Fig. 4: MELK augments the expression of the CRS gene DLAT through the PI3K/mTOR signaling pathway.

A DLAT was filtered out as the only candidate gene among the datasets of shMELK, cuproptosis-related signatures(CRS) genes and the datasets of DepMap. B–D Correlation of expression, gene effect and mRNA ratio between MELK and DLAT. The mRNA ratio was calculated by mRNA (tumor)/mRNA (adjacent tissue). E, F qPCR and immunofluorescence showed that MELK expression regulated the expression of DLAT in Huh7 cells. G, H WB showed that the PI3K signal pathway was regulated by MELK. I, J MELK-silenced or MELK-overexpressing Huh7 or Hep3B cells were incubated in PI3K agonist 740 Y-P (30 μM, 24 h) or PI3K inhibitor PF-04691502 (0.3 μM, 48 h), and DLAT expression was detected with WB. K, L Mitochondrial respiration was measured by OCR assay in Huh7 cells treated with MELK knockdown or/and 740 Y-P. M Intracellular copper contents with pulse treatment of elesclomol for 2 hr. N The expression of TOM20 and DLAT (monomer and oligomer) was detected by WB after treatment with elesclomol. O In MELK-overexpressed Huh7 cells, DLAT was knocked down or regulated by elesclomol, and the expression of TOM20 and DLAT were detected. n.s., *, **, *** and **** represented not signifificant, P < 0.05, <0.01, <0.001 and <0.0001, respectively. Analyzed data were from three independent experiments and shown as means ± SEM. Data were from three independent experiments and analyzed with the t test (E, L, and O). Spearman correlation analysis was performed in (B–D, F).