Figure 1.
Chemical structure of T-2 and HT-2 toxin.
Figure 2.
Influence of T-2 and HT-2 toxin on cell viability of PBCEC.
Cell viability of PBCEC depending on different T-2 and HT-2 toxin concentrations (1 nM – 10 µM) after 48 h incubation was determined by the CCK-8 assay. Number of analyzed samples n = 18 over three individual preparations. Data is given as mean±SEM.
Figure 3.
Effects of T-2 and HT-2 toxin on barrier function of PBCEC over 48 h.
A: TEER measurements after incubation of various concentrations of T-2 toxin from the apical side to PBCEC monolayers. B: Results of TEER measurements after incubation of different HT-2 toxin concentrations from the apical side to PBCEC monolayers. all: n = 9 with standard deviations 10 – 15% (not shown).
Figure 4.
Barrier integrity after toxin incubation as determined by [14C]sucrose permeation.
Permeation of [14C]sucrose was used as an additional indicator for barrier integrity after incubation of different concentrations of T-2 and HT-2 toxin for 12 h and 48 h from the apical side (a), the basolateral side (b) or from both sides (a+b); all: mean±SEM; n = 3.
Figure 5.
Immunocytochemical staining of occludin after T-2 and HT-2 toxin incubation from the apical side.
A: Immunocytochemical staining of tight junction protein occludin in PBCEC after T-2 or HT-2 toxin incubation for 48 h from the apical side. TEER of control (A, 1) and 50 nM T-2 toxin (A, 2) incubated cells>1000 Ω*cm2; TEER of 75 nM T-2 toxin (A, 3) incubated filters>50 Ω*cm2; TEER of 200 nM HT-2 toxin (A, 4) incubated filters>600 Ω*cm2. Regions of lost or impaired occludin are indicated by arrows. B: Western blotting of occludin with actin as loading control of control filters (B, 1), 50 nM T-2 toxin (B, 2), 75 nM T-2 toxin (B, 3) and 200 nM HT-2 toxin (B, 4) incubated PBCEC for 24 h.
Figure 6.
Permeation of T-2 and HT-2 toxin across PBCEC monolayers after various time points.
A: Recovery of T-2 and HT-2 toxin (% of initially applied toxin) in the basolateral compartment after application of toxin (T-2 toxin: 25 nM; HT-2 toxin: 50 nM) to the apical compartment for 2 h, 24 h and 48 h. B: Recovery of T-2 and HT-2 toxin (% of initially applied toxin) in the apical compartment after application of toxin (T-2 toxin: 10 nM; HT-2 toxin: 50 nM) to the basolateral compartment for 2 h, 24 h and 48 h. C: Recovery of T-2 and HT-2 toxin (% of initially applied toxin) in the apical and basolateral compartment after application of equimolar concentrations of T-2 (10 nM) and HT-2 (50 nM) toxin to both compartments (apical and basolateral) for 24 h and 48 h.* indicates only traces detected; all: mean±SEM with a minimum of n = 6.