Fig 1: Specific deletion of Casp11 in neutrophils inhibited NETs formation and renal fibrosis after UUO.A Western blot showing caspase11 activation in the kidneys after UUO. Kidney tissue lysates were isolated on day 0, 2, 5, and 10 after UUO. n = 4. B Representative images of Masson trichrome staining of kidney sections from Casp11wt/wtMrp8cre and Casp11fl/flMrp8cre mice on day 13 after UUO. Scale bar: 100 µm. n = 6. C, D Representative images of immunofluorescence staining of kidney sections from Casp11wt/wtMrp8cre and Casp11fl/flMrp8cre mice, showing the expression of a-SMA C and Col I D. DAPI (blue) was used for nuclear staining. Scale bar: 100 µm. E Representative images of immunofluorescence staining of kidney sections from Casp11wt/wtMrp8cre and Casp11fl/flMrp8cre mice on day 10 after UUO, showing the expression of Histone-H3 and MPO. Scale bar: 100µm. F, G Representative images of immunofluorescence staining of Cit-H3 and MPO and quantification of neutrophils undergoing NETs formation. The neutrophils were isolated from Casp11+/+ and Casp11-/- mice and treated as described in Fig. 7A. n = 6. Scale bar: 50 µm. H Schematic model of GSDMD-dependent NETs promoting MMT and renal fibrosis in obstructive nephropathy.
Fig 2: The GSDMD expression detected by immunofluorescence assay. The protein expressions of GSDMD were analyzed by immunofluorescence assay. GSDMD was probed by GSDMD antibody, The nucleus was stained by DAPI. (A1,A2) Raw 264.7 cells without any treatment, (A1) (× 200), (A2) (× 400); (B1,B2) Raw264.7 cells treated with LPS + ATP; (C1,C2) SENP7 stable knockdown Raw 264.7 cells treated with LPS + ATP. (A1,B1,C1) (× 200), (A2,B2,C2) (× 400). (D) The relative protein expression levels of GSDMD from three independent pictures were represented as the density of GSDMD fluorescence intensity normalized to numbers of cells. Compared with the NORMAL group, *p < 0.05, compared with the CONTROL group, **p < 0.05.
Fig 3: SENP7 knockdown inhibits pyroptosis in Raw 264.7 cells. (A) Cleaved-GSDMD, GSDMD, IL-1ß and IL-18 expression levels detected by western blot assay; (B–E) The relative protein expression levels from three independent experiments are represented as the density of Cleaved-GSDMD, GSDMD, IL-1ß and IL-18 bands normalized to that of GSDMD or ß-actin bands. (F,G) The expression levels of IL-1ß and IL-18 in supernatants detected by ELISA assay. NORMAL: Raw 264.7 cells without any treatment; CONTROL: Raw 264.7 cells treated with LPS + ATP; SENP7-shRNA: SENP7 stable knockdown Raw 264.7 cells treated with LPS + ATP. Compared with the NORMAL group, *p < 0.05, compared with the CONTROL group, **p < 0.05.
Fig 4: Protein levels of interleukin (IL)-1ß, IL-18 and gasdermin D (GSDMD) in perivascular adipose tissue from Zucker lean rats (controls) and male Zucker diabetic fatty rats, with or without liraglutide for 12 weeks (DM and DM+GLP-1 groups): (a) representative western blots; and (b) IL-1ß, IL-18 and GSDMD levels normalised to ß-actin. Data presented as mean ± SEM of 10 rats per group (samples assayed in triplicate); *P < 0.05, DM versus controls, or DM+GLP-1 versus DM group (analysis of variance).
Fig 5: Specific deletion of Gsdmd in neutrophils instead of macrophages protected the kidney from undergoing fibrosis after UUO.A Representative images of Masson trichrome staining of kidney sections. Kidneys were collected from Gsdmdfl/fl, Gsdmdwt/wtLyzcre, Gsdmdfl/flLyzcre, Gsdmdwt/wtMpr8cre and Gsdmdfl/flMpr8cre mice on day 13 after UUO. Scale bar: 100 µm. B, C Representative images of immunofluorescence staining of kidney sections, showing the expression of a-SMA B and Col I C. DAPI (blue) was used for nuclear staining. Scale bar: 100 µm. D–F Quantification of renal fibrosis scores evaluated by Masson trichrome staining D, a-SMA E, and Col I F expression by immunofluorescence. n = 6. **P < 0.01.
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