Fig. 6

PELP1 interactions play an essential role in SETDB1 mediated Akt methylation. A Interactome network of PELP1, SETDB1, AKT1, and related genes based on GeneMANIA database. Each node represents genes and edges represent possible interactions. B GSEA plots of ESR1 targets via AKT1 regulation signature in RNA-seq data of ER⁺ BC with PELP1 or SETDB1- KD. C Total lysates from MCF7 cells grown in 10% serum were subjected to GST pull-down assays using GST-vector or GST-Akt purified from HEK293T cells and its ability to form trimeric complex with SETDB1, PELP1 was analyzed by Western blotting. D HEK293T cells are transfected with vector or SETDB1-GFP. Cells were serum-starved and stimulated with serum for 15 min and total lysates were subjected to immunoprecipitation and formation of trimeric complex was analyzed by Western blotting. E HEK293T cells transfected with indicated constructs were serum-starved and stimulated with serum for 15 min before being subjected to IP analysis.Western blotting was used to confirm trimeric complex. F Total lysates from HEK293T cells transfected with indicated constructs was subjected to immunoprecipitation using tri-methylation K-me3 antibody. Cells were serum-starved for 24 h and stimulated with 10% serum for 15 min before being subjected to IP analysis. The level of methylated Akt in immunoprecipitate and Akt phosphorylation in total cell lysate was verified by Western blotting. G In vitro methylation assay was performed using GST-Akt protein derived from HEK293T cells as substrate and recombinant SETDB1 protein as the source of methyltransferase. Effect of purified full-length PELP1 derived from baculovirus on SETDB1 mediated Akt methylation was determined. H–J MCF7 and ZR75 cells stably transfected with indicated constructs were serum starved for 24 h or E2 deprived for 48 h, and then stimulated with E2 (10^–8 M, H) or 10% serum (I, J) for 15 min. Effect of PELP1 on SETDB1 induced Akt and ER phosphorylation was measured using Western blotting