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. Author manuscript; available in PMC: 2016 Dec 29.
Published in final edited form as: Cell Metab. 2015 Jan 6;21(1):65–80. doi: 10.1016/j.cmet.2014.12.005

Figure 6. Activation of PKM2 modulates anti-mycobacterial macrophage responses.

Figure 6

Cell lysates from FSL-1 and CpG (24hrs) treated BMDMs were analyzed for PKM2, IL-1β, Hif-1α or β-actin expression by western blotting (A), and pkm2 mRNA by qRT-PCR (B). BMDMs pretreated with TEPP-46 (50μM, 30min) were activated using FSL-1 (100ng/ml) and CpG (1μg/ml) for 24 hours. il1b was analyzed by qRT-PCR (C). Expression levels of IL-1β, Hif-1α, PKM2 and β-actin protein (D), pkm2 mRNA (E), IL-10 (F), TNFα and IL-6 protein (G, left and right) were measured in BMDMs ±TEPP-46 (30 min) stimulated using heat inactivated Mtb. (H) BMDMs ±TEPP-46 (25μM) were infected with live Mtb H37Ra (MOI 5 bacteria/cell, 3hrs) and gene expression of Il1b (left), tnf (middle) and Il10 (right) mRNA analysed (qRT-PCR). Data is mean ± SD for triplicate determinations, n=2. (I) BMDMs ±TEPP-46 (25μM) were infected as above (3 and 72hrs). IL-1β (left), TNFα (middle) and IL-10 (right) production were measured in supernatants of infected cells. BMDMs from (I) were lysed and CFU/ml determined (J). (K) BMDMs derived from wild type or IL-1 Type I Receptor knock out cells were infected as for (H) above. Cells were lysed at 72 hours post-infection and CFU/ml determined. Depicted as means ± SD of results from triplicate wells for one representative experiment n=2. *P<0.05, **P<0.01, ***P<0.001 (two-way analysis of variance with post-hoc Bonferroni correction).