Figure 3.

Phenylpyruvate is taken up by BMDMs and triggers a proinflammatory macrophage phenotype

(A–C) BMDMs were pretreated with dimethyl sulfoxide (DMSO, control) or increasing concentrations of phenylpyruvate for 4 h and then stimulated with media or LPS (100 ng/mL) for 24 h. The black triangle indicates increasing phenylpyruvate concentrations starting from the left: 200, 400, and 800 μM. The RT-qPCR results showing relative mRNA expression of Nos, Tnf, and Il6 normalized to Actb (n = 3).

(D) Immunostaining and statistical analysis of Nos in BMDMs after pretreatment with phenylpyruvate and stimulation with LPS (n = 3). Scale bar, 150 μm.

(E) BMDMs were treated as in (A). Flow cytometry data for the M1 surface marker CD86, showing MFI (left) and representative histograms of CD86 (right) (n = 3).

(F–H) BMDMs were pretreated with increasing phenylpyruvate concentrations for 4 h and then stimulated with LPS (100 ng/mL) for 24 h and ATP (3 mM) for 45 min. Inflammatory factors IL-1β, IL-18, and TNFα in the supernatant were measured using ELISA assay (n = 3).

(I) Immunoblots of supernatants (SN) and cell extracts (lysate) of BMDMs treated as in (F) (n = 3).

(J) Immunoblots of supernatants (SN) and cell extracts (lysate) of BMDMs, which were pretreated with increasing phenylpyruvate concentrations for 4 h and then stimulated with LPS (100 ng/mL) for 24 h and nigericin (10 μM) for 45 min (n = 3).

(K) Immunostaining showing the uptake of phenylpyruvate into BMDMs. The FITC antibody indicated phenylpyruvate, and the lipid raft antibody indicated plasma membrane. Scale bar, 50 μm.

(L) BMDMs were pretreated with phenylpyruvate (400 μM) with or without cytochalasin D (10 μM) and then stimulated with LPS (100 ng/mL) for 24 h and ATP (3 mM) for 45 min. Immunoblots of the supernatants (SN) and cell extracts (lysate) (n = 3). Data are shown as mean ± SD. ∗p < 0.05, ∗∗p < 0.01.