Fig. 3

Influence of CRISPR/Cas9 HDAC knockouts on proliferation and invasiveness. a Proliferation and Western blot analysis (insets) of one to three HDAC1 (top) or HDAC2 (bottom) CRISPR/Cas9 knockouts in EwS cell lines CHLA-10, EW7 and SK-N-MC in comparison to controls (Cas9) are shown. Protein levels of Western blot analysis were detected with antibodies against HDAC1 and HDAC2. β-actin antibodies served as loading control. Proliferation and cell impendence was measured by the xCELLigence assay every 4 h. Data are shown as mean ± SEM (hexaplicates/group; p-value < 0.0001). b and c Analysis of anchorage-independent colony formation in methylcellulose of EwS cell lines with CRISPR/Cas9 mediated knockout of HDAC1 or HDAC2. Macrographs show a representative experiment with CHLA-10 and SK-N-MC HDAC1 ko (b) or HDAC2 ko (c). The number of colonies in different CHLA-10 or SK-N-MC HDAC clones was summarized in two bar graphs. d Analysis of invasiveness through Matrigel of different HDAC1 and HDAC2 CRISPR/Cas9 knockouts. Left, representative micrographs of invasive HDAC2 knockout and control cells 48 h after incubation are shown. Right, quantitative results for two different EwS lines are shown. Data are mean ± SEM of two independent experiments