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. 2024 Jul 18;9(16):e183237. doi: 10.1172/jci.insight.183237

Figure 6. PI3Kγ promotes NETs’ formation via noncanonical pyroptosis pathways in vitro.

Figure 6

(A) Representative Western blot (A) and quantitative comparison of Cit H3 and GSDMD in WT neutrophils with different treatments: PBS, LPS (5 μg/mL), and LPS + DSF (30 μM). n = 3. (B) Representative Western blot and quantitative comparison of IL-1β and GSDMD in neutrophils with different treatments: PBS, LPS (5 μg/mL), LPS + KO (PI3Kγ–/–). n = 3. (C) Representative Western blot and quantitative comparison of Cit H3, IL-1β, GSDMD, caspase-11, and caspase-1 in the neutrophils with different treatments: PBS, LPS (100 ng/mL), LPS + nigericin (10 μM), LPS + nigericin + KO, PBS, Pam3CSK4 (1 μg/mL), Pam3CSK4 + transfer LPS (tLPS, 10 μg/mL), Pam3CSK4 + tLPS + KO. n = 3. (D) Representative immunofluorescence staining and quantitative comparison of NETs produced by neutrophils with different treatments: PBS, LPS (5 μg/mL), LPS + KO, LPS + H89 (20 μM) + KO, LPS + MDL12330A (10 μM) + KO. NETs were detected using immunofluorescence staining. NETs’ expression was calculated by NET-expressing cell numbers/total cell numbers per high-power field (original magnification, 40×). n = 6. Scale bars, 50 μm. (E) Comparison of cAMP concentration of neutrophils with different treatments as described in D by ELISA. n = 4. (F) Comparison of PKA kinase activity of neutrophils with different treatments as described in D by kinase activity assay. n = 4. (G) Representative Western blot and quantitative comparison of Cit H3, GSDMD, and caspase-11 in the neutrophils with different treatments as described in D. n = 3. (A–G) One-way ANOVA followed by Fisher’s least significant difference post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. GSDMD, gasdermin D; pro-, prosoma; cle-, cleaved; Casp1, caspase-1; Casp11, caspase-11; LPS, lipopolysaccharide; DSF, disulfiram; Nig, nigericin; Pam, Pam3CSK4; KO, phosphoinositide-3-kinase γ knockout; MDL, MDL12330A.