Fig 2: Silencing SMC2 phenocopies the nuclear defects induced by CDK7i.A, seventy-two hours after siRNA silencing of SMC2, MDA-MB-231 cells were stained with DAPI (white) and phalloidin (red). Micronuclei, dysmorphic nuclei, and multiple nuclei are indicated by arrowheads. B, quantitation of SMC2 protein following siRNA silencing relative to total protein in MDA-MB-231 cells. C, quantitation of SMC2 relative to β-actin 72 h after siRNA transfection in MDA-MB-231 cells. Quantitation of nuclear phenotypes in MDA-MB-231 cells transfected with siRNA targeting SMC2. D–F, the same as in A–C, but in HCC38 cells. Horizontal solid lines are means; dashed lines are quartiles. Significance was evaluated by log-rank test. Three biological replicates were completed per experiment. ∗ = p < 0.05. G, overall survival (OS) curve in patients with breast cancer (all subtypes) generated with cBioPortal, stratified based on SMC2 expression; 60 patients in the high mRNA group (μ = 11.04) and 1022 patients in the low mRNA group (μ = 10.79).

Fig 3: CDK7 is necessary to maintain expression of genes that control mitotic fidelity including those comprising condensin.A, Venn diagram of genes that are differentially downregulated (left) or upregulated (right) in MDA-MB-231 and MDA-MB-468 cells treated with THZ1 (75 nM and 50 nM, respectively) compared with vehicle (DMSO). B, Gene Set Enrichment Analysis of genes in the cell cycle G2/M phase was evaluated in MDA-MB-231 (left) and MDA-MB-468 (right) cells. Normalized enrichment score (NES) and false discovery rate (FDR) are shown. C, table of significantly downregulated Gene Ontology pathways as assessed by evaluating the shared 1367 significantly downregulated genes in MDA-MB-231 and MDA-MB-468 cells. D, volcano plot of significant differentially expressed genes (DEGs) in MDA-MB-231 cells. Genes comprising the condensin complex are indicated by red dots with labels. E, representative Western blot of SMC2 protein in MDA-MB-231 cells treated with vehicle (DMSO), THZ1 (75 nM), or CT7001 (CT7, 750 nM). Values are the average of three replicates representing SMC2/β-actin. F, RT-PCR validation of condensin gene suppression in MDA-MB-231 cells treated as in E. G and H, same as E and F but using MDA-MB-468 cells treated with vehicle or THZ1 (50 nM). I and J, same as E and F but using HCC38 cells treated with vehicle or THZ1 (50 nM). K, RT-PCR validation of condensin gene suppression in HCC38 cells treated with CT7001 (CT7, 750 nM). L and M, MDA-MB-231 Cas9-expressing cells were transfected with nonspecific sgRNA (NTS) or sgRNA targeted to CDK7 (sgCDK7 #1, sgCDK7 #2) for 72 h. L, representative blot of SMC2 protein expression following CDK7 gene disruption. Numbers represent relative SMC2/β-actin ratios. β-Actin blot is reused from Figure 3A. This Western was probed for both SMC2 and CDK7 (to confirm CRISPR-mediated knockout of CDK7) with β-actin as the loading control for each. M. quantitation of SMC2 mRNA relative to GAPDH 72 h after transfection of MDA-MB-231 Cas9 cells with sgCDK7 versus nontargeting control. Bars are means ± SEM. At least three biological replicates were completed per experiment in duplicate. ∗ = p < 0.05.