Figure 2.
Impairment of T-loop phosphorylation by CDK7 inhibition causes cell cycle arrest and Rb activation in MM cells. (A) MM cell lines (n = 8) were treated with the indicated concentrations of YKL-5-124 for 24 hours. Cell cycle was evaluated by propidium iodide staining followed by flow cytometric analysis and analyzed with ModFit LT 5.0 software. (B) Whole-cell lysates from H929 and AMO1 cells treated with the indicated concentrations of YKL-5-124 for 24 hours were subjected to WB analysis and probed with antibodies against Rb and p-Rb, and GAPDH as a loading control. The ratio of phosphorylated/total forms of indicated Rb was analyzed with Image J software and represented as fold change from untreated cells. Mean values ± SD in 4 MM cell lines is shown in the graph. (C) H929 cells stably expressing E2F1 luciferase reporter were treated with 50 nM YKl-5-124 for 24 hours. E2F1 activity was assessed using the Promega luciferase reporter assay system, and fold change of E2F1 activity compared with untreated cells is displayed (mean ± SD). ∗∗∗P < .001. (D) H929 and AMO1 cells were treated with 250 nM YKL-5-124 for 24 hours, and the nuclear extract was analyzed by EMSA. The shifted probe caused by E2F1 binding is indicated by the band labeled E2F1. (E) H929 and AMO1 cells were treated with 500 nM YKL-5-124 for 24 hours and chromatin immunoprecipitated using E2F1 or control mouse IgG antibodies. The crosslinked DNA was subjected to quantitative polymerase chain reaction using primers specific for a representative set of E2F1 target genes. Data are represented as the percentage of input. (F) E2F score was calculated by using E2F1 genes identified previously.11 After RNA-seq normalization, we converted expression values for each gene to z scores, with a mean of 0 and SD of 1. After scaling the expression, we calculated the total score as the sum of scaled scores from all the genes. A Pearson correlation coefficient was calculated between E2F score and CDK7 expression. (G) AMO1 cells expressing control vector (PCW) or T121 were treated with doxycycline for 24 hours, followed by YKL-5-124 (500 nM) for 6 hours, and then chromatin immunoprecipitated using E2F1 or control mouse IgG antibodies. The crosslinked DNA was subjected to quantitative polymerase chain reaction using primers specific for a representative set of E2F1 target genes. Data are represented as the percentage of input. (H) AMO1 cells expressing either PCW or T121 plasmid were treated with doxycycline for 24 hours, followed by YKL-5-124 for 24 hours. The cell cycle was evaluated by propidium iodide staining followed by flow cytometric analysis. (I-J) MM cells expressing either control or T121 plasmid were treated with doxycycline for 24 hours followed by YKL-5-124 treatment for 48 hours. Cell viability was measured by CTG assay (I) and apoptosis by annexin V+ staining (J). Data represent the mean of 4 independent experiments. IgG, immunoglobulin G; SD, standard deviation.

Impairment of T-loop phosphorylation by CDK7 inhibition causes cell cycle arrest and Rb activation in MM cells. (A) MM cell lines (n = 8) were treated with the indicated concentrations of YKL-5-124 for 24 hours. Cell cycle was evaluated by propidium iodide staining followed by flow cytometric analysis and analyzed with ModFit LT 5.0 software. (B) Whole-cell lysates from H929 and AMO1 cells treated with the indicated concentrations of YKL-5-124 for 24 hours were subjected to WB analysis and probed with antibodies against Rb and p-Rb, and GAPDH as a loading control. The ratio of phosphorylated/total forms of indicated Rb was analyzed with Image J software and represented as fold change from untreated cells. Mean values ± SD in 4 MM cell lines is shown in the graph. (C) H929 cells stably expressing E2F1 luciferase reporter were treated with 50 nM YKl-5-124 for 24 hours. E2F1 activity was assessed using the Promega luciferase reporter assay system, and fold change of E2F1 activity compared with untreated cells is displayed (mean ± SD). ∗∗∗P < .001. (D) H929 and AMO1 cells were treated with 250 nM YKL-5-124 for 24 hours, and the nuclear extract was analyzed by EMSA. The shifted probe caused by E2F1 binding is indicated by the band labeled E2F1. (E) H929 and AMO1 cells were treated with 500 nM YKL-5-124 for 24 hours and chromatin immunoprecipitated using E2F1 or control mouse IgG antibodies. The crosslinked DNA was subjected to quantitative polymerase chain reaction using primers specific for a representative set of E2F1 target genes. Data are represented as the percentage of input. (F) E2F score was calculated by using E2F1 genes identified previously.11 After RNA-seq normalization, we converted expression values for each gene to z scores, with a mean of 0 and SD of 1. After scaling the expression, we calculated the total score as the sum of scaled scores from all the genes. A Pearson correlation coefficient was calculated between E2F score and CDK7 expression. (G) AMO1 cells expressing control vector (PCW) or T121 were treated with doxycycline for 24 hours, followed by YKL-5-124 (500 nM) for 6 hours, and then chromatin immunoprecipitated using E2F1 or control mouse IgG antibodies. The crosslinked DNA was subjected to quantitative polymerase chain reaction using primers specific for a representative set of E2F1 target genes. Data are represented as the percentage of input. (H) AMO1 cells expressing either PCW or T121 plasmid were treated with doxycycline for 24 hours, followed by YKL-5-124 for 24 hours. The cell cycle was evaluated by propidium iodide staining followed by flow cytometric analysis. (I-J) MM cells expressing either control or T121 plasmid were treated with doxycycline for 24 hours followed by YKL-5-124 treatment for 48 hours. Cell viability was measured by CTG assay (I) and apoptosis by annexin V+ staining (J). Data represent the mean of 4 independent experiments. IgG, immunoglobulin G; SD, standard deviation.

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