Table 1.
Sequences of primers used for quantitative PCR analysis.
Figure 1.
mBSA displays features of amyloid.
(A) Fluorescence intensity of Thioflavin T at 480 nm in buffer, with BSA, mBSA, Aβ or with the reverse control peptide. Error bars are means ± SD of 3 independent experiments. **p<0.005. (B) Gel shift analysis of salmon sperm ssDNA pre-mixed with different amounts of BSA or mBSA. (C) Thioflavin S fluorescence in the presence of different BSA proteins or aggregates observed under fluorescence microscopy. Original magnification 100X. scale bar: 20 µm. (D) Fluorescence emission profiles of Congo Red in the presence of buffer, different proteins or aggregates. (E) Birefringence of Congo Red on mBSA or mBSA plus ssDNA aggregate observed by polarized light microscopy. Original magnification 40X. bar: 50 µm. (F) Transmission electron microscopy analysis of mBSA or mBSA plus ssDNA. Bar: 100 nm.
Figure 2.
mBSA shares properties with soluble amyloid precursor.
(A) Gel shift analysis of salmon sperm ssDNA that was pre-mixed with mBSA in the presence of different amounts of heparin. (B) Assessment of cell death of RPMI 8226 cells cultured 24 hrs with different amounts of BSA or mBSA. Shown are representative results of PI staining from 2 independent experiments. (C) Assessment of dead RPMI 8226 cell population cultured with different amounts of BSA or mBSA that were pre-incubated with medium, DNA or heparin. Results shown are representative of 2 independent experiments. (D) Fluorescence emission profiles of bis-ANS obtained after incubation in PBS, with BSA or with mBSA.
Figure 3.
Serum amyloid P binds to mBSA.
(A) Binding of SAP to different amounts of Aβ or the reverse control peptide was assessed by ELISA. Similar results were obtained from 2 independent experiments. (B) Binding of SAP to different amounts of BSA or mBSA with or without ssDNA was assessed by ELISA. Error bars are means ± SD of 2 independent experiments.
Figure 4.
mBSA elicits IL-1β production by activating inflammasome.
(A) Secretion of IL-1β by BMDM stimulated with different forms of BSA or Aβ peptides for 2 hours. Error bars are means ± SD of 5 independent experiments. ****p<0.0001. (B) Caspase 1 activation in BMDMs stimulated with different forms of BSA analyzed by FLICA assay. Representative result of 3 independent experiments is shown. Original magnification 100X. bar: 20 µm. (C) Acridine orange staining of BMDM stimulated by different forms of BSA to reveal lysosome integrity. Original magnification of fluorescence microscopy 100X. bar: 20 µm. (D) Secretion of IL-1β by BMDM induced by different forms of BSA in the presence of different inhibitors. Error bars are means ± SD of 2 independent experiments. *p<0.05, **p<0.005. (E) Secretion of IL-1β by BMDM induced by different forms of BSA or Aβ peptides in the presence or not of the NRLP3 inhibitor glybenclamide. Error bars are means ± SD of data obtained with cells from 4 different mice. *p<0.05, **p<0.005. (F) Secretion of IL-1β by BMDM induced by different forms of BSA in the presence of the caspase 1 inhibitor Ac-YVAD-CMK. Error bars are means ± SD of data obtained with cells from 3 different mice. *p<0.05, **p<0.005.
Figure 5.
mBSA triggers inflammation in vivo.
(A) Numbers of infiltrating macrophages (left), monocytes (middle) and neutrophils (right) in the peritoneum of mice 4 h after i.p. injection of different stimuli. Error bars are means ± SD of 4 mice per group. *p<0.05, **p<0.005. (B) Levels of IL-1α (left) and IL-1β (right) secreted in the peritoneal lavages. *p<0.05. (C) Gene expression of peritoneal exudate cells presented as a heat map. One BSA-injected animal was used as a reference. Each block represents one mouse. (D) Plot of induced transcript expression of the chemokines from the bottom cluster of *p<0.05, **p<0.005. (C). (E) Numbers of infiltrating macrophages (left), monocytes (middle) and neutrophils (right) in the peritoneum of wild-type or IL-1β−/− mice 4 h after i.p. injection of different stimuli. Error bars are means ± SD of 3 mice per group. **p<0.005.