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

Figure 2.

Figure 2

Lactate activates PKM2 in lipopolysaccharide-induced macrophages. (A) Naïve bone-marrow-derived macrophage (BMDM) cells were incubated with lipopolysaccharide (LPS) for 4 hr, followed by vehicle (LPS) or lactate (LPS-LA) for 20 hr. The mRNA was extracted for testing PKM1 and PKM2 expression (n=3). (B) BMDM cell proteins were analyzed by western blotting (WB) for the PKM2 level. (C) BMDM cell lysates were assayed for pyruvate kinase activity (n = 6). (D) BMDM cells were collected and crosslinked with disuccinimidyl suberate (DSS). The tetrameric (240KD), dimeric (120KD), and monomeric (60KD) forms of PKM2 were analyzed by WB. (E) Cytosolic and nuclear proteins were purified from the BMDM cells separately for PKM2 level analysis. (F) The localization of PKM2 was analyzed by immunofluorescence staining. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; blue). The line charts represent fluorescence intensity (MFI), presenting the distance from α to γ. WB data represents three independent experiments. Data represents mean ± SEM, Scale bar = 100 µm. ***p < 0.001, ****p < 0.0001.