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. 2020 Oct 5;130(11):5893–5908. doi: 10.1172/JCI136466

Figure 6. DCAF1 is required to prevent human T cell aging.

Figure 6

(A) The protein expression of DCAF1 and β-actin in human 293T cells transduced with lentivirus expressing 2 shRNAs targeting Dcaf1 and scrambled control for 4 days. The results are representative of 3 independent experiments. (B) Flow cytometry of ROS level in human T cells transduced with lentivirus expressing 2 shRNAs targeting Dcaf1 and scrambled control for 4 days, analyzed by 2′,7′-dichlorofluorescin diacetate (DCFDA) (gray area, no DCFDA; n = 4; representative results of 2 independent experiments are shown; means ± SD, **P < 0.01, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (C) SA-β-gal activity in human T cells transduced with lentivirus expressing 2 shRNAs targeting Dcaf1 and scrambled control for 6 days, assessed by flow cytometry with the fluorescent β-gal substrate C12FDG (gray area, no C12FDG; n = 4; representative flow cytometry results are shown; means ± SD, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (D) qRT-PCR analysis to determine mRNA expression of p16Ink4a in human T cells transduced with 2 shRNAs targeting Dcaf1 and scrambled control for 6 days (n = 6 from 2 independent experiments; means ± SD, **P < 0.01, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test).