Figure 1
Figure 1. S14161 inhibited D-cyclin expression. (A) The chemical structure of S14161. (B) S14161 inhibited cyclin D2 promoter transactivation. NIH3T3 cells were first transfected with pRSV.Luc and pCCD2.Luc, respectively. Twenty-four hours later, cells were treated with S14161 for 20 hours, followed by performance of a luciferase activity assay as described in “Luciferase assay.” Luc indicates luciferase; RSV, Rous sarcoma virus promoter; and CCND2, cyclin D2. (C) Myeloma (JJN3, KMS12, LP1, and RPMI-8226) and leukemia (OCI-AML2, K76A, U937, MDAY) cells were treated with 5μM S14161 (S1) or vehicle for 24 hours. After incubation, cells were harvested and total proteins isolated. Expression of cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin D3 (CCND3), β-actin, and tubulin was measured by immunoblotting. (D) KMS11 and K562 cells were treated with increasing concentrations of S1 for 24 hours. After incubation, cells were harvested and total proteins isolated. Expression of CCND2, CCND3, β-actin, and tubulin was measured by immunoblotting. (E) NIH3T3 cells were transfected with pcDNA3.1-CCND2 (under control of the cytomegalovirus promoter), followed by S1 treatment at indicated concentrations for 24 hours. Cells were harvested for CCND2 expression analysis. Tubulin was used as a loading control. (F) LP1 and AML2 cells were treated with 5μM S1 for 24 hours, and total mRNA was isolated. CCND2 (from LP1) and CCND3 (from AML2) expression was measured relative to 18S RNA by real-time reverse-transcription PCR. Data represent the mean ± SD percentage of D-cyclin expression relative to controls (ΔΔCT normalization; n = 3). (G) KMS11 cells were treated with increasing concentrations of S1. Twenty-four hours after incubation, cell cycle was measured by propidium iodide staining and flow cytometry. Data represent the mean ± SD percentage of cells at phases of the cell cycle (n = 3). A representative experiment is shown. (H) Myeloma cells OPM2 and LP1 were treated with S1 at indicated concentrations for 24 hours followed by cell lysates and immunoblotting assay against human CDK9–specific antibody. GAPDH was used as a loading control.

S14161 inhibited D-cyclin expression. (A) The chemical structure of S14161. (B) S14161 inhibited cyclin D2 promoter transactivation. NIH3T3 cells were first transfected with pRSV.Luc and pCCD2.Luc, respectively. Twenty-four hours later, cells were treated with S14161 for 20 hours, followed by performance of a luciferase activity assay as described in “Luciferase assay.” Luc indicates luciferase; RSV, Rous sarcoma virus promoter; and CCND2, cyclin D2. (C) Myeloma (JJN3, KMS12, LP1, and RPMI-8226) and leukemia (OCI-AML2, K76A, U937, MDAY) cells were treated with 5μM S14161 (S1) or vehicle for 24 hours. After incubation, cells were harvested and total proteins isolated. Expression of cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin D3 (CCND3), β-actin, and tubulin was measured by immunoblotting. (D) KMS11 and K562 cells were treated with increasing concentrations of S1 for 24 hours. After incubation, cells were harvested and total proteins isolated. Expression of CCND2, CCND3, β-actin, and tubulin was measured by immunoblotting. (E) NIH3T3 cells were transfected with pcDNA3.1-CCND2 (under control of the cytomegalovirus promoter), followed by S1 treatment at indicated concentrations for 24 hours. Cells were harvested for CCND2 expression analysis. Tubulin was used as a loading control. (F) LP1 and AML2 cells were treated with 5μM S1 for 24 hours, and total mRNA was isolated. CCND2 (from LP1) and CCND3 (from AML2) expression was measured relative to 18S RNA by real-time reverse-transcription PCR. Data represent the mean ± SD percentage of D-cyclin expression relative to controls (ΔΔCT normalization; n = 3). (G) KMS11 cells were treated with increasing concentrations of S1. Twenty-four hours after incubation, cell cycle was measured by propidium iodide staining and flow cytometry. Data represent the mean ± SD percentage of cells at phases of the cell cycle (n = 3). A representative experiment is shown. (H) Myeloma cells OPM2 and LP1 were treated with S1 at indicated concentrations for 24 hours followed by cell lysates and immunoblotting assay against human CDK9–specific antibody. GAPDH was used as a loading control.

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