDa), endogenous caspase-3 (∼35 KDa), activated caspase-3 (∼19 KDa and 17 KDa) in Caco-2 cells. Preconfluent Caco-2 cells were transiently transfected with 40 nM of GSTA1 siRNA or non-specific (NS) siRNA for 72 h and treated with NaB (10 mM) for 48 h. β-actin (∼42 KDa) was used as a protein loading control. Densitometric analysis of (B) GSTA1 levels and (C) caspase-3 activation in GSTA1 down-regulated cells with and without NaB (10 mM) treatment. Values represent the mean ± S.E of three independent experiments with three replicates each. Bars indicated by different letters differ significantly from one another (p≤0.05).</p

Modulation of GSTA1 levels mediate changes in Caco-2 cell growth.

<p>Effect of (A) GSTA1 down-regulation and (B) GSTA1-V5 overexpression on Caco-2 cell viability evaluated by MTS assay over three days. Asterisks depict significant differences between controls and the cells with GSTA1 modulated levels (*, <i>p</i>≤0.05; and **, <i>p</i>≤0.01). (C) Effect of GSTA1-V5 over-expression on cellular proliferation at 72 h as determined by BrdU incorporation in Caco-2 cells. Bars indicated by different letters differ significantly from one another (p≤0.001). Values represent the mean ± S.E. of four independent experiments with three replicates each.</p

GSTA1 over-expression does not interfere with NaB-induced apoptosis.

<p>(A) Representative western blots of V5 (∼26 KDa), endogenous caspase-3 (∼35 KDa) and activated caspase-3 (∼19 KDa and 17 KDa) in Caco-2 cells. Preconfluent Caco-2 cells were transiently transfected with one µg of either GSTA1-V5 or empty vector (EV) for 48 h and treated with NaB (10 mM) for 48 h. β-actin (∼42 KDa) was used as a protein loading control. (B) Densitometric analysis of caspase-3 activation in GSTA1 over-expressed cells with and without NaB (10 mM) treatment. Values represent the mean ± S.E of three independent experiments. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

Relative abundance and activity of GSTA1 in transiently transfected Caco-2 cells.

*<p>Each value represents the mean ± S.E of three independent experiments with three replicates each. ND, not determined; NS, non-specific. Values with different letters are significantly different from each other.</p

NaB (10 mM) causes GSTA1-JNK complex dissociation without activating JNK in Caco-2 cells.

<p>(A) Representative western blot of GSTA1 (∼25 KDa) and GST Pi (∼26 KDa) protein levels in GSTA1-JNK complexes. Cells were transiently transfected with GSTA1 siRNA and non-specific (NS) siRNA for 72 h and treated with 10 mM NaB. GSTA1-JNK complexes were then pulled-down from cell lysates using c-Jun fusion beads. (B) Representative western blot of phosphorylated JNK (∼54 KDa and 46 KDa) and phosphorylated p38 (∼43 KDa) protein expression in preconfluent Caco-2 cells with the treatment of 10 mM NaB. β-actin (∼42 KDa) was used as a protein loading control.</p

GSTA1 levels increase in differentiating Caco-2 cells.

<p>Preconfluent and 10 d postconfluent Caco-2 cells were assessed for: (A) protein expression of GSTA1 (∼25 KDa) and GSTP1 (∼26 KDa). β-actin was used as a protein loading control; (B) GSTA1 enzyme activity (nmol/mg/min); (C) mRNA levels of differentiation markers: AlkP, villin, DPP-4 and E-cadherin by real time RT-PCR; and (D) AlkP enzyme activity (µmol/mg/min). Values represent the mean ± S.E. of three independent experiments with three replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

GSTA1 down-regulation increases the percentage of Caco-2 cells in the S phase.

<p>(A) Changes of cell cycle phase distribution in GSTA1 down-regulated Caco-2 cells as compared to controls. (B) Graphic representation of percent of cells in G1, S and G2 phase of cell cycle in non-transfected control, GSTA1 siRNA and NS siRNA transfected Caco-2 cells. Asterisks depict significant differences between control and GSTA1 down-regulated cells (*, p≤0.05; and **, p≤0.01).</p

Modulation of GSTA1 does not affect NaB-induced differentiation.

<p>Preconfluent Caco-2 cells were transiently transfected with 40 nM of GSTA1 siRNA or non-specific (NS) siRNA and after 72 h, cells were treated with 1 mM NaB for 72 h; (A) GSTA1 activity (nmol/mg/min) was determined in GSTA1 down-regulated cells. (B) AlkP activity (µmol/mg/min) was measured to determine the effect of GSTA1 down-regulation on NaB-induced differentiation. (C) Preconfluent Caco-2 cells were transiently transfected with one µg of either GSTA1-V5 or empty vector (EV) for 48 h and were treated with 1 mM NaB for 48 h. AlkP activity (µmol/mg/min) was measured to determine the effect of GSTA1 over-expression on NaB-induced differentiation. Values represent the mean ± S.E. of three independent experiments with six replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p