Additional file 1: of Tissue transglutaminase in astrocytes is enhanced by inflammatory mediators and is involved in the formation of fibronectin fibril-like structures

TG2 and fibronectin protein expression in rat astrocytes and microglia. Untreated (Ctrl) and 48 h LPS-treated primary rat astrocytes (A) expressed more fibronectin (FN) and more TG2 compared to untreated (Ctrl) and LPS-treated primary rat microglia (M). Representative blot of three independent experiments is shown. (TIFF 195 kb

Combinations of primary and secondary antibodies used for immunoreactive labeling.

<p>FN: fibronectin.</p

β₁-integrin and fibronectin immunoreactivity show co-presence with TG2 positive cells in marmoset EAE lesions.

<p>β₁-integrin (A) and fibronectin (FN) (F) immunoreactivity (green) is found in normal appearing white matter (NAWM) near TG2 (red) in the endothelium of the vessel walls. β₁-integrin (green; B, C, C′, D) appears in early (EA), late (LA) active lesions and inactive (IA) white matter lesions on the cell surface of a subset of TG2 (red) positive cells. β₁-integrin also shows some co-localization with TG2 positive cells in cortical grey matter lesions (cGML) (E, E′). Arrows represent TG2/β₁-integrin double labeled cells. FN (green; G, H, H′, I) appears clearly in the extracellular matrix but also shows co-labeling with a subset of TG2 (red) positive cells in early and late active lesions. Hardly any FN immunoreactivity is present in grey matter lesions (J). Arrows represent TG2/FN double labeled cells. Scale bar is 20 µm. Inserts in figures C′, E′ and H′ represent higher magnifications in which the close association of TG2 positive cells with β₁-integrin or FN (C′, E′ or H′, respectively) can be appreciated. Scale bars in the inserts are 10 µm.</p

TG2 is not expressed in astrocytes, oligodendrocytes, T-cells and B-cells.

<p>TG2 immunoreactivity (red) is not present in GFAP (green; A, E, I), Olig2 (green; B, F, J), CD3 (green; C, G, K) or CD20 (green; D, H, L) positive cells in early (EA), late active (LA) and inactive (IA) lesions. Scale bar is 20 µm.</p

Secondary antibodies used.

<p>Secondary antibodies used.</p

Quantification of TG2 and Iba-1 positive cells in early/late active versus inactive white matter lesions.

<p>The number of TG2 (A) and Iba-1 positive cells (B) per white matter sample area of 0.1 mm² is significantly decreased in inactive lesions compared to (early/late) active lesions. Data are shown as mean + SEM, n = 8 for early/late active lesions, n = 12 for inactive lesions, *P<0.001.</p

Characterization of marmoset EAE lesions and TG2 immunoreactivity.

<p>The normal appearing white matter (NAWM) shows an intact LFB myelin staining (A) and few MRP14 positive macrophages (G). Early active (EA) lesions display myelin degradation (B) and foamy macrophages (H). Late active (LA) lesions show degradation of myelin (C) combined with less MRP14 positive macrophages (I). Inactive (IA) lesions are characterized by an absence of both myelin staining (D) and MRP14 positive macrophages (J). The normal appearing grey matter (NAGM) shows intact myelin fibers (E) and very few MRP14 positive macrophages (K). Cortical grey matter lesions (cGML) show an absence of myelin fibers (F) and presence of MRP14 positive microglia (L). TG2 immunoreactivity is present in endothelium of the vessel walls in NAWM (M). Early active and late active lesions display additional TG2 positive cells (N and O respectively). Inactive lesions show less additional TG2 immunoreactivity (P). Cortical grey matter lesions also show additional TG2 positive cells (R) compared to the endothelial staining in normal appearing grey matter (Q). Scale bar is 20 µm.</p

Lesion types per animal.

<p>MOG: myelin oligodendrocyte glycoprotein, EA: early active, LA: late active, IA: inactive, cGML: cortical grey matter lesion.</p

TG2 positive cells show co-labeling with Iba-1 in marmoset EAE lesions.

<p>Early (EA) (A-C) and late active (LA) (D-F) lesions show mostly cytoplasmic cellular localization of immunoreactive TG2 (A, D), and cells with membrane labeled Iba-1 (B, E). TG2 positive cells co-label with Iba-1 positive cells (C, F). Inactive (IA) (G-I) lesions show less TG2 positive cells and co-labeling with Iba-1 positive cells seems less apparent (I) Cortical grey matter lesions (cGML) (J-L) show TG2 positive cells co-labeling with Iba-1 positive cells that have radially projecting processes (L), instead of the more rounded morphology seen in white matter lesions (C, F). Arrows represent monocyte-like cells that are either single (top 2 rows) or double labeled (merge), arrowheads represent microglial cells that are either single (top 2 rows) or double labeled (merge). Scale bar is 20 µm.</p

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

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