TABLE 2

Effects of transfection of varying amounts of mutant I-κBα on barrier integrity and microtubule cytoskeleton following treatment regimes

Intestinal Caco-2 cells stably transfected with varying amounts (1, 2, 3, or 4 μg) of an I-κBα dominant-negative (Ser 32/36 mutant) were exposed to oxidant (H2O2, 0.5 mM). In separate studies, cells stably transfected with these varying amounts of mutant I-κBα were preincubated either with a low dose of EGF (1 ng/mL, known to be nonprotective in wild-type cells) or with a high dose of EGF (10 ng/ml, known to be protective in wild-type cells) prior to oxidant exposure. Select treatments from wild-type (WT) cell monolayers are also shown. Monolayer barrier function (fluorescein sulfonic acid, FSA clearance) and microtubule cytoskeletal integrity (%normal) were then assessed.

Treatment
FSA Clearance
% Normal Microtubules
nl/h/cm 3
Vehicle 19 ± 8 98 ± 2
H2O2 [WT] 815 ± 9* 40 ± 4*
H2O2 [1 μg mutant I-κBα] 723 ± 14*† 51 ± 2*†
H2O2 [2 μg mutant I-κBα] 486 ± 21*† 67 ± 2*†
H2O2 [3 μg mutant I-κBα] 201 ± 34*† 90 ± 3*†
H2O2 [4 μg mutant I-κBα] 225 ± 27*† 88 ± 4*†
EGF (1 ng/ml) + H2O2 [WT] 738 ± 17* 43 ± 3*
EGF (1 ng/ml) + H2O2 [1 μg mutant I-κBα] 712 ± 23*† 53 ± 2†*
EGF (1 ng/ml) + H2O2 [2 μg mutant I-κBα] 389 ± 35*† 72 ± 3†*
EGF (1 ng/ml) + H2O2 [3 μg mutant I-κBα] 205 ± 18*† 90 ± 2†
EGF (1 ng/ml) + H2O2 [4 μg mutant I-κBα] 200 ± 21*† 89 ± 4†
EGF (10 ng/ml) + H2O2 [WT] 41 ± 11† 91 ± 2†
EGF (10 ng/ml) + H2O2 [1 μg mutant I-κBα] 49 ± 20† 89 ± 3†
EGF (10 ng/ml) + H2O2 [2 μg mutant I-κBα] 56 ± 18† 88 ± 4†
EGF (10 ng/ml) + H2O2 [3 μg mutant I-κBα] 43 ± 9† 90 ± 5†
EGF (10 ng/ml) + H2O2 [4 μg mutant I-κBα]
58 ± 21†
87 ± 4†

  • ↵* p < 0.05 compared with vehicle.

  • ↵† p < 0.05 compared to H2O2 in WT cells.

  • ↵ p < 0.05 compared to corresponding cells transfected with lower amounts (1 or 2 μg) of mutant I-κBα. Mean ± S.E.M. n = 4 to 6/group.