Figure 1. THZ1 inhibition of CDK7 leads... | Rockefeller University Press

Figure 1.

$Figure 1. THZ1 inhibition of CDK7 leads to selective growth inhibition and apoptosis of TSC mutant cells. (A) Cell growth curves of TSC-deficient and TSC-intact cell lines treated with the indicated doses of THZ1. Cell number was calculated by measurement of double-stranded DNA (dsDNA) content using PicoGreen after 5 d in 96-well plate assays. Each data point represents the mean of four independent measurements. SEM is indicated. (B) Phase-contrast images of cells that were treated with vehicle control or THZ1 (30 nM) for 7 d. Note THZ1-induced death of TSC1 or TSC2 null cells, but not TSC wild-type cells (three independent experiments performed, and ≥10 images per field analyzed for each condition). Scale bar, 100 µm. (C) Images of crystal violet–stained dishes that were treated with vehicle control or 30 nM THZ1 for 10 d after plating 1,000 cells per well in a 12-well plate (three independent experiments performed). Scale bar, 2 mm. (D) Apoptotic cell fraction was determined after treatment with control (CTRL), rapamycin (RAP; 20 nM), THZ1 (30 nM), or a combination of both for 72 h. Apoptotic cell death was quantified by Annexin V/PI staining and flow cytometry and is shown as the percentage of cells that were PI positive. Each data point represents the mean ± SEM of three independent experiments. **, P < 0.01; ***, P < 0.001. (E) Immunoblot analysis shows that cleaved caspase 3 is increased in total protein lysates from the 621.101 TSC2-deficient cell line treated with THZ1 (30 nM) with or without rapamycin (Rap; 20 nM) for 72 h, but not in the TSC2-intact control line. β-Actin serves as a loading control (three independent experiments performed). (F) Immunoblot analysis shows that THZ1 inhibits RNAPII CTD phosphorylation in both TSC-deficient and TSC-intact cells. Cells were treated with vehicle control (first lane) or increasing concentrations of THZ1 (10, 30, 100, and 1,000 nM) for 4 h before lysates were prepared for immunoblotting (two independent experiments performed).$

THZ1 inhibition of CDK7 leads to selective growth inhibition and apoptosis of TSC mutant cells. (A) Cell growth curves of TSC-deficient and TSC-intact cell lines treated with the indicated doses of THZ1. Cell number was calculated by measurement of double-stranded DNA (dsDNA) content using PicoGreen after 5 d in 96-well plate assays. Each data point represents the mean of four independent measurements. SEM is indicated. (B) Phase-contrast images of cells that were treated with vehicle control or THZ1 (30 nM) for 7 d. Note THZ1-induced death of TSC1 or TSC2 null cells, but not TSC wild-type cells (three independent experiments performed, and ≥10 images per field analyzed for each condition). Scale bar, 100 µm. (C) Images of crystal violet–stained dishes that were treated with vehicle control or 30 nM THZ1 for 10 d after plating 1,000 cells per well in a 12-well plate (three independent experiments performed). Scale bar, 2 mm. (D) Apoptotic cell fraction was determined after treatment with control (CTRL), rapamycin (RAP; 20 nM), THZ1 (30 nM), or a combination of both for 72 h. Apoptotic cell death was quantified by Annexin V/PI staining and flow cytometry and is shown as the percentage of cells that were PI positive. Each data point represents the mean ± SEM of three independent experiments. **, P < 0.01; ***, P < 0.001. (E) Immunoblot analysis shows that cleaved caspase 3 is increased in total protein lysates from the 621.101 TSC2-deficient cell line treated with THZ1 (30 nM) with or without rapamycin (Rap; 20 nM) for 72 h, but not in the TSC2-intact control line. β-Actin serves as a loading control (three independent experiments performed). (F) Immunoblot analysis shows that THZ1 inhibits RNAPII CTD phosphorylation in both TSC-deficient and TSC-intact cells. Cells were treated with vehicle control (first lane) or increasing concentrations of THZ1 (10, 30, 100, and 1,000 nM) for 4 h before lysates were prepared for immunoblotting (two independent experiments performed).