Fig 1: Expression of NLRP3, ASC, caspase-1, IL-1ß, and IAPP/amylin in Langerhans’ islets of pancreas. Formalin-fixed and paraffin-embedded specimens of nine autopsy cases were immunostained with specific antibodies to ASC, NLRP3, caspase-1, IL-1ß, amylin, and insulin as indicated. Representative Langerhans’ islets in each case are presented. Parallel hematoxylin and eosin staining is presented on the left. The bar is 100 µm. The cases are summarized in Table 1.
Fig 2: IAPP/amylin-induced interaction between NLRP3 and ASC in a cell-free system. (a) Schematic representations of NLRP3 and ASC. Synthetic C-terminal biotinylated full-length NLRP3 (NLRP3-FL-Btn) and N-terminal FLAG-tagged ASC (FLAG-ASC-FL) are indicated. The pyrin domain (PYD) is indicated by black boxes. The caspase recruitment domain (CARD) is indicated by a dark gray box. The nucleotide-binding oligomerization domain (NOD) is indicated by a light gray box. Leucine-rich repeats are indicated by a striped box. Amino acid sequence numbers are indicated under each schema. (b) Interaction between NLRP3-FL-Btn and FLAG-ASC-FL was detected by an amplified luminescent proximity homogeneous assay (Alpha). Responses (counts) were measured using an EnSpire™ Multimode Plate Reader. The results are given as means ± standard deviation from triplicate wells. *P-value < 0.05 was considered statistically significant using Mann–Whitney U test. (c) IAPP/amylin directly interact with NLRP3 in a dose-dependent manner. Representative results are shown from three independent experiments.
Fig 3: Correlation diagram comparing IAPP/amylin-positive scores and each value. (a) Correlation analysis between ASC-positive score and IL-1ß-positive score. (b) Correlation analysis between IAPP/amylin-positive score and ASC-positive score. (c) Correlation analysis between IAPP/amylin-positive score and IL-1ß-positive score. (d) Correlation analysis between IAPP/amylin-positive score and NLRP3-positive score. (e) Correlation analysis between IAPP/amylin-positive score and caspase-1-positive score. (f) Correlation analysis between IAPP/amylin-positive score and serum HbA1c (%).
Fig 4: IAPP/amylin directly interacted with LRR domain of NLRP3. (a) A schematic representation of the NLRP3 structure depicts Exon components and truncated proteins. (b) Synthetic protein-protein interactions between biotinylated IAPP/amylin and FLAG-NLRP3-FL, FLAG-NLRP3-(Exons 1-2), FLAG-NLRP3-(Exons 3-9), or FLAG-NLRP3-(Exons 4-9) were assessed by amplified luminescent proximity homogeneous assay (Alpha). The results are given as means ± standard deviation from triplicate wells. *P-value < 0.05 was considered statistically significant using Mann–Whitney U test. (c) Interactions between biotinylated IAPP/amylin and FLAG-NLRP3-FL, FLAG-NLRP3-(Exons 1-2), FLAG-NLRP3-(Exons 3-9), or FLAG-NLRP3-(Exons 4-9) were assessed by immunoprecipitation. The asterisk indicates a non-specific band.WT: wild-type; P: precipitation; WB: western blot; Lysate: lysate after immunoprecipitation.
Fig 5: Pathology of pancreatic islets of Langerhans.(A)Hematoxylin-eosin (HE) staining of pancreatic islets of animals in the four groups. Magnification: 200×. Scale bar = 150 µm. Immunohistochemistry of pancreatic islets for insulin, IAPP and caspase 3. All sections were obtained from the pancreatic tail. Tg: n = 5; Nc: n = 3–6. Magnification: 200×. Scale bar = 100 µm. (B) The pancreatic islet average areas (sum pixels of each) from the four groups were calculated. (C–E) The AODs include pancreatic insulin expression density (C); total IAPP expression density (D); and caspase 3 expression density (E). The significance levels are * p < 0.05 or ** p < 0.01. P values were calculated using Student’s t-test.
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