Fig. 4. NSA does not inhibit other innate immune pathways.

(A) iBMDM cells were treated with TNF-α, zVAD-fmk, and GDC-0152 for 24 hours in the presence or absence of 20 μM NSA. Cell death was measured by LDH release. (B and C) The impact of NSA on TLR pathways was assessed using RT-PCR of inflammatory gene transcripts after stimulation of TLR1/2 with Pam3CSK or TLR4 with LPS in the presence or absence of 20 μM NSA. (D) qRT-PCR was used to measure CXCL10 and IL-6 expression after infection with S. Typhimurium. Log phase S. Typhimurium was used to infect iBMDM cells at an MOI of 50:1 with induction of IL-6 and CXCL10 genes measured after 0 and 2 hours. Relative induction was based off of GAPDH as the housekeeping reference gene. Data are taken from technical triplicates and are representative of three independent experiments. **P < 0.01. (E) Cell death as measured by LDH assay in macrophages treated for 8 hours with 100 μM etoposide and DMSO, 10 μM NSA, or 20 μM NSA. **P < 0.01. (F) Pyroptotic pore formation as assessed by PI uptake in iBMDM cells after activation of the NLRP3 inflammasome with LPS and nigericin and treatment with 20 μM NSA, TCEP, iodoacetamide, or DTT. (G) iBMDM cells expressing mCerulean-ASC that form specks upon activation of the inflammasome were stimulated with LPS and nigericin in the presence of DMSO or NSA. Formation of specks was visualized using epifluorescent microscopy. Scale bars, 100 μm. (A to F) Data are means ± SE. n.s., not significant.