Fig. 5
Nuclear histone deacetylase activity is reduced in IPF. a Western blot (WB) using the indicated antibodies and whole-cell protein extracts from Ctrl and IPF primary human lung fibroblasts (hLF) derived from three different donors, each. b HDAC1-specific activity assay using nuclear or cytosolic protein extracts from Ctrl or IPF hLF that were transfected with control miRNA (MIRCTRL) or MIRLET7D probes. c WB using antibodies specific for MiCEE components (top) or HDAC1, or 2 (middle), MIRLET7D-specific TaqMan assay (middle), and fluorometric HDAC activity assay (bottom) of fractions after sucrose gradient ultracentrifugation (SGU) of nuclear extracts from Ctrl and IPF hLF transfected as in b. Squares show fractions where MiCEE is expected. In, Input (5% of material prior SGU); MW, molecular weight; Rel exp, relative expression. d WB using HDAC1 (top) or HDAC2 (bottom)-specific antibodies after immunoprecipitation using either pan-acetyl-lysine (AcK) or phospho-serine (PhS)-specific antibodies to precipitate endogenous inactive HDAC1 and HDAC2 from selected SGU fractions analyzed in c. Input, 5% of IP starting material. e WB using the indicated antibodies and whole-cell protein extracts from Ctrl and IPF hLF that were transfected with an empty plasmid or expression constructs containing inactive (ina) or active (act) HDAC1 as indicated. In all bar plots, data are shown as mean (SD) (n = 3 biologically independent experiments for a and c; n = 2 biologically independent experiments for b). Asterisks in all plots, P-values after unpaired t-test, two tailed, ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant. Source data are provided as a Source Data file. See Supplementary Fig. 4
