Fig. 1: GATA2 is retained at mitotic chromatin independently of cell context.

a Experimental approach to address mitotic bookmarking function of the hemogenic reprogramming transcription factors (TFs) GATA2, GFI1B and FOS, and their role in vivo for definitive haematopoiesis. b TF expression in the cytoplasmic (Cy) and chromatin-bound (Chr) protein fractions of mitotic human dermal fibroblasts (HDFs) expressing the indicated TF. Histone 3 (H3) and calnexin (CANX) were used as loading controls. kDA, kilodaltons. c Live-cell images of HDFs overexpressing mCherry (mCh)-TFs fusion proteins (red) during mitosis (Pro – prophase, Meta – metaphase, Telo/Ck – Telophase/Cytokinesis). Histone 2B (H2B)-mTurquoise (blue) signal shows DNA content. Mitotic events: n(GATA2) = 48, n(GFI1B) = 24, n(FOS) = 30, n(mCherry) = 137. Scale bars, 10 µm. d Quantification of the ratio between signal intensity of chromatin-retained and cytoplasm-localised TFs in metaphasic HDFs. n(mCherry)=30, n(GATA2) = 14, n(GFI1B) = 22, n(FOS) = 30. Mean ± SD is represented. Statistical significance was analysed by one-way ANOVA followed by Bonferroni’s multiple comparison test. a and b, p < 0.001. e Endogenous expression of GATA2, GFI1B and FOS in cytoplasmic and chromatin-bound fractions of mitotic K562 cells. H3 and CANX were used as loading controls. f Protein expression from asynchronous HDFs (overexpressed TFs, right panel) and K562 (endogenous TFs, left panel) whole-cell extracts. HDFs were transduced with mCh-TFs and the same number of mCh⁺ cells were FACS-purified for western blotting. CANX and Actin were used as loading controls. g Transcript levels of each endogenous TF in K562 cells. n = 3 biological independent samples per condition. Expression was normalised to GAPDH. h Transcript levels for overexpressed mCh-TFs in mCh⁺ FACS-purified HDFs. n(mCh-GATA2) = 3 technical replicates, n(mCh-GFI1B) = 2 technical replicates. (mCh-FOS) = 3 technical replicates. g, h Mean ± SD is represented. Expression was normalised to GAPDH. i, j Protein levels of GATA2, GFI1B and FOS in Cy and Chr fractions of mitotic HEK 293T cells when GFI1B (i) or FOS (j), were transduced individually or in combination with one or two additional TFs. Western blots were performed twice. Source data are provided as a Source Data file.