Fig. 2: MED1 IDR deacetylation is induced by multiple stresses and regulated by SEC-associated SIRT1.
From: MED1 IDR deacetylation controls stress responsive genes through RNA Pol II recruitment
a, Immunoblotting showing enhanced acetylation of endogenous MED1 in MCF7 cells treated with Ex527, where the effect was attenuated by cotreatment with A485 (6 μM, 24 h). b, Immunoblotting showing enhanced acetylation of WT but not 6KR FLAG–MED1 in MCF7 cells treated with Ex527 (100 μM, 24 h). A control sample (ctrl IP) was included, in which the lysate of normal MCF7 cells was immunoprecipitated with anti-FLAG. c, Immunoblotting showing enhanced acetylation of WT but not 6KR MED1 in T47D cells where endogenous MED1 was depleted by CRISPR–Cas9 (referred to as WT/KO and 6KR/KO) treated with Ex527 (25 and 100 μM for 4 h). Unmodified T47D cells with IgG used as the IP control. d, Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells treated with various concentrations of DFOM (24 h). HIF1α was detected to demonstrate the induction of hypoxic response. e, Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells treated with DFOM (100 μM, 24 h), while cotreatment of Ex527 (50 μM) prevented this effect. f, Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells under 42 °C heat shock for various durations. g, Immunoblotting showing reduced acetylation of WT FLAG–MED1 in MCF7 cells treated with various concentrations of H2O2 (4 h). h, Immunoblotting showing reduced acetylation of endogenous MED1 in MCF7 cells treated with H2O2 or DFOM (either with 200 μM, 24 h), while cotreatment of Ex527 (50 μM, 24 h) prevented this effect. i, Immunoblotting showing elevated SIRT1 detected for association with endogenous AFF4 in MCF7 cells treated with DFOM (16 h), while equivalent association of cyclin T1 with AFF4 with or without DFOM treatment was detected. j, AlphaFold 3 prediction of structure of the SEC associated with SIRT1 (green). The overall structure (top) is shown in cartoon form and the close-up views of the SIRT1–SEC interface (bottom left and bottom right) are shown in cartoon form and surface representation. The extended fraction of SIRT1 inserting into the pocket formed by AFF4 (dark blue) and CDK9 (light blue) is indicated with a dashed circle. For a,h, IgG was used as an IP control. For a–i, densitometry analysis was performed on immunoblotting images and the normalized protein levels (relative to the IP target protein, FLAG tag of MED1 or endogenous MED1 or AFF4) are shown; these immunoblots are representative of three independent experiments.
