Supplementary material for #IFLA-D-14-00341
NOD1 and NOD2 interact with the phagosome cargo in mast cells: a detailed morphological evidence
Corresponding author: Giuliano Zabucchi
Legends to supplementary figures
Fig. 1s
Evaluation of the specificity of the immunogold analysis.
The specificity of the immunogold reaction was evaluated using as a primary antibody a rabbit-anti-rat mesothelin, a molecule which is absent in mast cells. a and b, show that the positive signal for this antigen is hardly detectable both in resting and phagocytizing RPMC. The same result was obtained using non-immune mouse IgG in both resting (c) and phagocytizing RPMC.
Fig.2s
Subcellular distribution of phagosome markers as evaluated by immunogold analysis.
The figure shows additional pictures of the immunogold analysis to indicate the different subcellular compartments with which the phagosome markers associate.
a-d LAMP1 gold particles 20nm, bar = 0.5 m; b-e vATPase gold particles 20nm, bar = 0.5 m; c-f CATD gold particles 10nm, bar = 0.5 m. a, the subcellular distribution of LAMP1 in resting RPMC is shown. It appears that most of the gold particles are associated with the granule surface and matrix, while only few seem to be free in the cytosol. d, the subcellular distribution of LAMP1 in phagocytizing mast cell is shown. Many gold particles label the ingested E.coli at phagosome membrane level, on the bacterial surface and even within the bacterial cell. Some gold particles are associated with the granules while only a few seem to be free in the cytosol. b, the subcellular distribution of vATPase in resting RPMC is shown. It appears that the labelling, albeit weaker than that observed for LAMP1, is mostly associated with granules. However, in this case some vesicles seem to be positive (arrows). e, the subcellular distribution of vATPase in phagocytizing mast cell is shown. Some gold particles label the ingested E.coli, some gold particles label the two ingested E.coli at phagosome membrane level and within the bacterial cell. Some gold particles are associated with the granules while only few seem to be free in the cytosol (arrows). Very few of them are associated with the granules. The white arrows shows two putative positive vesicles. c, the subcellular distribution of CATD in resting RPMC is shown. It appears that most of the gold particles are associated with the granule surface and matrix, few of them, if any, seem to be free in the cytosol. A positive small vesicle is also shown (arrow). f, the subcellular distribution of CATD in phagocytizing mast cell is shown. Many gold particles label the ingested E.coli, at phagosome membrane level, on the bacterial surface and even within the bacterial cell. Some gold particles are associated with the granules while only few seem to be free in the cytosol. Putative positive vesicles are also shown.
Fig. 3s
Subcellular distribution of NOD1as evaluated by immunogold analysis.
The figure shows additional pictures of the immunogold analysis to indicate the different subcellular compartments to which NOD1 (labelled with 10 nm GP) associates. a-c Resting RPMC, bar = 0.5 m; d-f phagocytizing RPMC, bar = 0.2 m. a-c, in resting cells NOD1 frequently labels the granule membrane, which sometimes leaves a space between itself and the granule matrix, less frequently the gold particles are found within the granule matrix. In some resting cells NOD1 appears to heavily label putative small cytoplasmic vesicles (arrows). d-f In phagocytizing cells the NOD1 labelling gold particles are clearly reduced in number on the granule surface, while they label the phagosome membrane (arrows in d) , and both the bacterial cell wall (arrows in e) and the bacterial cell interior (arrows in f). In these pictures the interruptions of the phagosome membrane are remarkably evident (arrowheads in d-f).
Fig. 4s
Subcellular distribution of NOD2 as evaluated by immunogold analysis.
The figure shows additional pictures of the immunogold analysis to indicate the different subcellular compartments with which NOD2 (labelled with 10 nm GP) is associated. a-b, resting RPMC, bar = 0.2 m; c phagocytizing RPMC, bar = 0.2 m. a-b, in resting cells NOD2 labels the granule membrane; less frequently gold particles are found within the granule matrix. In resting cells NOD2 appears to heavily label putative cytoplasmic vesicles. In phagocytizing cell, the gold particles label the phagosome membrane. In this picture the interruption of the phagosome membrane is evident (arrowhead in c).