Fig. 6
Active EP300 inactivates HDAC1 in IPF. a WB using the indicated antibodies and whole-cell protein extracts from Ctrl and IPF lung fibroblasts (hLF) derived from three different donors each. ActEP300, active EP300. See Supplementary Fig. 6a–c. b Top: HDAC1-specific activity assay using nuclear or cytosolic protein extracts from Ctrl or IPF hLF treated with placebo (vehicle solution) or 10 µM EP300 inhibitor (EP300ihn, CBP30). Bottom: WB monitoring the quality of the fractions used in the HDAC1 activity assay. LMNB1, nuclear marker. GAPDH, cytosolic marker. See Supplementary Figs. 6d and 7a. c Sequential ChIP (ChIP–reChIP) analysis of representative MIRLET7D target promoters using HDAC1- or HDAC2-specific antibodies, followed by AcK- or PhS-specific antibodies, respectively, and chromatin isolated from Ctrl or IPF hLF after treatment with EP300inh or transfection of a dominant-negative EP300 (dnEP300) construct. IgG, immunoglobulin G (negative control). See Supplementary Fig. 7. d WB using the indicated antibodies after Co-IP assay using immunoglobulin G (IgG; negative control) or HDAC1-specific antibodies to immunoprecipitate endogenous HDAC1 from nuclear protein extracts of Ctrl hLF. Input, 5% of IP starting material. e WB after Co-IP assay as in d using C1D-specific antibodies to immunoprecipitate endogenous C1D from nuclear protein extracts of Ctrl hLF that were transfected with full-length (fl) or deletion mutant 3 (del3, lacking C1D interaction domain on amino acids 1–285) HA-tagged EXOSC10 constructs25. f WB after Co-IP assay as in d using C1D-specific antibodies to immunoprecipitate endogenous C1D from nuclear protein extracts of IPF hLF that were either transfected with empty plasmid or with dnEP300 expression construct or treated with EP300inh as indicated. In all plots, data are shown as mean (SD) (n = 3 biologically independent experiments in b; n = 2 biologically independent experiments in c); asterisks, P-values after unpaired t-test, two tailed, ***P < 0.001; **P < 0.01; *P < 0.05. Source data are provided as a Source Data file
