Fig. 4

Gfi1 is a target for acetylation and binds p53 in MM cells to prevent apoptosis. HEK293-T cells were transfected with either Flag- or Myc-Gfi1 and HA-P300, incubated 8 h with 10 mM NAM and/or 5 μM TSA (HDACi), and cell lysates were immunoprecipitated (IP) using either an anti-Flag or anti-Myc antibody and analyzed by Western blot (WB) (a, top). MM.1S cells, treated with HDACi (8 h), were IP with Gfi1 antibodies and analyzed by WB (a, bottom). H929 cells were treated with HDACi (8 h) (b) or with actinomycin D (1 and 10 nM; 16 h) (c) and analyzed by WB. Chromatin from H929 cells was analyzed using SimpleChIP ® kit and qPCR with ChIP-qPCR primers for BAX and NOXA. The bar graph represents the fold enrichment of p53 protein at BAX and PUMA promoters (d). H929 cell’s viability was measured by MTT assay after 24 and 48 h treatment with HDACi or vehicle control (e). H929 cells were treated with HDACi (8 h), IP with anti-Gfi1 antibody, and analyzed by WB (f). Paraformaldehyde-fixed cytospins of H929 cells (HDACi for 8 h) were stained for p53 (red-Alexa Fluor® 594), Gfi1 (green-Alexa Fluor® 488), and DAPI for nuclei (Magnification × 40, bar 30 mm). The images are representative of three independent experiments (g). H929 cell’s (HDACi; 8 h) cytosolic and nuclear fraction’s (Cell Fractionation kit-Abcam, Cambridge, MA, US) lysates were analyzed by WB with anti-β actin and Lamin B1 used as fraction specific loading controls (h). For identifying the acetylation regions in Gfi1, HEK293-T cells were transfected with different forms of Myc-Gfi1 (aa1-261, aa1-300, aa239-423, aa291-423, aa341-423, and aa1-423) and HA-P300, incubated with HDACi (4 h), IP with anti-Myc antibody, and analyzed by WB (i). HEK293-T cells were transfected with Myc-Gfi1 (wt) or the Myc-Gfi1 K292R mutant with or without the HA-P300 plasmid, treated with HDACi (8 h), and cell lysates were IP with anti-Myc antibody and analyzed by WB (j)