Supplementary Figure 1. Representative photomicrographs showing each grade of staining for JAM-A protein. Bars=50 μm.
Grade 3
Grade 2 Grade 4
Grade 1
Supplementary Figure 2. Expression of junctional proteins (CLD-3, occludin, CLD-2, E-cadherin) in Caco-2 monolayers incubated with tryptase, T, (3 mU or 15 mU) for up to 24 hours. A) Blots show a representative of a minimum of two independent experiments. B) The results are expressed as a change relative to untreated controls.
The density of each individual band was compared with the corresponding control band and normalised against GAPDH by densitometry. *P<0.05 (Student’s t-test). C) Expression of junctional proteins in the apical region of Caco-2 monolayers compared to untreated controls. Bars=20 μm.
CLD-3 GAPDH
Occludin GAPDH
CLD-2 GAPDH
E-cadherin
GAPDH
0 1 2
E-cadherin (% control) 0 1 2
CLD-2 (% control) 0 1 2 3
Occludin (% control) 0 2 4
CLD-3 (% control)
Time (h) C 1 2 4 1 2 4 C 24 24
Time (h) C 1 2 4 1 2 4 C 24 24
Time (h) C 1 2 4 1 2 4 C 24 24
Time (h) C 1 2 4 1 2 4 C 24 24
*
* *
Control 3 mU T 1 h 15 mU T 1 h
3 mU T 2 h 3 mU T 4 h 15 mU T 2 h 15 mU T 4 h 15 mU T 24 h
3 mU T 24 h
Control
A) B)
3 mU T
Control 15 mU T
Control 15 mU tryptase 4 h
CLD-3 Occludin
Control 15 mU tryptase 4 h C)
Supplementary Table 1. Expression of JAM-A in control group according to gender and age (years) of patients at the time of biopsy. The expression was assessed semiquantitatively using a 4-grade scale. A) Analysis of controls according to gender and age < 32 years (the average age of IBS) and ≥ 32 years and resultant JAM-A expression (grade). Data were analysed with Mann-Whitney U test. B) Data were analysed with Spearman correlation and J-T test.
Gender, median (range) Age at the time of biopsy (years)
Males (n=7) Females (n=5) 26±2.7 51.6±4.4
JAM-A 4 (3-4) 4 (1-4) 4 (3-4) 4 (1-4)
P=0.63 P=0.61
Age at the time of biopsy (years)
Spearman correl. J-T test
rs P P
JAM-A -0.33 0.3 0.275
A)
B)