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. 2019 May 30;13(1):76–90. doi: 10.1016/j.stemcr.2019.04.023

Figure 6.

Figure 6

CCL5 Promotes Cell Cycling and Cell Survival after Irradiation

(A) Left, representative flow cytometric analysis of KSL cells in G0 (Ki67−7-AAD−), G1 (Ki67+7-AAD−), and G2/S/M (Ki67+7-AAD+) phase at 72 h following 300 cGy in culture with CCL5 + TSF (CCL5) compared with TSF alone.

(B) Cell-cycle analysis of KSL cells at 72 h following 300 cGy in culture with CCL5 + TSF (CCL5) compared with TSF alone. n = 7–8 per group, ∗p = 0.009 and p = 0.005 for G0/G1 and G2/S/M, respectively.

(C) RT-PCR analysis of Cdk2, Cdk4, and Cdk6 mRNA expression from KSL cells at 72 h following 300 cGy and treatment with TSF or 30 ng/mL CCL5. n = 3 per group, ∗p = 0.0002, p = 0.004, and p = 0.004 for Cdk2, Cdk4, and Cdk6, respectively. Data are normalized to GAPDH and TSF for each Cdk.

(D) Schematic diagram of study design for Figures 6E–6G. At 24 h after 500 cGy TBI, mice were injected with saline or 0.1 μg/g body weight of CCL5 and analyzed at 2 h postinjection.

(E) RT-PCR analysis of Cdk2, Cdk4, and Cdk6 of BM Lin− cells. n = 4 per group, ∗p = 0.003, p < 0.0001, and p < 0.0001 for Cdk2, Cdk4, and Cdk6, respectively. Data are normalized to GAPDH and saline for each Cdk.

(F) Representative flow cytometric Ki67/7-AAD cell-cycle analysis from KSL cells following 500 cGy TBI and treatment with CCL5 or saline. Nonirradiated control is shown for comparison.

(G) Levels of G0/G1 and G2/S/M of KSL cells from irradiated CCL5- or saline-treated C57BL/6 mice. n = 5 per group, ∗p < 0.001 for G0/G1 and G2/S/M.

(H) Left, representative flow cytometric analysis of annexin V+ KSL cells and progeny at 72 h following 300 cGy in culture with CCL5 (blue line) compared with TSF alone (gray line). Right, quantification of total annexin V+ cells day 7 following 300 cGy and treatment with CCL5 or TSF alone. n = 3 per group, ∗p < 0.0001.

(I) Representative flow cytometric analysis and quantification of γ-H2AX in KSL and progeny following 300 cGy irradiation and 24 h culture with CCL5 (blue) or TSF alone (gray). Isotype is shown as black curve. n = 6 per group, ∗p = 0.02.

(J) Flow cytometric analysis of annexin V+ from KSL cells and progeny of Ccr5+/+ and Ccr5−/− mice at 72 h following 300 cGy. n = 6 per group, ∗p = 0.02. Data are shown as means ± SEM. Student's t test (two-tailed with unequal variance) was applied to the data.