Journal: Oxidative Medicine and Cellular Longevity

Article Title: Recurrent Hypoglycemia Impaired Vascular Function in Advanced T2DM Rats by Inducing Pyroptosis

doi: 10.1155/2022/7812407

Figure Lengend Snippet: NF- κ B, NLRP3, and cGAS–STING pathway activity of the aortas in response to acute and recurrent hypoglycemia in aged T2DM rats. (a) p-p65, NLRP3, ASC, (b) cleaved caspase-1, cGAS, and (e) STING expression were assayed by western blotting. The expression and location of NLRP3 were determined by (c) immunohistochemistry and (d) immunofluorescence; ∗ p < 0.05 DM vs. control; # p < 0.05 H-DM, RH-DM vs. DM; & p < 0.05 H-DM vs. RH-DM.

Article Snippet: Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and incubated with primary antibodies: eNOS (1 : 1000, ab300071, Abcam), iNOS (1 : 1000, ab283655, Abcam), NOX2 (1 : 2000, 19013-1-AP, ProteinTech), NOX4 (1 : 2000, 14347-1-AP, ProteinTech), p-p65 (1 : 1000, 3033, Cell Signaling Technology), p65 (1 : 1000, 8242, Cell Signaling Technology), NLRP3 (1 : 1000, NBP2-12446, NOVUS), ASC (1 : 1000, sc-514414, Santa Cruz), Caspase-1 (1 : 1000, bs-10743R, Bioss), cGAS (1 : 1000, NBP3-16666, NOVUS), STING (1 : 1000, CST50494, Cell Signaling Technology), GSDMD (1 : 1000, NBP2-33422, NOVUS), Bax (1 : 1000, 50599-2-Ig, ProteinTech), Bcl-2 (1 : 1000, 26593-1-AP, ProteinTech), and β -actin (1 : 5000, 20536-1-AP, ProteinTech).

Techniques: Activity Assay, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3-mediated activation of superoxide dismutase 2

doi: 10.1074/jbc.M117.791715

Figure Lengend Snippet: SIRT3 blunts the NLRP3 inflammasome. A, IL-1β release into the supernatant of cultured peritoneal macrophages obtained from fed or 48-h–fasted SIRT3+/+ or SIRT3−/− mice left untreated or primed with LPS and stimulated with 5 mm ATP was assessed by ELISA (top panel) and immunoblot analysis (bottom panel). Error bars represent mean ± S.E. (n = 9–17). *, p < 0.05. Sup, supernatant. B and C, ratio of release of IL-1β, IL-6, and TNF comparing SIRT3−/− with SIRT3+/+ 48-h–fasted mouse peritoneal macrophages primed with LPS and stimulated with 5 mm ATP (B) or SIRT3−/− with SIRT3+/+ BMDMs primed with LPS and stimulated with 3 mm ATP (C). Cytokines were measured by ELISA. Error bars represent mean ± S.E. (n = 9–21). *, p < 0.05. D, immunoblot of release of active caspase-1 (p10 subunit) and mature IL-1β (p17 subunit) into the supernatants of cultured BMDMs left untreated or primed with LPS and stimulated with nigericin. E, ROS levels in LPS-treated BMDMs. Error bars represent mean ± S.E. (n = 3). A.U., arbitrary units. F, SOD2 activity in LPS-treated BMDM mitochondria. Error bars represent mean ± S.E. (n = 3). G, measurement of mitochondrial genomic DNA extrusion in SIRT3+/+ and SIRT3−/− BMDMs left untreated or primed with LPS and stimulated with 3 mm ATP. Error bars represent mean ± S.E. (n = 8).

Article Snippet: The following antibodies were used: caspase-1 (2225, Cell Signaling Technology; sc-514, Santa Cruz Biotechnology; AG-20B-0042-C100, Adipogen), IL-1β (ab9722, Abcam), NLRP3 (AG-20B-0014-C100, Adipogen), ASC (sc-22514, Santa Cruz Biotechnology), SOD2 (sc-18504, Santa Cruz Biotechnology), SIRT3 (5490S, Cell Signaling Technology), β-actin (A1978, Sigma-Aldrich), β-tubulin (2146S, Cell Signaling Technology), and Tom20 (sc-11415, Santa Cruz Biotechnology).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3-mediated activation of superoxide dismutase 2

doi: 10.1074/jbc.M117.791715

Figure Lengend Snippet: SOD2 blunts the NLRP3 inflammasome. A, immunoblot analysis of relative SOD2 protein acetylation in peritoneal macrophages obtained from fed or 48-h–fasted SIRT3+/+ or SIRT3−/− mice. B, immunoblot analysis of relative SOD2 protein acetylation in cultured BMDMs obtained from SIRT3+/+ or SIRT3−/− mice. Inset numbers show the average relative intensity in two independent experiments ± S.D., and the arrow shows the acetylated Lys-68 residue on SOD2. C, immunoblot analysis of cell lysates from untreated or LPS-treated human THP-1 macrophages transfected with control siRNA or siRNA targeting SOD2 or SIRT3. D, immunoblot analysis of release of active caspase-1 (Casp1, p20 subunit) and mature IL-1β (p17) into supernatants (Sup) from human THP-1 macrophages transfected with control siRNA or siRNA targeting SOD2 or SIRT3, left untreated or primed with LPS and stimulated with nigericin (Nig), and lysates of the same cells solubilized with Triton X-100–containing buffer, followed by cross-linkage of insoluble pellets with DSS (I. Pellet + DSS) to capture ASC dimers and oligomers. E and F, ELISA of IL-1β (E) and TNF (F) release into supernatants of human THP-1 macrophages transfected with control siRNA or siRNA targeting SOD2, left untreated or primed with LPS and stimulated with 5 mm ATP. Error bars represent mean ± S.E. (n = 9). ns, not significant.

