Fig. 5: SPI1 inhibits ACSL4 expression by recruiting EZH2-mediated H3K27me3.

A Western blot analysis of ACSL4 protein in 786-O and ACHN cells treated with shCtrl, shSPI1, UNC1999 (5 μM) and Entinostat (300 nM). B Western blot analysis of ACSL4 protein expression levels with different time gradients of 5uM concentration of UNC1999 treated 786-O and ACHN cells. C qRT-PCR for mRNA expression of EZH2 and ACSL4 in si-Ctrl and si-EZH2 groups (n = 3). D Western bolt for protein expression of EZH2, H3K27me3 and ACSL4 in si-Ctrl and si-EZH2 groups. E Western bolt for protein expression of EZH2 and ACSL4 in 786-O and ACHN. F Prediction of EZH2 binding site upstream of ACSL4 promoter based on HumanTFDB. G ChIP-PCR analysis of the binding of EZH2 and H3K27me3 to the promoter of ACSL4 in 786-O and ACHN. H Immunofluorescence staining of SPI1 (red), EZH2 (green) and DAPI (blue) (n = 3). I Lysates from 786-O and ACHN cells were immunoprecipitated (Ip) with an anti-SPI1 antibody or immunoglobulin G (IgG) as indicated. Immunoprecipitates were analysed by immunoblotting with anti-EZH2 and anti-SPI1 antibodies. J, K Schematic representation of the ACSL4 promoter–luciferase reporter constructs. Two binding sites of the ACSL4 promoter (wild type or mutant) were cloned into the pGL4 reporter vector. The SPI1 binding site and EZH2 binding site are indicated (n = 3). Data are presented as the mean ± SD and analyzed by t -test or Kruskal-Wallis test. *: P < 0.05, **: P < 0.01, ***: P < 0.001.