Figure 1. Distinct dynamics of MOF, MSL and NSL complexes during differentiation from ESCs to NPCs.
(A) We monitored the cell morphology during differentiation of mouse embryonic stem cells into neuronal progenitor cells (NPC) via embryoid body formation (EB) with bright field microscopy. The day of differentiation is indicated in white boxes. (B) Western blot analysis for ESC to NPC differentiation. Stages of differentiation together with the day of differentiation (d0–d15) are indicated on top. GAPDH and histone 3 (H3) were used as loading controls. For expression analysis see Figure 1—figure supplement 1.
Figure 1—figure supplement 1. Monitoring RNA and protein levels in ESCs and NPCs.
(A) We monitored the expression dynamics during ESC differentiation for markers of pluripotency (Oct4, Nanog, Rex1, Klf4), embryoid body formation (Fgf5), differentiation (Sox2), and NPC (Nestin). Panels 3 and 4 contain the expression profiles for members of the MSL complex (Msl1, Msl2), Mof, and the NSL complex (Kansl1, Kansl3, Mcrs1), respectively. All results are represented as relative values individually normalized to Rplp0 expression levels (panel 2) on a given day and to the highest expression level of a given gene during the entire differentiation process (highest expression level of each gene = 1). The x-axes show days of differentiation. All results are expressed as means ± SD for technical replicates. For primers see Supplementary file 3C. (B) Bright field images illustrate the cell morphology before and after the process of differentiation. The immunofluorescence analysis indicates the specific staining for the NESTIN (green) in neuronal progenitors (NPC); DNA is counterstained with DAPI (blue). (C) Expression changes for selected ESC-specific and NPC-specific markers before and after differentiation of wild-type WT26 cells using RT-PCR analysis and RNA-seq. (D) Western blots for proteins from two ES cell lines and their NPC derivatives. Different dilutions were loaded (100%, 30%, 10%) with the order indicated on top of the blots. Anti-GAPDH was used as loading control; arrows indicate the protein of interest.
Figure 1—figure supplement 2. Verification of antibodies used in this study.
(A) Immunoprecipitations from ESC nuclear extracts with antibodies specific for KANSL1, KANSL3 or MOF, and rabbit or rat antisera. The blot was probed with indicated antibodies showing the co-immunoprecipitation of several NSL complex members. Pol II = RNA Polymerase II. (B) and (C) same as (A) except that immunoprecipitations were performed with antibodies specific to MSL1 (B) and MSL2 (C). Asterisks represent the IgG signal.