Figure 6
Figure 6. CDK9 inhibition or shRNA decrease MCL-1 levels and restore sensitivity to ABT-737. (A) Immunoblot analysis of OCI-LY1 R10 whole-cell lysates after 4 hours of flavopiridol treatment with different doses. Viability was assayed by annexin-V–FITC staining and flow cytometry. (B) OCI-LY1 and OCI-LY1 R10 cells were treated for 4 hours with DMSO or 300nM flavopiridol in combination with 0 or 250nM ABT-737, stained with annexin-V–FITC, and analyzed by flow cytometry. This experiment was performed in quadruplicate. (C) Immunoblot analysis of SU-DHL-4 R2 whole-cell lysates after 4 hours of flavopiridol treatment with different doses. (D) SU-DHL-4 and SU-DHL-4 R2 cells were treated for 12 hours with DMSO or 500nM ABT-737. During the last 4 hours of ABT-737 treatment, cells were treated with DMSO or 300nM flavopiridol. Cells were then stained with annexin-V–FITC and PI and analyzed by flow cytometry. The experiment was performed in triplicate. (E) Immunoblot analysis of MCL-1 knockdown by shRNA in single-cell clones. (F) Clones were chosen based on knockdown in panel E and tested for sensitivity by treatment with 500nM ABT-737 or DMSO. This experiment was performed in quadruplicate. All P values were determined by 2-tailed t tests. (G) Immunoblot analysis of OCI-LY1 R10 whole-cell lysates after 4 hours of PHA 767491 treatment with different doses. (H) OCI-LY1 and OCI-LY1 R10 cells were treated for 4 hours with DMSO or 3000nM PHA 767491 in combination with 0 or 1μM ABT-737, stained with annexin-V–FITC, and analyzed by flow cytometry. (I) Immunoblot analysis of SU-DHL-4 R2 whole-cell lysates after 4 hours of PHA 767491 treatment with different doses. (J) SU-DHL-4 and SU-DHL-4 R2 cells were treated for 12 hours with DMSO or 1μM ABT-737. During the last 4 hours of ABT-737 treatment, cells were treated with DMSO or 3000nM PHA 76741. Cells were then stained with annexin-V–FITC and PI and analyzed by flow cytometry. Error bars for panels B, D, and F represent SEM of independent quadruplicate, triplicate, and quadruplicate experiments, respectively. Error bars in panels H and J are representative of technical replicates, and the graphs presented are representative of 2 independent experiments.

CDK9 inhibition or shRNA decrease MCL-1 levels and restore sensitivity to ABT-737. (A) Immunoblot analysis of OCI-LY1 R10 whole-cell lysates after 4 hours of flavopiridol treatment with different doses. Viability was assayed by annexin-V–FITC staining and flow cytometry. (B) OCI-LY1 and OCI-LY1 R10 cells were treated for 4 hours with DMSO or 300nM flavopiridol in combination with 0 or 250nM ABT-737, stained with annexin-V–FITC, and analyzed by flow cytometry. This experiment was performed in quadruplicate. (C) Immunoblot analysis of SU-DHL-4 R2 whole-cell lysates after 4 hours of flavopiridol treatment with different doses. (D) SU-DHL-4 and SU-DHL-4 R2 cells were treated for 12 hours with DMSO or 500nM ABT-737. During the last 4 hours of ABT-737 treatment, cells were treated with DMSO or 300nM flavopiridol. Cells were then stained with annexin-V–FITC and PI and analyzed by flow cytometry. The experiment was performed in triplicate. (E) Immunoblot analysis of MCL-1 knockdown by shRNA in single-cell clones. (F) Clones were chosen based on knockdown in panel E and tested for sensitivity by treatment with 500nM ABT-737 or DMSO. This experiment was performed in quadruplicate. All P values were determined by 2-tailed t tests. (G) Immunoblot analysis of OCI-LY1 R10 whole-cell lysates after 4 hours of PHA 767491 treatment with different doses. (H) OCI-LY1 and OCI-LY1 R10 cells were treated for 4 hours with DMSO or 3000nM PHA 767491 in combination with 0 or 1μM ABT-737, stained with annexin-V–FITC, and analyzed by flow cytometry. (I) Immunoblot analysis of SU-DHL-4 R2 whole-cell lysates after 4 hours of PHA 767491 treatment with different doses. (J) SU-DHL-4 and SU-DHL-4 R2 cells were treated for 12 hours with DMSO or 1μM ABT-737. During the last 4 hours of ABT-737 treatment, cells were treated with DMSO or 3000nM PHA 76741. Cells were then stained with annexin-V–FITC and PI and analyzed by flow cytometry. Error bars for panels B, D, and F represent SEM of independent quadruplicate, triplicate, and quadruplicate experiments, respectively. Error bars in panels H and J are representative of technical replicates, and the graphs presented are representative of 2 independent experiments.

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