Loss of CDK7 but not CDK12 or CDK13 selectively reduces growth of TSC-deficient cell lines. (A) Immunoblot analysis of cell lines in which CDK7 has been knocked out by either CRISPR/Cas9 (KO, left and middle) or shRNA (right; three independent experiments performed). (B) Dilutional clonal growth assays (top) show reduction in colony growth of TSC1-null or TSC2-null cells with CDK7 loss compared with control and TSC-intact cells, crystal violet staining. Quantification of crystal violet staining is shown for three pairs of TSC-null and TSC-intact cell lines. Error bars indicate SEM of triplicate wells from a representative experiment (three independent experiments performed). Scale bar, 2 mm. (C) Tumor volume of xenografts derived from HCV.29 cells infected with EV or CDK7.KO.1 or CDK7.KO.2 (two different gRNAs) at 51 d after injection. Cells were infected with lentivirus, selected with puromycin for 2 d, and then harvested for subcutaneous injection. 3 million HCV.29 (viability >94% for all groups, assayed by trypan blue exclusion) were subcutaneously injected into flanks of nude mice. Each data point represents the mean of tumor volume determined by caliper measurements ± SEM (n = 5 per group, two tumors per mouse). (D) Phase-contrast images of cells infected with virus encoding EV, CDK.KO.12, and CDK.KO.13. After infection and selection with puromycin (1.5 mg/ml, 96 h), cells were seeded in 6-well plates (5,000 cells per well for HCV.29.TSC1⁻ and HCV.29.TSC1⁺) and imaged with an inverted microscope (three independent experiments performed; ≥10 images per field were analyzed for each condition). Scale bar, 200 µm. (E) Quantification of relative cell number by PicoGreen assay in cells with KO of CDK7, CDK12, or CDK13, grown for 5 d. Each data point represents the mean of four independent experiments ± SEM. N.S., nonsignificant; ***, P < 0.001.