Fig. 5

Sulfarotene inhibits SOS2 transcription via RARα. a Immunofluorescence showed that RARα (red) colocalized with SOS2 (green) in the nucleus of HCC TRCs treated with sulfarotene. DAPI: blue. b Map of the SOS2 5’ region from the + 1 transcription starting site. Amplicon 1 and 2, regions of interest within the SOS2 promoter in luciferase reporter experiments. Amplicons 3, region of interest in the exon 7 locus as revealed in ChIP-PCR experiments using anti-RARα antibody. c Heat map of RARα and H3K27ac ChIP-Seq signals in Hep3B-TRCs in response to sulfarotene treatment as compared to DMSO. Each row shows ± 5 kb centered on the RARα peak. The ChIP-Seq signal was depicted by color scaled intensity. d Gene tracks of ChIP-Seq signals for RNA-Seq, H3K27Ac and RARα signals around the SOS2 loci in Hep3B-TRCs after treatment with 5.0 µM sulfarotene. ChIP-Seq and RNA-Seq signals were visualized with the Integrated Genome Viewer (IGV). e-f Interactions of RARα with 2 promoter elements and 1 element on exon 7 of SOS2 gene were determined by ChIP-qPCR analysis with anti-RARα antibody and rat IgG as controls. Hep3B-TRCs and PLC/PRF/5-TRCs with stable overexpression of RARα (e) or treated with 5.0 µM sulfarotene in the presence or absence of BMS195614 (f) for 24 h were cultured for 5 more days, then ChIP-qPCR analysis was performed as described. g The H3K27ac signals indicated enhancers in Hep3B-TRCs treated with sulfarotene relative to DMSO. The super enhancer (SE) zone is illustrated by the dashed lines. h Metagene representations of RARα ChIP-Seq signals in units of read count per million mapped reads at a meta composite of SEs in Hep3B-TRCs treated with 5.0 µM sulfarotene compared to DMSO. i-j Gene tracks of ChIP-Seq signals for RNA-seq, H3K27Ac and RARα signals around the HSPB1 and SOS2 loci in Hep3B-TRCs treated with 5.0 µM sulfarotene compared to DMSO. Red bars show the sulfarotene-specific SEs and the typical enhancers (TEs)