Figure 2. LPS and Pam3CSK4 alter NALP7 protein stability and trafficking.

(a) NALP7 mRNA expression analysed by qPCR in THP-1 cells exposed to LPS or Pam3CSK4 for 16 h was unchanged compared to untreated control, whereas pro-IL-1β mRNA expression increased. Data shown as mean fold change±s.d. as determined by ΔΔCq analysis (n=3). (b) In cycloheximide (CHX) chase, NALP7 protein stability is increased in THP-1 cells exposed to LPS (200 ng ml⁻¹) or Pam3CSK4 (100 ng ml⁻¹) compared to vehicle control (CON) (c) NALP7 protein abundance increased over time when treated with leupeptin (50 μM) or bafilomycin (100 nM) but not MG132 (20 μM). (b,c) Densitometric analysis of the NALP7 signal versus time, normalized to β-actin. Data shown as mean±s.d. (n=3). *P<0.05 compared to 0 h or **P<0.01 versus CON. (d) Immunofluorescence co-staining of NALP7 (green), Lysotracker (red) and DAPI (blue) in Beas2B cells exposed to LPS (5 μg ml⁻¹), Pam3CSK4 (2 μg ml⁻¹), leupeptin (100 μM), or vehicle control for 2 h. Lysotracker (1:2,000) was added for the final 30 min of incubation before fixing and staining. Original magnification was × 100, Scale bar, 5 μm. Images are representative of three or more images captured for each condition (n=2). (a) One-sample t-test (hypothetical value=1). (b,c) Two-way analysis of variance (ANOVA) with post hoc Dunnett's multiple comparisons test.