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. 2020 Dec 5;11(12):1036. doi: 10.1038/s41419-020-03212-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — HEK293T cells were transfected with Flag- PKM2 / HA-HDAC8 (A) or Flag- HDAC8/HA- PKM2 (B). After 48 h, HA-tagged protein was immunoprecipitated from HEK293T cells lysates. Then, immunoprecipitates and whole-cell extracts were subsequently separated by SDS-PAGE and immunoblotted with Flag and HA antibodies. C HepG2 cells were transfected with HA- PKM2 for 48 h, Extracts were immunoprecipitated with anti-HA (IgG as control). Immunoprecipitates and whole-cell extracts were subsequently analyzed by western blotting with an anti-HDAC8 and HA antibody. D Lysates of HepG2 cells were immunoprecipitated with the antibody against PKM2. Immunoprecipitates and whole-cell extracts were analyzed by western blotting with anti-PKM2 and anti-HDAC8 antibodies. E The Duollink PLA technology showed the interaction(red) between HDAC8 and PKM2 in SMMC-7721 and LO2 cells (Scale bar, 50 µm). F The mixture of recombinant GST- PKM2(GST-vector as a control) and His- HDAC8 were rotated and incubated for 3 h. Bound proteins were eluted, then analyzed with Coomassie Blue staining. G FLAG-HDAC8 was co-expressed in HEK293T cells with HA-PKM2 fragments as indicated. Co-IP followed by western blotting was performed to determine their interaction. H, I After transfected with Flag- PKM2 24 h, PCI-34051 (25 µM) and TSA/NAM (1 µM /10 mM) (H) or different doses of PCI-34051 (I) are added for 24 h. Cell lysates were immunoprecipitated with Flag affinity gel. PKM2 acetylation and protein levels were analyzed by immunoblotting using the indicated antibodies. J–L Interfered by siHDAC8 (J), PCI-34051 (K), and HA-HDAC8 (L) in HepG2 cells or HDAC8-KO HepG2 cells, separately. After immunoprecipitated with Flag affinity gel, acetylation of K62 was determined by western blotting. M LC-MS-MS of PKM2 showing the acetylation of PKM2 K62 in HDAC8-KO HepG2 cells. HepG2 cells and HDAC8-KO HepG2 cells transfected with Flag-PKM2. The immunoprecipitates were sent for LC-MS-MS analysis of the acetylation intensity of PKM2 peptides.