Article Snippet: The following antibodies were used: caspase-1 (2225, Cell Signaling Technology; sc-514, Santa Cruz Biotechnology; AG-20B-0042-C100, Adipogen), IL-1β (ab9722, Abcam), NLRP3 (AG-20B-0014-C100, Adipogen), ASC (sc-22514, Santa Cruz Biotechnology), SOD2 (sc-18504, Santa Cruz Biotechnology), SIRT3 (5490S, Cell Signaling Technology), β-actin (A1978, Sigma-Aldrich), β-tubulin (2146S, Cell Signaling Technology), and Tom20 (sc-11415, Santa Cruz Biotechnology).

Techniques: Western Blot, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3-mediated activation of superoxide dismutase 2

doi: 10.1074/jbc.M117.791715

Figure Lengend Snippet: SOD2 inhibition of the NLRP3 inflammasome is acetylation-level dependent and modulates NLRP3 localization to mitochondria and its interaction with ASC. A and B, ELISA of IL-1β (A) and TNF (B) release into supernatants of human THP-1 macrophages stably transduced with control empty vector or with WT or mutant SOD2 vectors, left untreated or primed with LPS and stimulated with 5 mm ATP. Error bars represent mean ± S.E. (n = 23). *, p < 0.05. C, immunoblot analysis of release of active caspase-1 (Casp1, p20 subunit) and mature IL-1β (p17) to supernatants (Sup) from human THP-1 macrophages stably transduced with control empty vector or with wild-type or mutant SOD2 vectors, primed with LPS and stimulated with nigericin (Nig), and lysates of the same cells solubilized with Triton X-100–containing buffer, followed by cross-linkage of insoluble pellets with DSS (I. Pellet + DSS) to capture ASC dimers and oligomers. D, measurement of mitochondrial genomic DNA extrusion in human THP-1 macrophages stably transduced with control empty vector or with wild-type or mutant SOD2 vectors, left untreated or primed with LPS and stimulated with nigericin. Error bars represent mean ± S.E. (n = 6). *, p < 0.05. E, representative immunoblot analysis for NLRP3 and ASC on mitochondrial fractions from untreated or LPS-treated human THP-1 macrophages stably transduced with control empty vector or with SOD2. F, representative immunoprecipitation (IP) and immunoblot (IB) analysis of the interaction between NLRP3 and ASC in human THP-1 macrophages stably transduced with control empty vector or with wild-type or mutant SOD2 vectors, primed with LPS and left untreated or stimulated with nigericin. Inset numbers show the relative intensity of the bands compared with the control treated with LPS and nigericin ± S.E. (n = 3). The asterisks show significant (p < 0.05) reduction in WT and KR lanes compared with the control and KQ lanes. − indicates immunoprecipitation of a control sample primed with LPS and stimulated with nigericin but without an antibody, and IgG indicates immunoprecipitation of the same sample with an unspecific antibody. Input indicates immunoblot analysis of the same samples before immunoprecipitation. C, control vector; WT, wild-type SOD2 vector; KQ, mutant K68Q/K122Q SOD2 vector; KR, mutant K68R/K122R SOD2 vector.

Article Snippet: The following antibodies were used: caspase-1 (2225, Cell Signaling Technology; sc-514, Santa Cruz Biotechnology; AG-20B-0042-C100, Adipogen), IL-1β (ab9722, Abcam), NLRP3 (AG-20B-0014-C100, Adipogen), ASC (sc-22514, Santa Cruz Biotechnology), SOD2 (sc-18504, Santa Cruz Biotechnology), SIRT3 (5490S, Cell Signaling Technology), β-actin (A1978, Sigma-Aldrich), β-tubulin (2146S, Cell Signaling Technology), and Tom20 (sc-11415, Santa Cruz Biotechnology).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation

Journal: Cell Proliferation

Article Title: Combination of resolvin E1 and lipoxin A4 promotes the resolution of pulpitis by inhibiting NF‐κB activation through upregulating sirtuin 7 in dental pulp fibroblasts

doi: 10.1111/cpr.13227

Figure Lengend Snippet: Primers used for the selected human genes

Article Snippet: Anti‐NLRP3, anti‐caspase1, anti‐IL‐1β, anti‐IL‐18 (mentioned above) and anti‐OCN (PB1009, BOSTER) antibodies were used as primary antibodies at 1:200 dilutions.

Techniques: