ERW1041E and BJJF078: Name and chemical structure of the TG2 inhibitors.

<p>ERW1041E and BJJF078: Name and chemical structure of the TG2 inhibitors.</p

TG activity in spinal cord post-mortem tissue derived from EAE animals treated with vehicle, BJJF078 or ERW1041E.

<p>(a-f) <i>In situ</i> TG activity was detected in spinal cord sections derived from (a,d) vehicle, (b,e) BJJF078 and (c,f) ERW1041E treated mice. (d-f) Co-incubation of the sections with the TG2 inhibitor Z006 diminished TG activity. Scale bar: 50 μm. n = 11 or 12 animals/group.</p

qPCR analysis of gene expression associated with inflammation in the spinal cord of ERW1041E and vehicle only treated EAE mice.

<p>mRNA levels of inflammatory markers (a) IL-1ra, (b) IL-1β, (c) IFNγ, (d) TNFα and (e) iNOS were not significantly changed in spinal cord of ERW1041E animals compared to the vehicle treated group. n = 9–12 animals/group. Statistical analysis: student T-test or, if not normally distributed: Mann-Whitney U test.</p

Oligonucleotide primers used for cDNA amplification.

<p>Oligonucleotide primers used for cDNA amplification.</p

Inhibition of TG activity and TG2-fibronectin interaction by BJJF078 and ERW1041E in solution and <i>in vitro</i>.

<p>(a) BJFF078 and ERW1041E dose-dependently inhibited human (hTG2) and mouse (mTG2) recombinant TG2 activity. BJJF078 and ERW1041E inhibited also (b) human recombinant TG1 and (c) human recombinant FXIII. (d) Cellular activity of human TG2 in THP1 cells was dose-dependently inhibited by BJJF078 and Z006 but not by ERW1041E. (e,f) Effect of BJJF078 and ERW1041E on TG2 binding to fibronectin without (e) or with added calcium (f). n = 1–3 independent experiments with triplicates each.</p

Transglutaminases mRNA, and TG1 and TG2 immunoreactivity in post-mortem spinal cord tissue of EAE animals.

<p>(a) EAE spinal cord mRNA levels for Tgm1, Tgm2, Tgm3, Tgm6 (n.d. = not detectable) and F13A1, (b) TG2 immunoreactivity (green) associates with CD45 (red) stained EAE lesions whereas TG1 immunoreactivity (green) is found in some lesions (d) but not in others (d). Scale bar: 25 μm. n = 9 animals/group.</p

Cell infiltrates and TG2 expression in the spinal cord of BJJF078 or ERW1041E treated EAE mice.

<p>(a-d) BJJF078 and ERW1041E did not affect the infiltration of CD45 positive leukocytes into the spinal cord. Also (e-h), CD68 positive phagocytic cells and (i-l) CD3 positive T cells were observed in similar amount in vehicle and TG2 inhibitor treated animals. (b, f, j) show co-labelling of TG2 (green) with the immune cells makers (red) (b) CD45, (f) CD68 and (j) CD3. Scale bar: 100 μm, except for b,f,j: 50 μm. n = 9–12 animals/group.</p

Primary antibodies used for immunohistochemistry.

<p>Primary antibodies used for immunohistochemistry.</p

Semi-quantitative analysis of CD45, CD68 and CD3 expression in the spinal cord of EAE mice treated with vehicle or TG2 activity inhibitors.

<p>Semi-quantitative analysis of CD45, CD68 and CD3 expression in the spinal cord of EAE mice treated with vehicle or TG2 activity inhibitors.</p