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. 2022 Oct 24;18(16):6210–6225. doi: 10.7150/ijbs.75434

Figure 6.

Figure 6

The K62R mutant reverses the regulation of lactate on PKM2 pyruvate kinase activity and macrophage phenotype transition. (A) Wild-type flag-PKM2 overexpressed 293T cells were incubated with vehicle (WT-CTRL) or 20 mM lactate (WT-LA) for 24 hr. Then cell lysates were assayed for pyruvate kinase activity (n = 4). (B) WT (WT-LA) or K62R site mutation (K62R-LA) flag-PKM2 overexpressed 293T cells were incubated with 20 mM lactate for 24 hr. Then cell lysates were assayed for pyruvate kinase activity (n = 8). (C) 293T cells were collected and crosslinked with disuccinimidyl suberate (DSS). The tetrameric (240KD), dimeric (120KD), and monomeric (60KD) forms of PKM2 were analyzed by western blotting (WB). (D) Cytosolic and nuclear proteins were purified from 293T cells separately for PKM2 level analysis. WB data represents three independent experiments. (E) WT or K62R site mutation PKM2 overexpressed BMDM cells were constructed through the lentiviral vector. Then the PKM2-overexpressed BMDM cells were incubated with 100 ng/ml LPS ± 20 mM lactate for 24 hr. Cell proteins were analyzed by WB for the iNOS and ARG1 levels. The blots are representative of three independent experiments. Immunofluorescence staining of iNOS (F) or ARG1 (G) was performed. Data represents mean ± SEM. Scale bar = 100 µm. *p < 0.5, **p < 0.01, ***p < 0.001.