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. 2022 Apr 29;13(4):414. doi: 10.1038/s41419-022-04801-0

Fig. 1. Midkine suppresses AMPK activation in an intracellular localization-dependent manner.

Fig. 1

a MDK expression in the cell lysate and conditioned medium (CM) of HepG2, HCCLM3, Bel-7402, and SMMC-7721 cells was tested by immunoblotting. b CM from MDK-overexpressing MHCC97H cells was harvested and used to culture Bel-7402 and SMMC-7721 cells for 2 h, intracellular MDK was tested by western blotting. c Cytoplasmic and nuclear fractions of the MDK overexpressing MHCC97H cells and the control cells was prepared and the distribution of MDK was detected by immunoblotting. d Signaling pathway enrichment assay with MDK-correlated genes based on the TCGA database. We first defined MDK correlated genes by calculating the Pearson correlation coefficients between MDK and all other genes according to their mRNA expression levels (data from the TCGA RNA-seq datasets). Then the overlap significance between MDK correlated genes and pathway genes (data from KEGG) was examined. Then the GSEA-based pathway enrichment was performed. e–f Analysis of p-AMPKα Thr172 level affected by MDK. MDK overexpressing Bel-7402 cells and control cells were treated at different glucose concentration (25 mM and 1 mM) for 2 h (e), or with 10 mM 2DG for 4 h (f), then the cells was lysed and the indicated proteins were tested with immunoblotting. g MDK overexpressing cells and control cells were subjected to glucose starvation for the indicated time, then the cells were harvested for western blotting. h Knock-down of MDK in HCCLM3 cells with two independent shRNAs resulted in elevated p-AMPKα Thr172 level after 4 h treatment of 10 mM 2DG. i Western blotting of the MHCC97H cells at different time points of CM treatment. CM is from MDK-overexpressing MHCC97H cells. j MDK knocked-down HCCLM3 cells and the control cells were treated with or without heparin treatment (30 μg/ml) for 4 h under 10 mM 2DG culture conditions, following by immunoblotting to test the indicated protein level. k–l The Bel-7402 (k) and MHCC97H (l) cells were subjected to a combination treatment with or without MDK- CM and heparin (30 μg/ml) for 2 h, then western blotting was performed to test the p-AMPKα Thr172 level.