Antibody-Validated Proteins in Inflamed Islets of Fulminant Type 1 Diabetes Profiled by Laser-Capture Microdissection Followed by Mass Spectrometry
Figure 2
Immunohistochemical validation of the presence of LMD-LC-MS-identified proteins in FT1DM-affected islets.
(A)–(D), Triple immunostaining for plastin-2 (LCP1) expression in FT1DM-affected pancreas (A)–(C) and non-diabetic control (D) tissues. (A), Triple immunostaining for plastin-2 (LCP1) in FT1DM-affected pancreas. LCP1 (red) was over-expressed in mononuclear cells (MNCs) that aggressively infiltrated to or around the islets (green: insulin, blue: glucagon). (B), Merged image of triple immunostaining for LCP1 (green), CD68+ macrophages (red), and glucagon (blue). Many MNCs are positive for both CD68 and LCP1 and appear yellow (arrowheads). (C), Merged image of triple immunostaining for LCP1 (green), CD8 (red), and glucagon (blue). Many MNCs are positive for both CD8 and LCP1 and appear yellow (arrowheads). (D), Merged image of triple immunostaining for LCP1 (red), insulin (green), and glucagon (blue) in non-diabetic pancreas tissue. Few cells are positive for LCP1. (E)–(F), Expression of Ras GTPase-activating-like protein (IQGAP1) in the islets and MNCs in FT1DM-affected (E) and non-diabetic (F) islets. IQGAP1 (brown) was highly expressed in infiltrating MNCs and some FT1DM islet cells (insulin: red, glucagon: green). (G)–(H), DEAD box helicase5 (DDX5) expression in FT1DM-affected (G) and control (H) pancreas. DDX5 (brown) was hyper-expressed in the nucleus and cytoplasm of all subsets of islet cells, including beta cells (red) and alpha cells (green). Weak expression of DDX5 was observed in the nucleus of non-diabetic pancreas cells (H). (I)–(L), Expression of thymidine phosphorylase (TYMP) in FT1DM-affected (I) and control pancreas (J) tissues. TYMP was over-expressed in MNCs infiltrated to the islets in FT1DM tissue (I). No expression of TYMP was observed in non-diabetic control pancreas tissue (J). Triple immunostaining of FT1DM pancreatic tissue for TYMP (green), CD68+ (red), and insulin (blue). Merged image (K) shows that TYMP is expressed on CD68+ macrophages and appears yellow (arrowheads). Triple immunostaining of FT1DM-affected pancreatic tissue for TYMP (green), CD8+ (red), and insulin (blue). Merged image (L) shows that TYMP is localized on CD8+ T cells (arrowheads). (M)–(N), Expression of SAM domain and HD domain-containing protein 1 (SAMHD1) in FT1DM-affected pancreas (M) and control pancreas (N) tissues. Hyper-expression of SAMHD1 (brown) in the nucleus and cytoplasm of islet beta cells (red), alpha cells (green), and infiltrating MNCs is shown. No expression of SAMHD1 was observed in non-diabetic control pancreas tissue (N). (O)–(P), 6-Phosphogluconate dehydrogenase, decarboxylating (PGD) expression in FT1DM-affected pancreas tissue (O). PDG (brown) was over-expressed in the cytoplasm of islet-cells, and non-islet cells (arrowheads). PDG was only faintly expressed in the cytoplasm of non-diabetic islet cells (P). (Q)–(R), Signal transducer and activator of transcription-1 alpha/beta (STAT1) expression in FT1DM-affected pancreas (Q) and control pancreas (R) tissues. STAT1 was over-expressed in the cytoplasm and nucleus of islet beta cells (red), alpha cells (green), and MNCs (arrowheads). No staining for STAT1 was observed in control islet tissue (R). (S)–(T), Proteasome activator complex subunit 1 (PSME1, PA28a) expression in FT1DM-affected pancreas (S) and non-diabetic pancreas (T) tissues. PSME1 (brown) was over-expressed in the nucleus and cytoplasm of beta cells (red), alpha cells (green), and infiltrating MNCs (arrowheads) (S). PSME1 was not expressed in non-diabetic control pancreas tissue (T). (U)–(V), Tryptophanyl-tRNA synthetase (WARS) expression in the islets of FT1DM-affected (U) and non-diabetic control (V) pancreas tissues. Merged image shows expression of WARS (brown) in beta cells (red) and MNCs (arrowheads) in FT1DM-affected tissue (U). No staining of WARS was observed in non-diabetic control pancreas tissue (V). (W)–(X), Heat shock protein 70 kDa protein 1-like (HSPA1L) expression in the islets of FT1DM-affected (W) and non-diabetic pancreas (X) tissues. Strong expression of HSPA1L (brown) was observed in beta cells (red), alpha cells (green), and other subsets of endocrine cells (W). Weak expression of HSPA1L was observed in non-diabetic control islets (X). Positive staining for each identified protein was significantly more frequent (P = 0.048) in FT1DM-affected islets than control islets.