Supplementary Figure 2: Characterization of the clonal macroH2A-GFP inducible dKO iDF cell lines (related to Fig. 2).
From: Transcription-associated histone pruning demarcates macroH2A chromatin domains
(a) Western blots for indicated histone variants and histone modifications with isolated chromatin. Amido black staining of histones is used as loading control. (b) Immunofluorescence analysis of WT and dKO iDFs stained with antibodies against macroH2A1 or macroH2A2. (c) Proliferation curve of WT and dKO iDFs. Error bars represent s.d. from n = 3 independent experiments. (d) FACS analysis with live cells for the expression of macroH2A2-GFP and macroH2A1.1-GFP at different time points after dox induction. (e) Reverse transcription coupled with qPCR (RT-qPCR) analysis for macroH2A2 (H2afy2) gene expression level at indicated time points after dox induction. Error bars represent s.d. from n = 2 independent experiments. (f-h) Western blots for inducibly expressed macroH2A2-GFP and macroH2A1.1-GFP using isolated chromatin at indicated time points after dox addition with respective clonal cell lines. Chromatin extract from WT iDFs was used to show level of endogenous macroH2As. Amido black staining of histones and histone H3 are used as loading controls. Numbers below are quantifications of endogenous or inducibly expressed macroH2As. (i) Relative amount of endogenous macroH2A variants and inducibly expressed ones at 72 h post induction calculated based on quantifications from f to h. The amount of total endogenous macroH2A (sum of macroH2A1 and macroH2A2) in WT iDFs was set to 1. (j) Relative amount of macroH2A2-GFP expression level at indicated time points post induction in dKO iDFs compared to total macroH2A in WT iDFs. Calculation was based on f and i.
