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. 2021 Nov 9;12:700431. doi: 10.3389/fimmu.2021.700431

Figure 4.

Figure 4

Phosphorylation of PKM2 and STAT3 promote heightened IL-1β expression in macrophages with deletion of Bmal1. WT and Bmal1 -/- BMDMs were stimulated with LPS (100 ng/ml), RNA was isolated, and gene expression of (A) PKM2 and (F) IL-1β were analysed by RT-qPCR. Samples were normalized to their expression of the housekeeping gene 18S. Data presented is n=3 +/- SEM. Statistical analysis was performed by one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, ****p < 0.0001). Protein expression of (B) PKM2 and (E) pSTAT3 was analysed by Western blot using β-Actin as a loading control. (C) PKM2 tetramers, dimers, and monomers were resolved by crosslinking samples after LPS stimulation before Western blot analysis. Pro IL-1β protein expression was measured following pretreatment with (D) DASA-58 (25 µM) or (G) STATTIC (2.5 µM) before stimulation with LPS for 8 hours. Western immunoblot data presented is representative of n=3 independent experiments. PKM2 and pSTAT3 time course densitometry is relative to the Bmal1+/+ control band. Densitometry for Bmal1+/+ and Bmal1-/- DASA/LPS bands is relative to their LPS bands. These bands are indicated by * symbols. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001.