Effects of a new CDK9 inhibitor CDKI-73 on expression, proliferation, and cell cycle. (A) Analysis of expression of CDK9, CCNT1, and EWS-FLI1 24 h after treatment with 2 µM CDKI-73 in A673, EW7, and MHH-ES1 cell lines measured by qRT-PCR. Data are mean ± SEM; t-test. (B) Proliferation of EwS WT cell lines A673 and EW7 or JQ1-resistant A673 (A673r) and SKNMC (SKNMCr) cells was analyzed by xCELLigence assay. Upon reaching exponential growth rate, cells were treated with 2 µM CDKI-73/JQ1 or DMSO as negative control. Cellular impedance was measured every 4 h (relative cell index). Data are mean ± SEM (hexaplicates/group); *** p-value < 0.0005; 2-way ANOVA. (C) Cell cycle was analyzed 24 h after treatment with 1 µM JQ1, 1 µM CDKI-73, or both (each 1 µM) by flow cytometry of A673 and SKNMC. Cells were stained with propidium iodide. ** p-value < 0.005, * p-value < 0.05.