Extended Data Fig. 1: Characterization of hybrid WT and Msl2-KO ESC and NPCs.
(a, c, d, f, g-i) Western blot analysis of pluripotency factors, selected MSL and KANSL complex components and histone modifications in WT and Msl2-KO ESC and NPCs. ACTIN, DHX9, RNA POL II, histone H3 and H4 serve as loading controls across panels. All Western blot experiments have been performed twice and representative results of a single experiment are depicted. The same loading order and equal volumes of the same lysates were loaded on multiple gels for blots displayed in a given column. For gel source data, see Supplementary Fig. 1. (a) Male CaBl ESCs comparing the parental WT (lane 1) and 3 Msl2-KO clones (lane 2–4). Protein quantification of NANOG and OCT3/4 (middle panel) of the single experiment depicted in the left panel was performed relative to RNA POL II levels. Data of Msl2-KO clones (KO (n = 3), pink) are depicted as a fold change over the WT clone (WT (n = 1), grey) and data are represented as mean values +/− SEM. Sequencing experiments were performed on the parental WT and Msl2-KO clones 1 and 3. (b) RT-qPCR analyses of Msl2 exon1, Nanog and Oct4 mRNA levels in parental WT, 3 Msl2-KO male CaBl ESC clones. mRNA levels were normalized to Tbp. Results are represented as fold change over WT and data are represented as mean values +/− SEM. n = 4 independent experiments. (c) Male CaBl NPCs comparing the parental WT (lane 1) and 3 Msl2-KO clones (lane 2–4). Sequencing experiments were performed on the parental WT and Msl2-KO clone 1 and 2. (d) Male BlCa ESCs comparing the parental WT (lane 1) and 2 Msl2-KO clones (lane 2-3). Protein quantification of NANOG and OCT3/4 of the single experiment depicted in the left panel was performed relative to RNA POL II levels. Data of Msl2-KO clones (KO (n = 2), pink) are depicted as a fold change over the WT clone (WT (n = 1), grey) and data are represented as mean values +/− SEM. Sequencing experiments were performed on the parental WT and Msl2-KO clone 1. (e) RT-qPCR analyses of Msl2 exon1, Nanog and Oct4 mRNA levels in parental WT, 2 Msl2-KO male BlCa ESC clones. mRNA levels were normalized to Tbp. Results are represented as fold change over WT and data are represented as mean values +/− SEM. n = 4 independent experiments. (f) Male BlCa NPCs comparing the parental WT (lane 1) and 2 Msl2-KO clones (lane 2-3). Sequencing experiments were performed on the parental WT and Msl2-KO clone 1. (g) Female 9sCa ESCs comparing the parental WT (lane 1) and 3 Msl2-KO clones (lane 2–4). Asterisk indicates protein of interest. A background band (50 kDa) detected by MSL2 antibody was included to highlight KO specificity. Sequencing experiments were performed on the parental WT and Msl2-KO clones 1 and 2. (h) Female 9sCa NPCs comparing the parental WT (lane 1) and 2 Msl2-KO clones (lane 2-3). Sequencing experiments were performed on the parental WT and Msl2-KO clones 1 and 2. (i) Female CaBl NPCs comparing the parental WT (lane 1) and Msl2-KO clone (lane 2). Sequencing experiments were performed on the parental WT and Msl2-KO clone.
