American Society of Hematology

Figure 5.

$CDK4/6 inhibitor palbociclib inhibits ATLL cell proliferation and survival. (A) Viable cells were measured by the MTS assay for the indicated ATLL, ALK+ALCL, and MCL cell lines treated with palbociclib for 4 days. (B) Immunoblot analysis of phosphorylated Rb protein in ST1 and Su9T01 cells treated with palbociclib for 24 hours is shown. (C) DNA content was analyzed in ST1 ATLL cells treated with 1 μM of palbociclib for 24 hours. (D) Apoptotic cells were detected by analyzing Annexin V and propidium iodide (PI) on flowcytometry on day 4 after palbociclib treatment with 1 μM. (E) The ratio of PI(-)Annexin V(+) cells/PI(-) cells was monitored over time. (F) IC50 values for palbociclib in TP53-altered and TP53-intact ATLL cell lines are shown. (G) Viable cells were measured by the MTS assay for the indicated ATLL cell lines treated with the indicated concentrations of palbociclib and APR-246 for 4 days. (H) Cells were infected with a lentivirus expressing sgTP53 together with GFP, followed by treatment with palbociclib at the indicated concentrations. Shown is the fraction of GFP-positive cells at indicated time points normalized to day 0. (I) Cells were infected with a lentivirus expressing sgTP53 or control sgAAVS1 together with GFP, then treated with 1 μM of palbociclib for 24 hours. DNA content was analyzed in the GFP-positive fraction (blue, the cells have sgRNA) and GFP-negative cell fraction (red, the cells don’t have sgRNA). (J-K) Cells were infected with a lentivirus that expresses sgTP53 or control sgAAVS1 together with puromycin-resistant gene. After puromycin selection of the transduced cells, cells were grown in the culture media without puromycin for several days to recover the cell condition well. Then the cells were treated with 1 μM of palbociclib, and apoptotic cells were detected by analyzing Annexin V and propidium iodide (PI) on flowcytometry over time (J). Representative density plot illustrating the apoptotic cells on day 6 after palbociclib treatment (K). (L) Immunoblot analysis of p21 and p27 proteins in sgCDK6-transduced ST1. (M) Immunoblot analysis of p21 and p27 proteins in palbociclib-treated ATLL cell lines. TP53 status for each line is shown below the cell line name. (N) KK1 cells were infected with a lentivirus that expresses sgCDK2 or control sgAAVS1 together with GFP, followed by treatment with palbociclib (0.25 μM). Shown is the fraction of GFP-positive cells over time relative to the GFP-positive fraction on day 0. (O) ST1 cells were infected with a lentivirus expressing sgTP53 or control sgAAVS1 together with puromycin-resistance gene. After puromycin selection, cells were infected with a lentivirus expressing sgCDK2 or control sgAAVS1 together with GFP, followed by treatment with palbociclib (0.25 μM). Shown is the fraction of GFP-positive cells over time relative to the GFP-positive fraction on day 0. Error bars represent the SEM of replicates (A,E-H,J,N-O). **P < .01.$

CDK4/6 inhibitor palbociclib inhibits ATLL cell proliferation and survival. (A) Viable cells were measured by the MTS assay for the indicated ATLL, ALK+ALCL, and MCL cell lines treated with palbociclib for 4 days. (B) Immunoblot analysis of phosphorylated Rb protein in ST1 and Su9T01 cells treated with palbociclib for 24 hours is shown. (C) DNA content was analyzed in ST1 ATLL cells treated with 1 μM of palbociclib for 24 hours. (D) Apoptotic cells were detected by analyzing Annexin V and propidium iodide (PI) on flowcytometry on day 4 after palbociclib treatment with 1 μM. (E) The ratio of PI(-)Annexin V(+) cells/PI(-) cells was monitored over time. (F) IC50 values for palbociclib in TP53-altered and TP53-intact ATLL cell lines are shown. (G) Viable cells were measured by the MTS assay for the indicated ATLL cell lines treated with the indicated concentrations of palbociclib and APR-246 for 4 days. (H) Cells were infected with a lentivirus expressing sgTP53 together with GFP, followed by treatment with palbociclib at the indicated concentrations. Shown is the fraction of GFP-positive cells at indicated time points normalized to day 0. (I) Cells were infected with a lentivirus expressing sgTP53 or control sgAAVS1 together with GFP, then treated with 1 μM of palbociclib for 24 hours. DNA content was analyzed in the GFP-positive fraction (blue, the cells have sgRNA) and GFP-negative cell fraction (red, the cells don’t have sgRNA). (J-K) Cells were infected with a lentivirus that expresses sgTP53 or control sgAAVS1 together with puromycin-resistant gene. After puromycin selection of the transduced cells, cells were grown in the culture media without puromycin for several days to recover the cell condition well. Then the cells were treated with 1 μM of palbociclib, and apoptotic cells were detected by analyzing Annexin V and propidium iodide (PI) on flowcytometry over time (J). Representative density plot illustrating the apoptotic cells on day 6 after palbociclib treatment (K). (L) Immunoblot analysis of p21 and p27 proteins in sgCDK6-transduced ST1. (M) Immunoblot analysis of p21 and p27 proteins in palbociclib-treated ATLL cell lines. TP53 status for each line is shown below the cell line name. (N) KK1 cells were infected with a lentivirus that expresses sgCDK2 or control sgAAVS1 together with GFP, followed by treatment with palbociclib (0.25 μM). Shown is the fraction of GFP-positive cells over time relative to the GFP-positive fraction on day 0. (O) ST1 cells were infected with a lentivirus expressing sgTP53 or control sgAAVS1 together with puromycin-resistance gene. After puromycin selection, cells were infected with a lentivirus expressing sgCDK2 or control sgAAVS1 together with GFP, followed by treatment with palbociclib (0.25 μM). Shown is the fraction of GFP-positive cells over time relative to the GFP-positive fraction on day 0. Error bars represent the SEM of replicates (A,E-H,J,N-O). **P < .01.

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