FIGURE 6.

Microcystin (MC)-RR reduces the level of pyruvate kinase M2 (PKM2) via interaction with PKM2. NRK-49F cells were treated as explained in Figure 4. (A) Total protein extracted from the cultured NRK-49F cells pretreated with TGF-β1 and MC-RR was immunoprecipitated with the PKM2 antibody. A protein band was subsequently identified as MC-RR by western blot. (B) Examination on colocalization of MC-RR (green) with PKM2 (red) or with p-PKM2 (red) was performed by immunofluorescence staining. DAPI was used for nuclear staining (blue) (scale bar: 20 μm). (C) Interaction between PKM2 and MC-RR was predicted with molecular docking conducted using the Yinfo Cloud Platform. The hydrogen bonds (showed in broken blue line) and ionic bonds (in broken yellow line) were formed between the amino acid residues of PKM2 and MC-RR. (D) PKM2 rescue assay was performed by transfecting NRK-49F cells with the recombinant PKM2 expression plasmid (pcDNA3.1-PKM2). Control cells without transfection, vector (pcDNA3.1), and the pcDNA3.1-PKM2 transfected cells were treated as indicated for 48 h. The expressions of PKM2, α-smooth muscle actin (α-SMA), and fibronectin were detected by western blot.