Fig 1: HSV-1 infection induces pyroptosis and triggers interleukin 1ß (IL-1ß) maturation. (A) Representative micrographs of BV2 cells infected with HSV-1 (MOI = 5, 24 hpi) or mock infection. Yellow corner mark: pyroptotic cells (Scale bar, 100 µm, 40×). (B) Relative qRT-PCR analysis of IL-1ß, IL-18, TNF-a, and IL-6 mRNA levels in BV2 cells infected with HSV-1 and Mock infection (MOI = 5, 24 hpi). (C) IL-1ß was measured with ELISA in BV2 with HSV-1 and mock infection (MOI = 5, 24 hpi). (D) LDH release was measured in supernatant taken from cultured mock- and HSV-1-infected BV2. (E,F) Examination of the proteolytic cleavage of GSDMD in BV2 infected by HSV-1 for the indicated MOI (24 hpi), Western blot analysis of indicated proteins in the cell lysates. GSDMD-FL, full-length GSDMD; GSDMD-NT, the N-terminal cleavage product of GSDMD; GSDMD-CT, the C-terminal cleavage product of GSDMD. ß-actin was used as a loading control. All data are presented as mean ± SD, Student’s t-test, ***P < 0. 001.
Fig 2: NLRP3 Inflammasome is involved in HSV-1-induced pyroptosis. (A) Western blot analysis of indicated proteins in the supernatants (Sup), BV2 infected with HSV-1 (MOI = 5, 24 hpi). (B) BV2 cells treated with 1 µg/mL lipopolysaccharides (LPS) for 4 h followed by 2 µg/mL nigericin for 2 h or HSV-1 infection (MOI = 5, 24 hpi), the level of NLRP3, pro-caspase-1, gB, and IL-1ß was detected by Western blot. LPS + Nigericin in uninfected cells served as a positive control. (C,D) Immunoblot analysis of extracts of BV2 cells infected by HSV-1 (MOI = 5) for the indicated time points by the indicated antibodies. (E) The expression levels of NLRP3, caspase-1(p10), and cleavage of GSDMD expression in RAW264.7 were detected by Western blot. ß-actin was used as a loading control.
Fig 3: TLYS decoction suppresses the pyroptosis associated protein expressions in UUO rats. (A–E) Expression levels of NLRP3, GSDMD, IL-18 and IL-1ß in the kidney were detected by immunohistochemistry and the optical intensity of the abovementioned proteins was measured (n = 6). (F–K) The protein levels of NLRP3, caspase-1, GSDMD, IL-18 and IL-1ß in the kidney were assayed by Western blot and analyzed semi-quantitatively (n = 3). The magnification of the images is ×200, scale bar = 50 µm. Data were presented as means ± SD. *p < 0.05, **p < 0.01.
Fig 4: TLYS decoction suppresses the pyroptosis associated protein expressions of NRK-52E cells induced by hypoxia. (A–F) The protein levels of NLRP3, caspase-1, GSDMD, IL-18 and IL-1ß in NRK-52E cells induced by hypoxia were assayed by Western blot and analyzed semi-quantitatively (n = 3). (G) The positions of GSDMD and IL-18 was labeled by immunofluorescence (n = 6). The magnification of the images is ×400, scale bar = 50 µm. Data were presented as means ± SD. *p < 0.05, **p < 0.01.
Fig 5: GSDMD is important for the restriction of Leishmania species in vivo.WT and Gsdmd–/– mice were infected with 106 stationary-phase L. mexicana, L. major, and L. braziliensis promastigotes in the ear, and the ear thicknesses were followed for 8 (a–h) or 6 weeks (i–l). a, e, i Lesion development; b, f, j images of infected ears; c, d, g, h, k, l limiting dilution assay to quantify parasite burden in the infected ears (c, g, k) and draining lymph nodes (d, h, l) at 8 or 6 weeks of infection. Each dot in the bar graphics represents the value obtained from an individual mouse. Data are presented as mean values ± SD. *P < 0.05 comparing the indicated groups, as determined by the Student T test two-sided. Shown is one representative experiment of two independent experiments performed. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-cleaved C-terminal GSDMD antibody [EPR20859-147]