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. 2022 Mar 10;139(10):1541–1556. doi: 10.1182/blood.2021012734

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Figure 4. — CDK6 and CCND2 are essential molecules in G1/S transition in ATLL cells. (A) Immunoblot analysis of CDK6 and RB protein in sgCDK6-transduced ST1. (B) The indicated cell lines were infected with a lentivirus that expresses sgCDK6_1 and sgCCND2_1 together with GFP. The GFP-positive cell fraction was monitored as in Figure 2A. (C) ST1 cells were transduced with retroviruses expressing an sgRNA-resistant CDK6, CDK4 together with puromycin-resistance gene or with an empty vector expressing puromycin-resistance gene. After puromycin selection of the transduced cells, cells were subsequently transduced with lentiviruses coexpressing GFP and either sgCDK6_1 or sgCDK6_2. sgAAVS1 and sgRPL6 were used as a negative and positive control sgRNA, respectively. The GFP-positive cell fraction was monitored as in Figure 2A. (D) Su9T01 cells were transduced with vectors coexpressing GFP and either sgCDK6_1 and sgCCND2_1. 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) on day 5 after sgRNA transduction. (E) The percentages of apoptotic cells were detected by analyzing Annexin V and propidium iodide (PI) on flowcytometry on day 5 after sgRNA transduction. (F) The ratio of PI(-)Annexin V(+) cells/PI(-) Su9T01 cells transduced with indicated sgRNAs were monitored over time. (G) ST1 cells were transduced with retroviruses expressing an sgRNA-resistant CCND2, CCND1, CCND3 together with puromycin-resistance gene or with an empty vector expressing puromycin-resistance gene. After puromycin selection of the transduced cells, cells were subsequently transduced with lentiviruses coexpressing GFP and either sgCCND2_1 or sg CCND2_2. sgAAVS1 and sgRPL6 were used as a negative and positive control sgRNA, respectively. The GFP-positive cell fraction was monitored as in Figure 2A. (H) Immunoblot analysis of CCND1, CCND2, CCND3, CDK4, and CDK6 proteins in ATLL and MCL cell lines. (I) CCND1, CCND2, CCND3, CDK4, and CDK6 mRNA expression values on indicated Affymetrix microarray probe in primary ATLL, ALK+ALCL, and PTCL-NOS samples. Error bars represent mean with the SD. Error bars represent the SEM of replicates (B-C,F-G). *P < .05, **P < .01, ***P < .001.

Figure 4. — CDK6 and CCND2 are essential molecules in G1/S transition in ATLL cells. (A) Immunoblot analysis of CDK6 and RB protein in sgCDK6-transduced ST1. (B) The indicated cell lines were infected with a lentivirus that expresses sgCDK6_1 and sgCCND2_1 together with GFP. The GFP-positive cell fraction was monitored as in Figure 2A. (C) ST1 cells were transduced with retroviruses expressing an sgRNA-resistant CDK6, CDK4 together with puromycin-resistance gene or with an empty vector expressing puromycin-resistance gene. After puromycin selection of the transduced cells, cells were subsequently transduced with lentiviruses coexpressing GFP and either sgCDK6_1 or sgCDK6_2. sgAAVS1 and sgRPL6 were used as a negative and positive control sgRNA, respectively. The GFP-positive cell fraction was monitored as in Figure 2A. (D) Su9T01 cells were transduced with vectors coexpressing GFP and either sgCDK6_1 and sgCCND2_1. 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) on day 5 after sgRNA transduction. (E) The percentages of apoptotic cells were detected by analyzing Annexin V and propidium iodide (PI) on flowcytometry on day 5 after sgRNA transduction. (F) The ratio of PI(-)Annexin V(+) cells/PI(-) Su9T01 cells transduced with indicated sgRNAs were monitored over time. (G) ST1 cells were transduced with retroviruses expressing an sgRNA-resistant CCND2, CCND1, CCND3 together with puromycin-resistance gene or with an empty vector expressing puromycin-resistance gene. After puromycin selection of the transduced cells, cells were subsequently transduced with lentiviruses coexpressing GFP and either sgCCND2_1 or sg CCND2_2. sgAAVS1 and sgRPL6 were used as a negative and positive control sgRNA, respectively. The GFP-positive cell fraction was monitored as in Figure 2A. (H) Immunoblot analysis of CCND1, CCND2, CCND3, CDK4, and CDK6 proteins in ATLL and MCL cell lines. (I) CCND1, CCND2, CCND3, CDK4, and CDK6 mRNA expression values on indicated Affymetrix microarray probe in primary ATLL, ALK+ALCL, and PTCL-NOS samples. Error bars represent mean with the SD. Error bars represent the SEM of replicates (B-C,F-G). *P < .05, **P < .01, ***P < .001.