Figure 4

Garcinol reprograms H4K20 trimethylation by inducing SUV420H2. (A) Immunodetection of H4K20Me3 in MCF7 cells in response to garcinol treatment. Scalebar: 10 μm. (B) Western blots showing relative levels of H4K20Me3 and H3K9Me3 in acid-extracted histones prepared from MCF7 cells following treatment for 24 hours with HAT inhibitors at the indicated concentrations or control (DMSO). (C) Western blots showing induction of SUV420H2 and H4K20Me3 in MCF7 cells in response to garcinol exposure as in (B). (D) Quantitative analysis of the relative levels of SUV420H2 and H4K20Me3 in MCF7 cells in response to garcinol treatment as detected by flow cytometry. Cells were exposed to garcinol as in (B) and then fixed and permeabilised before incubation with primary and secondary (543 fluorophore-conjugated) antibodies. The data shown is the number of cells scoring positive for the indicated antigens from a total of 4000 scanned cells. (E) Relative levels of SUV420H2 transcripts in MCF7 cells at 24 hours post-transfection with siRNA duplexes targeting SUV420H2 (siSUV420H2) or a scramble control (siCTRL) siRNA mediated knockdown. (F) Western blots on MCF7 cell extracts following siRNA targeting as in (E) followed by exposure to garcinol (20 μM) for a further 24 hrs. The blot shows detection of H4K20Me3 levels following garcinol treatment in the control or SUV420H2 depleted cells.